register interest

Professor Sarah C Gilbert

Research Area: Immunology
Technology Exchange: Cellular immunology and Vaccine production and evaluation
Scientific Themes: Immunology & Infectious Disease
Keywords: vaccine, T cell, immunology, malaria, tuberculosis and influenza
Web Links:
How the 'flu vaccine works - as shown on the BBC news website.

How the 'flu vaccine works - as shown on the BBC news website.

The Clinical Biomanufacturing Facility, University of Oxford

The Clinical Biomanufacturing Facility, University of Oxford

For more than ten years we have been making and testing vaccines designed to induce T cell responses to the antigens we encode, chiefly using antigens from malaria and tuberculosis. We have had most success with heterologous prime-boost regimes using either a DNA vaccine or recombinant fowlpox or adenovirus to prime a response and recombinant MVA to boost it. Several of the vaccines we have developed have progressed into clinical trials. We continue to work on finding the most efficient way to induce a protective T cell response by vaccination and are exploring a number of novel ways to do this.

Recombinant adenoviruses for clinical trials can now be produced to GMP by the University's Clinical Biomanufacturing Facility. Staff at the CNF work closely with academics to prepare batches of new vaccines for clinical trials.

In 2008 clinical trials of a new 'flu vaccine began in Oxford. Most adults already have memory T cell responses to 'flu antigens, but over time these fall below protective levels. The new vaccine, MVA-NP+M1 boosts these low level responses to very high levels. The next stage will be to test if these T cell responses can prevent people from being infected with 'flu.

Name Department Institution Country
Professor Adrian VS Hill Jenner Institute Oxford University, Old Road Campus Research Building United Kingdom
Dr Alfredo Nicosia Okairos Italy
Professor Glyn Hewinson Animal and Plant Health Agency United Kingdom
Rampling T, Ewer KJ, Bowyer G, Bliss CM, Edwards NJ, Wright D, Payne RO, Venkatraman N et al. 2016. Safety and High Level Efficacy of the Combination Malaria Vaccine Regimen of RTS,S/AS01B With Chimpanzee Adenovirus 63 and Modified Vaccinia Ankara Vectored Vaccines Expressing ME-TRAP. J Infect Dis, 214 (5), pp. 772-781. | Show Abstract | Read more

BACKGROUND: The need for a highly efficacious vaccine against Plasmodium falciparum remains pressing. In this controlled human malaria infection (CHMI) study, we assessed the safety, efficacy and immunogenicity of a schedule combining 2 distinct vaccine types in a staggered immunization regimen: one inducing high-titer antibodies to circumsporozoite protein (RTS,S/AS01B) and the other inducing potent T-cell responses to thrombospondin-related adhesion protein (TRAP) by using a viral vector. METHOD: Thirty-seven healthy malaria-naive adults were vaccinated with either a chimpanzee adenovirus 63 and modified vaccinia virus Ankara-vectored vaccine expressing a multiepitope string fused to TRAP and 3 doses of RTS,S/AS01B (group 1; n = 20) or 3 doses of RTS,S/AS01B alone (group 2; n = 17). CHMI was delivered by mosquito bites to 33 vaccinated subjects at week 12 after the first vaccination and to 6 unvaccinated controls. RESULTS: No suspected unexpected serious adverse reactions or severe adverse events related to vaccination were reported. Protective vaccine efficacy was observed in 14 of 17 subjects (82.4%) in group 1 and 12 of 16 subjects (75%) in group 2. All control subjects received a diagnosis of blood-stage malaria parasite infection. Both vaccination regimens were immunogenic. Fourteen protected subjects underwent repeat CHMI 6 months after initial CHMI; 7 of 8 (87.5%) in group 1 and 5 of 6 (83.3%) in group 2 remained protected. CONCLUSIONS: The high level of sterile efficacy observed in this trial is encouraging for further evaluation of combination approaches using these vaccine types. CLINICAL TRIALS REGISTRATION: NCT01883609.

Vanderven HA, Ana-Sosa-Batiz F, Jegaskanda S, Rockman S, Laurie K, Barr I, Chen W, Wines B et al. 2016. What Lies Beneath: Antibody Dependent Natural Killer Cell Activation by Antibodies to Internal Influenza Virus Proteins. EBioMedicine, 8 pp. 277-290. | Show Abstract | Read more

The conserved internal influenza proteins nucleoprotein (NP) and matrix 1 (M1) are well characterised for T cell immunity, but whether they also elicit functional antibodies capable of activating natural killer (NK) cells has not been explored. We studied NP and M1-specific ADCC activity using biochemical, NK cell activation and killing assays with plasma from healthy and influenza-infected subjects. Healthy adults had antibodies to M1 and NP capable of binding dimeric FcγRIIIa and activating NK cells. Natural symptomatic and experimental influenza infections resulted in a rise in antibody dependent NK cell activation post-infection to the hemagglutinin of the infecting strain, but changes in NK cell activation to M1 and NP were variable. Although antibody dependent killing of target cells infected with vaccinia viruses expressing internal influenza proteins was not detected, opsonising antibodies to NP and M1 likely contribute to an antiviral microenvironment by stimulating innate immune cells to secrete cytokines early in infection. We conclude that effector cell activating antibodies to conserved internal influenza proteins are common in healthy and influenza-infected adults. Given the significance of such antibodies in animal models of heterologous influenza infection, the definition of their importance and mechanism of action in human immunity to influenza is essential.

Colston JM, Bolinger B, Cottingham MG, Gilbert S, Klenerman P. 2016. Modification of Antigen Impacts on Memory Quality after Adenovirus Vaccination. J Immunol, 196 (8), pp. 3354-3363. | Show Abstract | Read more

The establishment of robust T cell memory is critical for the development of novel vaccines for infections and cancers. Classical memory generated by CD8(+)T cells is characterized by contracted populations homing to lymphoid organs. T cell memory inflation, as seen for example after CMV infection, is the maintenance of expanded, functional, tissue-associated effector memory cell pools. Such memory pools may also be induced after adenovirus vaccination, and we recently defined common transcriptional and phenotypic features of these populations in mice and humans. However, the rules that govern which epitopes drive memory inflation compared with classical memory are not fully defined, and thus it is not currently possible to direct this process. We used our adenoviral model of memory inflation to first investigate the role of the promoter and then the role of the epitope context in determining memory formation. Specifically, we tested the hypothesis that conventional memory could be converted to inflationary memory by simple presentation of the Ag in the form of minigene vectors. When epitopes from LacZ and murine CMV that normally induce classical memory responses were presented as minigenes, they induced clear memory inflation. These data demonstrate that, regardless of the transgene promoter, the polypeptide context of a CD8(+)T cell epitope may determine whether classical or inflating memory responses are induced. The ability to direct this process by the use of minigenes is relevant to the design of vaccines and understanding of immune responses to pathogens.

Mullin J, Ahmed MS, Sharma R, Upile N, Beer H, Achar P, Puksuriwong S, Ferrara F et al. 2016. Activation of cross-reactive mucosal T and B cell responses in human nasopharynx-associated lymphoid tissue in vitro by Modified Vaccinia Ankara-vectored influenza vaccines. Vaccine, 34 (14), pp. 1688-1695. | Show Abstract | Read more

UNLABELLED: Recent efforts have been focused on the development of vaccines that could induce broad immunity against influenza virus, either through T cell responses to conserved internal antigens or B cell response to cross-reactive haemagglutinin (HA). We studied the capacity of Modified Vaccinia Ankara (MVA)-vectored influenza vaccines to induce cross-reactive immunity to influenza virus in human nasopharynx-associated lymphoid tissue (NALT) in vitro. Adenotonsillar cells were isolated and stimulated with MVA vaccines expressing either conserved nucleoprotein (NP) and matrix protein 1 (M1) (MVA-NP-M1) or pandemic H1N1 HA (MVA-pdmH1HA). The MVA vaccine uptake and expression, and T and B cell responses were analyzed. MVA-vectored vaccines were highly efficient infecting NALT and vaccine antigens were highly expressed by B cells. MVA-NP-M1 elicited T cell response with greater numbers of IFNγ-producing CD4+ T cells and tissue-resident memory T cells than controls. MVA-pdmH1HA induced cross-reactive anti-HA antibodies to a number of influenza subtypes, in an age-dependent manner. The cross-reactive antibodies include anti-avian H5N1 and mainly target HA2 domain. CONCLUSION: MVA vaccines are efficient in infecting NALT and the vaccine antigen is highly expressed by B cells. MVA vaccines expressing conserved influenza antigens induce cross-reactive T and B cell responses in human NALT in vitro, suggesting the potential as mucosal vaccines for broader immunity against influenza.

Sebastian S, Gilbert SC. 2016. Recombinant modified vaccinia virus Ankara-based malaria vaccines Expert Review of Vaccines, 15 (1), pp. 91-103. | Show Abstract | Read more

© 2015 Taylor & Francis.A safe and effective malaria vaccine is a crucial part of the roadmap to malaria elimination/eradication by the year 2050. Viral-vectored vaccines based on adenoviruses and modified vaccinia virus Ankara (MVA) expressing malaria immunogens are currently being used in heterologous prime-boost regimes in clinical trials for induction of strong antigen-specific T-cell responses and high-titer antibodies. Recombinant MVA is a safe and well-tolerated attenuated vector that has consistently shown significant boosting potential. Advances have been made in large-scale MVA manufacture as high-yield producer cell lines and high-throughput purification processes have recently been developed. This review describes the use of MVA as malaria vaccine vector in both preclinical and clinical studies in the past 5 years.

Alharbi NK, Spencer AJ, Salman AM, Tully CM, Chinnakannan SK, Lambe T, Yamaguchi Y, Morris SJ et al. 2016. Enhancing cellular immunogenicity of MVA-vectored vaccines by utilizing the F11L endogenous promoter. Vaccine, 34 (1), pp. 49-55. | Show Abstract | Read more

Modified vaccinia virus Ankara (MVA)-vectored vaccines against malaria, influenza, tuberculosis and recently Ebola virus are in clinical development. Although this vector is safe and immunogenic in humans, efforts remain on-going to enhance immunogenicity through various approaches such as using stronger promoters to boost transgene expression. We previously reported that endogenous MVA promoters such as pB8 and pF11 increased transgene expression and immunogenicity, as compared to the conventional p7.5 promoter. Here, we show that both promoters also rivalled the mH5 promoter in enhancing MVA immunogenicity. We investigated the mechanisms behind this improved immunogenicity and show that it was a result of strong early transgene expression in vivo, rather than in vitro as would normally be assessed. Moreover, keeping the TK gene intact resulted in a modest improvement in immunogenicity. Utilizing pB8 or pF11 as ectopic promoters at the TK locus instead of their natural loci also increased transgene expression and immunogenicity. In addition to a reporter antigen, the pF11 promoter was tested with the expression of two vaccine antigens for which cellular immunogenicity was significantly increased as compared to the p7.5 promoter. Our data support the use of the pF11 and pB8 promoters for improved immunogenicity in future MVA-vectored candidate vaccines.

Lambe T, Rampling T, Samuel D, Bowyer G, Ewer KJ, Venkatraman N, Edmans M, Dicks S, Hill AV, Tedder RS, Gilbert SC. 2016. Detection of Vaccine-Induced Antibodies to Ebola Virus in Oral Fluid. Open Forum Infect Dis, 3 (1), pp. ofw031. | Show Abstract | Read more

Blood sampling to assess production of antigen-specific antibodies after immunization is commonly performed, but it presents logistical difficulties for trials carried out during an infectious disease outbreak. In this study, we show that antibodies may be reliably detected in oral fluid collected in a minimally invasive manner without use of sharps. Clinical Trials Registration. NCT02240875.

Warimwe GM, Gesharisha J, Carr BV, Otieno S, Otingah K, Wright D, Charleston B, Okoth E et al. 2016. Chimpanzee Adenovirus Vaccine Provides Multispecies Protection against Rift Valley Fever. Sci Rep, 6 pp. 20617. | Show Abstract | Read more

Rift Valley Fever virus (RVFV) causes recurrent outbreaks of acute life-threatening human and livestock illness in Africa and the Arabian Peninsula. No licensed vaccines are currently available for humans and those widely used in livestock have major safety concerns. A 'One Health' vaccine development approach, in which the same vaccine is co-developed for multiple susceptible species, is an attractive strategy for RVFV. Here, we utilized a replication-deficient chimpanzee adenovirus vaccine platform with an established human and livestock safety profile, ChAdOx1, to develop a vaccine for use against RVFV in both livestock and humans. We show that single-dose immunization with ChAdOx1-GnGc vaccine, encoding RVFV envelope glycoproteins, elicits high-titre RVFV-neutralizing antibody and provides solid protection against RVFV challenge in the most susceptible natural target species of the virus-sheep, goats and cattle. In addition we demonstrate induction of RVFV-neutralizing antibody by ChAdOx1-GnGc vaccination in dromedary camels, further illustrating the potency of replication-deficient chimpanzee adenovirus vaccine platforms. Thus, ChAdOx1-GnGc warrants evaluation in human clinical trials and could potentially address the unmet human and livestock vaccine needs.

Dicks MD, Spencer AJ, Coughlan L, Bauza K, Gilbert SC, Hill AV, Cottingham MG. 2015. Differential immunogenicity between HAdV-5 and chimpanzee adenovirus vector ChAdOx1 is independent of fiber and penton RGD loop sequences in mice. Sci Rep, 5 pp. 16756. | Show Abstract | Read more

Replication defective adenoviruses are promising vectors for the delivery of vaccine antigens. However, the potential of a vector to elicit transgene-specific adaptive immune responses is largely dependent on the viral serotype used. HAdV-5 (Human adenovirus C) vectors are more immunogenic than chimpanzee adenovirus vectors from species Human adenovirus E (ChAdOx1 and AdC68) in mice, though the mechanisms responsible for these differences in immunogenicity remain poorly understood. In this study, superior immunogenicity was associated with markedly higher levels of transgene expression in vivo, particularly within draining lymph nodes. To investigate the viral factors contributing to these phenotypes, we generated recombinant ChAdOx1 vectors by exchanging components of the viral capsid reported to be principally involved in cell entry with the corresponding sequences from HAdV-5. Remarkably, pseudotyping with the HAdV-5 fiber and/or penton RGD loop had little to no effect on in vivo transgene expression or transgene-specific adaptive immune responses despite considerable species-specific sequence heterogeneity in these components. Our results suggest that mechanisms governing vector transduction after intramuscular administration in mice may be different from those described in vitro.

Sebastian S, Gilbert SC. 2016. Recombinant modified vaccinia virus Ankara-based malaria vaccines. Expert Rev Vaccines, 15 (1), pp. 91-103. | Show Abstract | Read more

A safe and effective malaria vaccine is a crucial part of the roadmap to malaria elimination/eradication by the year 2050. Viral-vectored vaccines based on adenoviruses and modified vaccinia virus Ankara (MVA) expressing malaria immunogens are currently being used in heterologous prime-boost regimes in clinical trials for induction of strong antigen-specific T-cell responses and high-titer antibodies. Recombinant MVA is a safe and well-tolerated attenuated vector that has consistently shown significant boosting potential. Advances have been made in large-scale MVA manufacture as high-yield producer cell lines and high-throughput purification processes have recently been developed. This review describes the use of MVA as malaria vaccine vector in both preclinical and clinical studies in the past 5 years.

Gilbert SC. 2015. Adenovirus-vectored Ebola vaccines. Expert Rev Vaccines, 14 (10), pp. 1347-1357. | Show Abstract | Read more

The 2014 outbreak of Ebola virus disease in West Africa has highlighted the need for the availability of effective vaccines against outbreak pathogens that are suitable for use in frontline workers who risk their own health in the course of caring for those with the disease, and also for members of the community in the affected area. Along with effective contact tracing and quarantine, use of a vaccine as soon as an outbreak is identified could greatly facilitate rapid control and prevent the outbreak from spreading. This review describes the progress that has been made in producing and testing adenovirus-based Ebola vaccines in both pre-clinical and clinical studies, and considers the likely future use of these vaccines.

Gilbert SC. 2015. Adenovirus-vectored Ebola vaccines. Expert Rev Vaccines, 14 (10), pp. 1-11. | Show Abstract | Read more

The 2014 outbreak of Ebola virus disease in West Africa has highlighted the need for the availability of effective vaccines against outbreak pathogens that are suitable for use in frontline workers who risk their own health in the course of caring for those with the disease, and also for members of the community in the affected area. Along with effective contact tracing and quarantine, use of a vaccine as soon as an outbreak is identified could greatly facilitate rapid control and prevent the outbreak from spreading. This review describes the progress that has been made in producing and testing adenovirus-based Ebola vaccines in both pre-clinical and clinical studies, and considers the likely future use of these vaccines.

Hodgson SH, Douglas AD, Edwards NJ, Kimani D, Elias SC, Chang M, Daza G, Seilie AM et al. 2015. Increased sample volume and use of quantitative reverse-transcription PCR can improve prediction of liver-to-blood inoculum size in controlled human malaria infection studies. Malar J, 14 (1), pp. 33. | Show Abstract | Read more

BACKGROUND: Controlled human malaria infection (CHMI) studies increasingly rely on nucleic acid test (NAT) methods to detect and quantify parasites in the blood of infected participants. The lower limits of detection and quantification vary amongst the assays used throughout the world, which may affect the ability of mathematical models to accurately estimate the liver-to-blood inoculum (LBI) values that are used to judge the efficacy of pre-erythrocytic vaccine and drug candidates. METHODS: Samples were collected around the time of onset of pre-patent parasitaemia from subjects who enrolled in two different CHMI clinical trials. Blood samples were tested for Plasmodium falciparum 18S rRNA and/or rDNA targets by different NAT methods and results were compared. Methods included an ultrasensitive, large volume modification of an established quantitative reverse transcription PCR (qRT-PCR) assay that achieves detection of as little as one parasite/mL of whole blood. RESULTS: Large volume qRT-PCR at the University of Washington was the most sensitive test and generated quantifiable data more often than any other NAT methodology. Standard quantitative PCR (qPCR) performed at the University of Oxford and standard volume qRT-PCR performed at the University of Washington were less sensitive than the large volume qRT-PCR, especially at 6.5 days after CHMI. In these trials, the proportion of participants for whom LBI could be accurately quantified using parasite density value greater than or equal to the lower limit of quantification was increased. A greater improvement would be expected in trials in which numerous subjects receive a lower LBI or low dose challenge. CONCLUSIONS: Standard qPCR and qRT-PCR methods with analytical sensitivities of ~20 parasites/mL probably suffice for most CHMI purposes, but the newly developed large volume qRT-PCR may be able to answer specific questions when more analytical sensitivity is required.

Ewer K, Rampling T, Venkatraman N, Bowyer G, Wright D, Lambe T, Imoukhuede EB, Payne R et al. 2016. A Monovalent Chimpanzee Adenovirus Ebola Vaccine Boosted with MVA. N Engl J Med, 374 (17), pp. 1635-1646. | Show Abstract | Read more

BACKGROUND: The West African outbreak of Ebola virus disease that peaked in 2014 has caused more than 11,000 deaths. The development of an effective Ebola vaccine is a priority for control of a future outbreak. METHODS: In this phase 1 study, we administered a single dose of the chimpanzee adenovirus 3 (ChAd3) vaccine encoding the surface glycoprotein of Zaire ebolavirus (ZEBOV) to 60 healthy adult volunteers in Oxford, United Kingdom. The vaccine was administered in three dose levels--1×10(10) viral particles, 2.5×10(10) viral particles, and 5×10(10) viral particles--with 20 participants in each group. We then assessed the effect of adding a booster dose of a modified vaccinia Ankara (MVA) strain, encoding the same Ebola virus glycoprotein, in 30 of the 60 participants and evaluated a reduced prime-boost interval in another 16 participants. We also compared antibody responses to inactivated whole Ebola virus virions and neutralizing antibody activity with those observed in phase 1 studies of a recombinant vesicular stomatitis virus-based vaccine expressing a ZEBOV glycoprotein (rVSV-ZEBOV) to determine relative potency and assess durability. RESULTS: No safety concerns were identified at any of the dose levels studied. Four weeks after immunization with the ChAd3 vaccine, ZEBOV-specific antibody responses were similar to those induced by rVSV-ZEBOV vaccination, with a geometric mean titer of 752 and 921, respectively. ZEBOV neutralization activity was also similar with the two vaccines (geometric mean titer, 14.9 and 22.2, respectively). Boosting with the MVA vector increased virus-specific antibodies by a factor of 12 (geometric mean titer, 9007) and increased glycoprotein-specific CD8+ T cells by a factor of 5. Significant increases in neutralizing antibodies were seen after boosting in all 30 participants (geometric mean titer, 139; P<0.001). Virus-specific antibody responses in participants primed with ChAd3 remained positive 6 months after vaccination (geometric mean titer, 758) but were significantly higher in those who had received the MVA booster (geometric mean titer, 1750; P<0.001). CONCLUSIONS: The ChAd3 vaccine boosted with MVA elicited B-cell and T-cell immune responses to ZEBOV that were superior to those induced by the ChAd3 vaccine alone. (Funded by the Wellcome Trust and others; ClinicalTrials.gov number, NCT02240875.).

Dicks MD, Guzman E, Spencer AJ, Gilbert SC, Charleston B, Hill AV, Cottingham MG. 2015. The relative magnitude of transgene-specific adaptive immune responses induced by human and chimpanzee adenovirus vectors differs between laboratory animals and a target species. Vaccine, 33 (9), pp. 1121-1128. | Show Abstract | Read more

Adenovirus vaccine vectors generated from new viral serotypes are routinely screened in pre-clinical laboratory animal models to identify the most immunogenic and efficacious candidates for further evaluation in clinical human and veterinary settings. Here, we show that studies in a laboratory species do not necessarily predict the hierarchy of vector performance in other mammals. In mice, after intramuscular immunization, HAdV-5 (Human adenovirus C) based vectors elicited cellular and humoral adaptive responses of higher magnitudes compared to the chimpanzee adenovirus vectors ChAdOx1 and AdC68 from species Human adenovirus E. After HAdV-5 vaccination, transgene specific IFN-γ(+) CD8(+) T cell responses reached peak magnitude later than after ChAdOx1 and AdC68 vaccination, and exhibited a slower contraction to a memory phenotype. In cattle, cellular and humoral immune responses were at least equivalent, if not higher, in magnitude after ChAdOx1 vaccination compared to HAdV-5. Though we have not tested protective efficacy in a disease model, these findings have important implications for the selection of candidate vectors for further evaluation. We propose that vaccines based on ChAdOx1 or other Human adenovirus E serotypes could be at least as immunogenic as current licensed bovine vaccines based on HAdV-5.

Coughlan L, Mullarkey C, Gilbert S. 2015. Adenoviral vectors as novel vaccines for influenza. J Pharm Pharmacol, 67 (3), pp. 382-399. | Show Abstract | Read more

OBJECTIVES: Influenza is a viral respiratory disease causing seasonal epidemics, with significant annual illness and mortality. Emerging viruses can pose a major pandemic threat if they acquire the capacity for sustained human-to-human transmission. Vaccination reduces influenza-associated mortality and is critical in minimising the burden on the healthcare system. However, current vaccines are not always effective in at-risk populations and fail to induce long-lasting protective immunity against a range of viruses. KEY FINDINGS: The development of 'universal' influenza vaccines, which induce heterosubtypic immunity capable of reducing disease severity, limiting viral shedding or protecting against influenza subtypes with pandemic potential, has gained interest in the research community. To date, approaches have focused on inducing immune responses to conserved epitopes within the stem of haemagglutinin, targeting the ectodomain of influenza M2e or by stimulating cellular immunity to conserved internal antigens, nucleoprotein or matrix protein 1. SUMMARY: Adenoviral vectors are potent inducers of T-cell and antibody responses and have demonstrated safety in clinical applications, making them an excellent choice of vector for delivery of vaccine antigens. In order to circumvent pre-existing immunity in humans, serotypes from non-human primates have recently been investigated. We will discuss the pre-clinical development of these novel vectors and their advancement to clinical trials.

Rimmelzwaan G, Leroux-Roels I, Gilbert S, Thomson A. 2015. Influenza vaccines: Where do we stand? Where do we go?: A bumpy but steady road upwards. Vaccine, 33 (49), pp. 7026-7028. | Read more

Alharbi NK, Spencer AJ, Hill AV, Gilbert SC. 2015. Deletion of Fifteen Open Reading Frames from Modified Vaccinia Virus Ankara Fails to Improve Immunogenicity. PLoS One, 10 (6), pp. e0128626. | Show Abstract | Read more

Modified vaccinia virus Ankara (MVA) is a highly attenuated strain of vaccinia virus, which has been used as a recombinant vaccine vector in many vaccine development programmes. The loss of many immunosuppressive and host-range genes resulted in a safe and immunogenic vaccine vector. However it still retains some immunomodulatory genes that may reduce MVA immunogenicity. Earlier reports demonstrated that the deletion of the A41L, B15R, C6L, or C12L open reading frames (ORFs) enhanced cellular immune responses in recombinant MVA (rMVA) by up to 2-fold. However, previously, we showed that deletion of the C12L, A44L, A46R, B7R, or B15R ORFs from rMVA, using MVA-BAC recombineering technology, did not enhance rMVA immunogenicity at either peak or memory cellular immune responses. Here, we extend our previous study to examine the effect of deleting clusters of genes on rMVA cellular immunogenicity. Two clusters of fifteen genes were deleted in one rMVA mutant that encodes either the 85A antigen of Mycobacterium tuberculosis or an immunodominant H2-Kd-restricted murine malaria epitope (pb9). The deletion mutants were tested in prime only or prime and boost vaccination regimens. The responses showed no improved peak or memory CD8+ T cell frequencies. Our results suggest that the reported small increases in MVA deletion mutants could not be replicated with different antigens, or epitopes. Therefore, the gene deletion strategy may not be taken as a generic approach for improving the immunogenicity of MVA-based vaccines, and should be carefully assessed for every individual recombinant antigen.

Kapulu MC, Da DF, Miura K, Li Y, Blagborough AM, Churcher TS, Nikolaeva D, Williams AR et al. 2015. Comparative assessment of transmission-blocking vaccine candidates against Plasmodium falciparum. Sci Rep, 5 pp. 11193. | Show Abstract | Read more

Malaria transmission-blocking vaccines (TBVs) target the development of Plasmodium parasites within the mosquito, with the aim of preventing malaria transmission from one infected individual to another. Different vaccine platforms, mainly protein-in-adjuvant formulations delivering the leading candidate antigens, have been developed independently and have reported varied transmission-blocking activities (TBA). Here, recombinant chimpanzee adenovirus 63, ChAd63, and modified vaccinia virus Ankara, MVA, expressing AgAPN1, Pfs230-C, Pfs25, and Pfs48/45 were generated. Antibody responses primed individually against all antigens by ChAd63 immunization in BALB/c mice were boosted by the administration of MVA expressing the same antigen. These antibodies exhibited a hierarchy of inhibitory activity against the NF54 laboratory strain of P. falciparum in Anopheles stephensi mosquitoes using the standard membrane feeding assay (SMFA), with anti-Pfs230-C and anti-Pfs25 antibodies giving complete blockade. The observed rank order of inhibition was replicated against P. falciparum African field isolates in A. gambiae in direct membrane feeding assays (DMFA). TBA achieved was IgG concentration dependent. This study provides the first head-to-head comparative analysis of leading antigens using two different parasite sources in two different vector species, and can be used to guide selection of TBVs for future clinical development using the viral-vectored delivery platform.

Alharbi NK, Chinnakannan SK, Gilbert SC, Draper SJ. 2015. Investigation of IRES Insertion into the Genome of Recombinant MVA as a Translation Enhancer in the Context of Transcript Decapping. PLoS One, 10 (5), pp. e0127978. | Show Abstract | Read more

Recombinant modified vaccinia virus Ankara (MVA) has been used to deliver vaccine candidate antigens against infectious diseases and cancer. MVA is a potent viral vector for inducing high magnitudes of antigen-specific CD8+ T cells; however the cellular immune responses to a recombinant antigen in MVA could be further enhanced by increasing transgene expression. Previous reports showed the importance of utilizing an early poxviral promoter for increasing transgene expression and therefore enhancing cellular immune responses. However, the vaccinia D10 decapping enzyme is reported to target and decap vaccinia virus early transcripts - a mechanism that could limit the usefulness of early promoters in MVA viral vectors if this enzyme shows the same activity in this closely related virus. Therefore, we attempted to increase transgene expression in recombinant MVA by inserting the encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES) upstream of a transgene sequence that is controlled by the B8R early promoter, and assessed D10 enzyme decapping activity in MVA. The aim of the IRES element was to initiate translation of the transgene transcript (after the removal of the cap structure by the D10 decapping protein) in a cap-independent manner. Here, we report that overexpression of the D10 decapping protein, in trans, in MVA reduced growth and transgene expression; however, the IRES element was not able to compensate for the negative effect of the D10 decapping protein. Recombinant MVA with EMCV IRES induced levels of both gene expression and transcription that were similar to the control recombinant MVA, encoding the same transgene but without the IRES element. Both viruses were tested in BALB/c mice and induced similar magnitudes of epitope-specific CD8+ T cells. This work indicates that the MVA version of the D10 decapping enzyme, overexpressed using a plasmid, is functional, but its negative effect on transgene expression by recombinant MVA cannot be overcome by the use of the EMCV IRES inserted upstream of the transgene initiation codon.

Tully CM, Lambe T, Gilbert SC, Hill AV. 2015. Emergency Ebola response: a new approach to the rapid design and development of vaccines against emerging diseases. Lancet Infect Dis, 15 (3), pp. 356-359. | Show Abstract | Read more

The epidemic of Ebola virus disease has spread at an alarming rate despite containment efforts. As a result, unprecedented large-scale international response efforts have been made in an attempt to gain control of the outbreak and reduce transmission. Several international consortia have been formed in a remarkable worldwide collaborative effort to expedite trials of two candidate Ebola virus vaccines: cAd3-EBOZ and rVSV-EBOV. In parallel, both vaccines are being manufactured in large amounts to enable future rapid deployment for management of the crisis.

Mullin J, Ahmed MS, Upile N, Vaughan C, McNamara P, Beer H, McCormick M, Lambe T, Gilbert SC, Zhang Q. 2014. Evaluating the potential of novel virus-vectored vaccines as intranasal vaccine candidates for broad immunity against influenza IMMUNOLOGY, 143 pp. 78-78.

Pavot V, Berthet M, Rességuier J, Legaz S, Handké N, Gilbert SC, Paul S, Verrier B. 2014. Poly(lactic acid) and poly(lactic-co-glycolic acid) particles as versatile carrier platforms for vaccine delivery. Nanomedicine (Lond), 9 (17), pp. 2703-2718. | Show Abstract | Read more

The development of safe and effective vaccines for cancer and infectious diseases remains a major goal in public health. Over the last two decades, controlled release of vaccine antigens and immunostimulant molecules has been achieved using nanometer or micron-sized delivery vehicles synthesized using biodegradable polymers. In addition to achieving a depot effect, enhanced vaccine efficacy using such delivery vehicles has been attributed to efficient targeting of antigen presenting cells such as dendritic cells. Biodegradable and biocompatible poly(lactic acid) and poly(lactic-co-glycolic acid) polymers belong to one such family of polymers that have been a popular choice of material used in the design of these delivery vehicles. This review summarizes research findings from ourselves and others highlighting the promise of poly(lactic acid)- and poly(lactic-co-glycolic acid)-based vaccine carriers in enhancing immune responses.

Bull TJ, Vrettou C, Linedale R, McGuinnes C, Strain S, McNair J, Gilbert SC, Hope JC. 2014. Immunity, safety and protection of an Adenovirus 5 prime--Modified Vaccinia virus Ankara boost subunit vaccine against Mycobacterium avium subspecies paratuberculosis infection in calves. Vet Res, 45 (1), pp. 112. | Show Abstract | Read more

Vaccination is the most cost effective control measure for Johne's disease caused by Mycobacterium avium subspecies paratuberculosis (MAP) but currently available whole cell killed formulations have limited efficacy and are incompatible with the diagnosis of bovine tuberculosis by tuberculin skin test. We have evaluated the utility of a viral delivery regimen of non-replicative human Adenovirus 5 and Modified Vaccinia virus Ankara recombinant for early entry MAP specific antigens (HAV) to show protection against challenge in a calf model and extensively screened for differential immunological markers associated with protection. We have shown that HAV vaccination was well tolerated, could be detected using a differentiation of infected and vaccinated animals (DIVA) test, showed no cross-reactivity with tuberculin and provided a degree of protection against challenge evidenced by a lack of faecal shedding in vaccinated animals that persisted throughout the 7 month infection period. Calves given HAV vaccination had significant priming and boosting of MAP derived antigen (PPD-J) specific CD4+, CD8+ IFN-γ producing T-cell populations and, upon challenge, developed early specific Th17 related immune responses, enhanced IFN-γ responses and retained a high MAP killing capacity in blood. During later phases post MAP challenge, PPD-J antigen specific IFN-γ and Th17 responses in HAV vaccinated animals corresponded with improvements in peripheral bacteraemia. By contrast a lack of IFN-γ, induction of FoxP3+ T cells and increased IL-1β and IL-10 secretion were indicative of progressive infection in Sham vaccinated animals. We conclude that HAV vaccination shows excellent promise as a new tool for improving control of MAP infection in cattle.

Davenport EE, Antrobus RD, Lillie PJ, Gilbert S, Knight JC. 2014. Transcriptomic profiling facilitates classification of response to influenza challenge Journal of Molecular Medicine, 93 (1), pp. 105-114. | Show Abstract | Read more

© The Author(s) 2014.Despite increases in vaccination coverage, reductions in influenza-related mortality have not been observed. Better vaccines are therefore required and influenza challenge studies can be used to test the efficacy of new vaccines. However, this requires the accurate post-challenge classification of subjects by outcome, which is limited in current methods that use artificial thresholds to assign ‘symptomatic’ and ‘asymptomatic’ phenotypes.We present data from an influenza challenge study in which 22 healthy adults (11 vaccinated) were inoculated with H3N2 influenza (A/Wisconsin/67/2005). We generated genome-wide gene expression data from peripheral blood taken immediately before the challenge and at 12, 24 and 48 h post-challenge. Variation in symptomatic scoring was found amongst those with laboratory confirmed influenza. By combining the dynamic transcriptomic data with the clinical parameters this variability can be reduced. We identified four subjects with severe laboratory confirmed influenza that show differential gene expression in 1103 probes 48 h post-challenge compared to the remaining subjects. We have further reduced this profile to six genes (CCL2, SEPT4, LAMP3, RTP4, MT1G and OAS3) that can be used to define these subjects. We have used this gene set to predict symptomatic infection from an independent study. This analysis gives further insight into host-pathogen interactions during influenza infection. However, the major potential value is in the clinical trial setting by providing a more quantitative method to better classify symptomatic individuals post influenza challenge. Key message & Differential gene expression signatures are seen following influenza challenge. & Expression of six predictive genes can classify response to influenza challenge. & The genomic influenza response classification replicates in an independent dataset.

Davenport EE, Antrobus RD, Lillie PJ, Gilbert S, Knight JC. 2015. Transcriptomic profiling facilitates classification of response to influenza challenge. J Mol Med (Berl), 93 (1), pp. 105-114. | Show Abstract | Read more

UNLABELLED: Despite increases in vaccination coverage, reductions in influenza-related mortality have not been observed. Better vaccines are therefore required and influenza challenge studies can be used to test the efficacy of new vaccines. However, this requires the accurate post-challenge classification of subjects by outcome, which is limited in current methods that use artificial thresholds to assign 'symptomatic' and 'asymptomatic' phenotypes. We present data from an influenza challenge study in which 22 healthy adults (11 vaccinated) were inoculated with H3N2 influenza (A/Wisconsin/67/2005). We generated genome-wide gene expression data from peripheral blood taken immediately before the challenge and at 12, 24 and 48 h post-challenge. Variation in symptomatic scoring was found amongst those with laboratory confirmed influenza. By combining the dynamic transcriptomic data with the clinical parameters this variability can be reduced. We identified four subjects with severe laboratory confirmed influenza that show differential gene expression in 1103 probes 48 h post-challenge compared to the remaining subjects. We have further reduced this profile to six genes (CCL2, SEPT4, LAMP3, RTP4, MT1G and OAS3) that can be used to define these subjects. We have used this gene set to predict symptomatic infection from an independent study. This analysis gives further insight into host-pathogen interactions during influenza infection. However, the major potential value is in the clinical trial setting by providing a more quantitative method to better classify symptomatic individuals post influenza challenge. KEY MESSAGE: Differential gene expression signatures are seen following influenza challenge. Expression of six predictive genes can classify response to influenza challenge. The genomic influenza response classification replicates in an independent dataset.

Busquets N, Lorenzo G, Lopez-Gil E, Rivas R, Solanes D, Galindo-Cardiel I, Xavier Abad F, Rodriguez F et al. 2014. Efficacy assessment of an MVA vectored Rift Valley Fever vaccine in lambs ANTIVIRAL RESEARCH, 108 pp. 165-172. | Read more

Kimani D, Jagne YJ, Cox M, Kimani E, Bliss CM, Gitau E, Ogwang C, Afolabi MO et al. 2014. Translating the immunogenicity of prime-boost immunization with ChAd63 and MVA ME-TRAP from malaria naive to malaria-endemic populations. Mol Ther, 22 (11), pp. 1992-2003. | Show Abstract | Read more

To induce a deployable level of efficacy, a successful malaria vaccine would likely benefit from both potent cellular and humoral immunity. These requirements are met by a heterologous prime-boost immunization strategy employing a chimpanzee adenovirus vector followed by modified vaccinia Ankara (MVA), both encoding the pre-erythrocytic malaria antigen ME-thrombospondin-related adhesive protein (TRAP), with high immunogenicity and significant efficacy in UK adults. We undertook two phase 1b open-label studies in adults in Kenya and The Gambia in areas of similar seasonal malaria transmission dynamics and have previously reported safety and basic immunogenicity data. We now report flow cytometry and additional interferon (IFN)-γ enzyme-linked immunospot (ELISPOT) data characterizing pre-existing and induced cellular immunity as well as anti-TRAP IgG responses. T-cell responses induced by vaccination averaged 1,254 spot-forming cells (SFC) per million peripheral blood mononuclear cells (PBMC) across both trials and flow cytometry revealed cytokine production from both CD4(+) and CD8(+) T cells with the frequency of CD8(+) IFN-γ-secreting monofunctional T cells (previously shown to associate with vaccine efficacy) particularly high in Kenyan adults. Immunization with ChAd63 and MVA ME-TRAP induced strong cellular and humoral immune responses in adults living in two malaria-endemic regions of Africa. This prime-boost approach targeting the pre-erythrocytic stage of the malaria life-cycle is now being assessed for efficacy in a target population.

Busquets N, Lorenzo G, López-Gil E, Rivas R, Solanes D, Galindo-Cardiel I, Abad FX, Rodríguez F et al. 2014. Efficacy assessment of an MVA vectored Rift Valley Fever vaccine in lambs. Antiviral Res, 108 (1), pp. 165-172. | Show Abstract | Read more

The present study has evaluated the protection conferred by a single subcutaneous dose of a modified vaccinia virus Ankara (MVA) vectored vaccine encoding the Rift Valley Fever virus (RVFV) glycoproteins Gn and Gc in lambs. Three groups of six to seven lambs were immunized as follows: one group received the vaccine (termed rMVA-GnGc), a second group received an MVA vector (vector control) and a third group received saline solution (non-vaccinated control). Fourteen days later, all animals were subcutaneously challenged with 10(5) TCID50 of the virulent RVFV isolate 56/74 and vaccine efficacy assessed using standard endpoints. Two lambs (one from the vaccine group and one from the vector control group) succumbed to RVFV challenge, showing characteristic liver lesions. Lambs from both the vector control and non-vaccinated groups were febrile from days 2 to 5 post challenge (pc) while those in the rMVA-GnGc group showed a single peak of pyrexia at day 3 pc. RVFV RNA was detected in both nasal and oral swabs from days 3 to 7 pc in some lambs from the vector control and non-vaccinated groups, but no viral shedding could be detected in the surviving lambs vaccinated with rMVA-GnGc. Together, the data suggest that a single dose of the rMVA-GnGc vaccine may be sufficient to reduce RVFV shedding and duration of viremia but does not provide sterile immunity nor protection from disease. Further optimization of this vaccine approach in lambs is warranted.

Gilbert S. 2014. Rapid vaccine development from concept to phase I HUMAN GENE THERAPY, 25 (5), pp. A3-A3.

Dean G, Clifford D, Gilbert S, McShane H, Hewinson RG, Vordermeier HM, Villarreal-Ramos B. 2014. Effect of dose and route of immunisation on the immune response induced in cattle by heterologous Bacille Calmette-Guerin priming and recombinant adenoviral vector boosting Veterinary Immunology and Immunopathology, 158 (3-4), pp. 208-213. | Show Abstract | Read more

BCG is used experimentally as a vaccine against tuberculosis (TB), induced by Mycobacterium bovis, in cattle (bTB). However, the efficacy of BCG is variable in humans, cattle and guinea pigs. An adenoviral vector expressing Antigen 85A (Ad5Ag85A) has enhanced protection against TB in mice when used in combination with BCG for prime-boost experiments. However, the route of immunisation affects the degree of protection seen. This work examines the immunogenicity of a new vectored vaccine (Ad5-TBF) that expresses Ag85A, Rv0287, Rv0288 and Rv0251c to explore the effects of dose of adenoviral boost and route of inoculation on immunogenicity. We found that 2×109 infectious units (iu) delivered intradermally conferred the most consistent and strongest responses of the different regimes tested. © 2014 .

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Dean G, Whelan A, Clifford D, Salguero FJ, Xing Z, Gilbert S, McShane H, Hewinson RG, Vordermeier M, Villarreal-Ramos B. 2014. Comparison of the immunogenicity and protection against bovine tuberculosis following immunization by BCG-riming and boosting with adenovirus or protein based vaccines Vaccine, 32 (11), pp. 1304-1310. | Show Abstract | Read more

There is a requirement for vaccines or vaccination strategies that confer better protection against TB than the current live attenuated Mycobacterium bovis Bacillus Calmette-Guerin (BCG) vaccine for use in cattle. Boosting with recombinant viral vectors expressing mycobacterial proteins, such as Ag85A, has shown a degree of promise as a strategy for improving on the protection afforded by BCG. Experiments in small animal models have indicated that broadening the immune response to include mycobacterial antigens other than Ag85A, such as Rv0288, induced by boosting with Ad5 constructs has a direct effect on the protection afforded against TB. Here, we compared the immunogenicity and protection against challenge with M. bovis afforded by boosting BCG-vaccinated cattle with a human type 5 (Ad5)-based vaccine expressing the mycobacterial antigens Ag85A (Ad5-85A); or Ag85A, Rv0251, Rv0287 and Rv0288 (Ad5-TBF); or with protein TBF emulsified in adjuvant (Adj-TBF). Boosting with TBF broaden the immune response. The kinetics of Ad5-TBF and Adj-TBF were shown to be different, with effector T cell responses from the latter developing more slowly but being more durable than those induced by Ad5-TBF. No increase in protection compared to BCG alone was afforded by Ad5-TBF or Adj-TBF by gross pathology or bacteriology. Using histopathology, as a novel parameter of protection, we show that boosting BCG vaccinated cattle with Ad5-85A induced significantly better protection than BCG alone. © 2013.

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Antrobus RD, Coughlan L, Berthoud TK, Dicks MD, Hill AVS, Lambe T, Gilbert SC. 2014. Clinical assessment of a novel recombinant simian adenovirus ChAdOx1 as a vectored vaccine expressing conserved influenza a antigens Molecular Therapy, 22 (3), pp. 668-674. | Show Abstract | Read more

Adenoviruses are potent vectors for inducing and boosting cellular immunity to encoded recombinant antigens. However, the widespread seroprevalence of neutralizing antibodies to common human adenovirus serotypes limits their use. Simian adenoviruses do not suffer from the same drawbacks. We have constructed a replication-deficient chimpanzee adenovirus-vectored vaccine expressing the conserved influenza antigens, nucleoprotein (NP), and matrix protein 1 (M1). Here, we report safety and T-cell immunogenicity following vaccination with this novel recombinant simian adenovirus, ChAdOx1 NP+M1, in a first in human dose-escalation study using a 3+3 study design, followed by boosting with modified vaccinia virus Ankara expressing the same antigens in some volunteers. We demonstrate ChAdOx1 NP+M1 to be safe and immunogenic. ChAdOx1 is a promising vaccine vector that could be used to deliver vaccine antigens where strong cellular immune responses are required for protection. © The American Society of Gene & Cell Therapy.

Dean G, Clifford D, Gilbert S, McShane H, Hewinson RG, Vordermeier HM, Villarreal-Ramos B. 2014. Effect of dose and route of immunisation on the immune response induced in cattle by heterologous Bacille Calmette-Guerin priming and recombinant adenoviral vector boosting. Vet Immunol Immunopathol, 158 (3-4), pp. 208-213. | Show Abstract | Read more

BCG is used experimentally as a vaccine against tuberculosis (TB), induced by Mycobacterium bovis, in cattle (bTB). However, the efficacy of BCG is variable in humans, cattle and guinea pigs. An adenoviral vector expressing Antigen 85A (Ad5Ag85A) has enhanced protection against TB in mice when used in combination with BCG for prime-boost experiments. However, the route of immunisation affects the degree of protection seen. This work examines the immunogenicity of a new vectored vaccine (Ad5-TBF) that expresses Ag85A, Rv0287, Rv0288 and Rv0251c to explore the effects of dose of adenoviral boost and route of inoculation on immunogenicity. We found that 2×10(9) infectious units (iu) delivered intradermally conferred the most consistent and strongest responses of the different regimes tested.

de Barra E, Hodgson SH, Ewer KJ, Bliss CM, Hennigan K, Collins A, Berrie E, Lawrie AM et al. 2014. A phase Ia study to assess the safety and immunogenicity of new malaria vaccine candidates ChAd63 CS administered alone and with MVA CS. PLoS One, 9 (12), pp. e115161. | Show Abstract | Read more

BACKGROUND: Plasmodium falciparum (P. falciparum) malaria remains a significant cause of mortality and morbidity throughout the world. Development of an effective vaccine would be a key intervention to reduce the considerable social and economic impact of malaria. METHODOLOGY: We conducted a Phase Ia, non-randomized, clinical trial in 24 healthy, malaria-naïve adults of the chimpanzee adenovirus 63 (ChAd63) and modified vaccinia virus Ankara (MVA) replication-deficient viral vectored vaccines encoding the circumsporozoite protein (CS) of P. falciparum. RESULTS: ChAd63-MVA CS administered in a heterologous prime-boost regime was shown to be safe and immunogenic, inducing high-level T cell responses to CS. With a priming ChAd63 CS dose of 5×109 vp responses peaked at a mean of 1947 SFC/million PBMC (median 1524) measured by ELIspot 7 days after the MVA boost and showed a mixed CD4+/CD8+ phenotype. With a higher priming dose of ChAd63 CS dose 5×1010 vp T cell responses did not increase (mean 1659 SFC/million PBMC, median 1049). Serum IgG responses to CS were modest and peaked at day 14 post ChAd63 CS (median antibody concentration for all groups at day 14 of 1.3 µg/ml (range 0-11.9), but persisted throughout late follow-up (day 140 median antibody concentration groups 1B & 2B 0.9 µg/ml (range 0-4.7). CONCLUSIONS: ChAd63-MVA is a safe and highly immunogenic delivery platform for the CS antigen in humans which warrants efficacy testing. TRIAL REGISTRATION: ClinicalTrials.gov NCT01450280.

Spencer AJ, Furze J, Honeycutt JD, Calvert A, Saurya S, Colloca S, Wyllie DH, Gilbert SC, Bregu M, Cottingham MG, Hill AV. 2014. 4-1BBL enhances CD8+ T cell responses induced by vectored vaccines in mice but fails to improve immunogenicity in rhesus macaques. PLoS One, 9 (8), pp. e105520. | Show Abstract | Read more

T cells play a central role in the immune response to many of the world's major infectious diseases. In this study we investigated the tumour necrosis factor receptor superfamily costimulatory molecule, 4-1BBL (CD137L, TNFSF9), for its ability to increase T cell immunogenicity induced by a variety of recombinant vectored vaccines. To efficiently test this hypothesis, we assessed a number of promoters and developed a stable bi-cistronic vector expressing both the antigen and adjuvant. Co-expression of 4-1BBL, together with our model antigen TIP, was shown to increase the frequency of murine antigen-specific IFN-γ secreting CD8(+) T cells in three vector platforms examined. Enhancement of the response was not limited by co-expression with the antigen, as an increase in CD8(+) immunogenicity was also observed by co-administration of two vectors each expressing only the antigen or adjuvant. However, when this regimen was tested in non-human primates using a clinical malaria vaccine candidate, no adjuvant effect of 4-1BBL was observed limiting its potential use as a single adjuvant for translation into a clinical vaccine.

Murphy SC, Hermsen CC, Douglas AD, Edwards NJ, Petersen I, Fahle GA, Adams M, Berry AA et al. 2014. External quality assurance of malaria nucleic acid testing for clinical trials and eradication surveillance. PLoS One, 9 (5), pp. e97398. | Show Abstract | Read more

Nucleic acid testing (NAT) for malaria parasites is an increasingly recommended diagnostic endpoint in clinical trials of vaccine and drug candidates and is also important in surveillance of malaria control and elimination efforts. A variety of reported NAT assays have been described, yet no formal external quality assurance (EQA) program provides validation for the assays in use. Here, we report results of an EQA exercise for malaria NAT assays. Among five centers conducting controlled human malaria infection trials, all centers achieved 100% specificity and demonstrated limits of detection consistent with each laboratory's pre-stated expectations. Quantitative bias of reported results compared to expected results was generally <0.5 log10 parasites/mL except for one laboratory where the EQA effort identified likely reasons for a general quantitative shift. The within-laboratory variation for all assays was low at <10% coefficient of variation across a range of parasite densities. Based on this study, we propose to create a Molecular Malaria Quality Assessment program that fulfills the need for EQA of malaria NAT assays worldwide.

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Antrobus RD, Berthoud TK, Mullarkey CE, Hoschler K, Coughlan L, Zambon M, Hill AVS, Gilbert SC. 2014. Coadministration of seasonal influenza vaccine and MVA-NP+M1 simultaneously achieves potent humoral and cell-mediated responses Molecular Therapy, 22 (1), pp. 233-238. | Show Abstract | Read more

Current seasonal influenza vaccines have reduced immunogenicity and are of suboptimal efficacy in older adults. We have previously shown that the novel candidate vaccine MVA-NP+M1 is able to boost memory T cell responses in adults aged 50-85 years. Preclinical studies have demonstrated that viral vectored vaccines can act as adjuvants when coadministered with protein-based vaccines. We have conducted a phase I clinical trial to compare the coadministration of seasonal influenza vaccine and MVA-NP+M1 with seasonal influenza vaccine alone in adults aged 50 years and above. This combination of vaccines was safe and well tolerated. T cell responses to internal influenza proteins were boosted to significantly higher levels in the group receiving MVA-NP+M1 compared with the group receiving seasonal influenza vaccine alone. Rates of seroprotection and seroconversion against the three vaccine strains were similar in both groups; however, there was a significant increase in the geometric mean titer ratio for the H3N2 component of seasonal influenza vaccine in the coadministration group. While some vaccine combinations result in immune interference, the coadministration of MVA-NP+M1 alongside seasonal influenza vaccine is shown here to increase some influenza strain-specific antibody responses and boost memory T cells capable of recognizing a range of influenza A subtypes. © The American Society of Gene & Cell Therapy.

Antrobus RD, Coughlan L, Berthoud TK, Dicks MD, Hill AV, Lambe T, Gilbert SC. 2014. Clinical assessment of a novel recombinant simian adenovirus ChAdOx1 as a vectored vaccine expressing conserved Influenza A antigens. Mol Ther, 22 (3), pp. 668-674. | Show Abstract | Read more

Adenoviruses are potent vectors for inducing and boosting cellular immunity to encoded recombinant antigens. However, the widespread seroprevalence of neutralizing antibodies to common human adenovirus serotypes limits their use. Simian adenoviruses do not suffer from the same drawbacks. We have constructed a replication-deficient chimpanzee adenovirus-vectored vaccine expressing the conserved influenza antigens, nucleoprotein (NP), and matrix protein 1 (M1). Here, we report safety and T-cell immunogenicity following vaccination with this novel recombinant simian adenovirus, ChAdOx1 NP+M1, in a first in human dose-escalation study using a 3+3 study design, followed by boosting with modified vaccinia virus Ankara expressing the same antigens in some volunteers. We demonstrate ChAdOx1 NP+M1 to be safe and immunogenic. ChAdOx1 is a promising vaccine vector that could be used to deliver vaccine antigens where strong cellular immune responses are required for protection.

Warimwe GM, Lorenzo G, Lopez-Gil E, Reyes-Sandoval A, Cottingham MG, Spencer AJ, Collins KA, Dicks MD et al. 2013. Immunogenicity and efficacy of a chimpanzee adenovirus-vectored Rift Valley fever vaccine in mice. Virol J, 10 (1), pp. 349. | Show Abstract | Read more

BACKGROUND: Rift Valley Fever (RVF) is a viral zoonosis that historically affects livestock production and human health in sub-Saharan Africa, though epizootics have also occurred in the Arabian Peninsula. Whilst an effective live-attenuated vaccine is available for livestock, there is currently no licensed human RVF vaccine. Replication-deficient chimpanzee adenovirus (ChAd) vectors are an ideal platform for development of a human RVF vaccine, given the low prevalence of neutralizing antibodies against them in the human population, and their excellent safety and immunogenicity profile in human clinical trials of vaccines against a wide range of pathogens. METHODS: Here, in BALB/c mice, we evaluated the immunogenicity and efficacy of a replication-deficient chimpanzee adenovirus vector, ChAdOx1, encoding the RVF virus envelope glycoproteins, Gn and Gc, which are targets of virus neutralizing antibodies. The ChAdOx1-GnGc vaccine was assessed in comparison to a replication-deficient human adenovirus type 5 vector encoding Gn and Gc (HAdV5-GnGc), a strategy previously shown to confer protective immunity against RVF in mice. RESULTS: A single immunization with either of the vaccines conferred protection against RVF virus challenge eight weeks post-immunization. Both vaccines elicited RVF virus neutralizing antibody and a robust CD8+ T cell response. CONCLUSIONS: Together the results support further development of RVF vaccines based on replication-deficient adenovirus vectors, with ChAdOx1-GnGc being a potential candidate for use in future human clinical trials.

Ewer KJ, O'Hara GA, Duncan CJ, Collins KA, Sheehy SH, Reyes-Sandoval A, Goodman AL, Edwards NJ et al. 2013. Protective CD8+ T-cell immunity to human malaria induced by chimpanzee adenovirus-MVA immunisation. Nat Commun, 4 pp. 2836. | Show Abstract | Read more

Induction of antigen-specific CD8(+) T cells offers the prospect of immunization against many infectious diseases, but no subunit vaccine has induced CD8(+) T cells that correlate with efficacy in humans. Here we demonstrate that a replication-deficient chimpanzee adenovirus vector followed by a modified vaccinia virus Ankara booster induces exceptionally high frequency T-cell responses (median >2400 SFC/10(6) peripheral blood mononuclear cells) to the liver-stage Plasmodium falciparum malaria antigen ME-TRAP. It induces sterile protective efficacy against heterologous strain sporozoites in three vaccinees (3/14, 21%), and delays time to patency through substantial reduction of liver-stage parasite burden in five more (5/14, 36%), P=0.008 compared with controls. The frequency of monofunctional interferon-γ-producing CD8(+) T cells, but not antibodies, correlates with sterile protection and delay in time to patency (P(corrected)=0.005). Vaccine-induced CD8(+) T cells provide protection against human malaria, suggesting that a major limitation of previous vaccination approaches has been the insufficient magnitude of induced T cells.

Pérez de Val B, Vidal E, Villarreal-Ramos B, Gilbert SC, Andaluz A, Moll X, Martín M, Nofrarías M, McShane H, Vordermeier HM, Domingo M. 2013. A multi-antigenic adenoviral-vectored vaccine improves BCG-induced protection of goats against pulmonary tuberculosis infection and prevents disease progression. PLoS One, 8 (11), pp. e81317. | Show Abstract | Read more

The "One world, one health" initiative emphasizes the need for new strategies to control human and animal tuberculosis (TB) based on their shared interface. A good example would be the development of novel universal vaccines against Mycobacterium tuberculosis complex (MTBC) infection. This study uses the goat model, a natural TB host, to assess the protective effectiveness of a new vaccine candidate in combination with Bacillus Calmette-Guerin (BCG) vaccine. Thirty-three goat kids were divided in three groups: Group 1) vaccinated with BCG (week 0), Group 2) vaccinated with BCG and boosted 8 weeks later with a recombinant adenovirus expressing the MTBC antigens Ag85A, TB10.4, TB9.8 and Acr2 (AdTBF), and Group 3) unvaccinated controls. Later on, an endobronchial challenge with a low dose of M. caprae was performed (week 15). After necropsy (week 28), the pulmonary gross pathology was quantified using high resolution Computed Tomography. Small granulomatous pulmonary lesions (< 0.5 cm diameter) were also evaluated through a comprehensive qualitative histopathological analysis. M. caprae CFU were counted from pulmonary lymph nodes. The AdTBF improved the effects of BCG reducing gross lesion volume and bacterial load, as well as increasing weight gain. The number of Ag85A-specific gamma interferon-producing memory T-cells was identified as a predictor of vaccine efficacy. Specific cellular and humoral responses were measured throughout the 13-week post-challenge period, and correlated with the severity of lesions. Unvaccinated goats exhibited the typical pathological features of active TB in humans and domestic ruminants, while vaccinated goats showed only very small lesions. The data presented in this study indicate that multi-antigenic adenoviral vectored vaccines boosts protection conferred by vaccination with BCG.

Colston J, Bolinger B, Turner A, Gilbert S, Klenerman P. 2013. AN ADENOVIRAL MODEL TO UNLOCK THE SECRETS OF MEMORY INFLATION? JOURNAL OF INFECTION, 67 (4), pp. 345-345. | Read more

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Gilbert SC. 2013. Clinical development of Modified Vaccinia virus Ankara vaccines Vaccine, 31 (39), pp. 4241-4246. | Show Abstract | Read more

The smallpox vaccine Vaccinia was successfully used to eradicate smallpox, but although very effective, it was a very reactogenic vaccine and responsible for the deaths of one or two people per million vaccinated. Modified Vaccinia virus Ankara (MVA) is a replication-deficient and attenuated derivative, also used in the smallpox eradication campaign and now being developed as a recombinant viral vector to produce vaccines against infectious diseases and cancer. Many clinical trials of these new vaccines have been conducted, and the findings of these trials are reviewed here. The safety of MVA is now well documented, immunogenicity is influenced by the dose and vaccination regimen, and information on the efficacy of MVA-vectored vaccines is now beginning to accumulate. © 2013 Elsevier Ltd.

Kreijtz JHCM, Gilbert SC, Sutter G. 2013. Poxvirus vectors Vaccine, 31 (39), pp. 4217-4219. | Read more

Gilbert SC. 2013. Advances in the development of universal influenza vaccines Influenza and other Respiratory Viruses, 7 (5), pp. 750-758. | Show Abstract | Read more

Despite the widespread availability and use of influenza vaccines, influenza still poses a considerable threat to public health. Vaccines against seasonal influenza do not offer protection against pandemic viruses, and vaccine efficacy against seasonal viruses is reduced in seasons when the vaccine composition is not a good match for the predominant circulating viruses. Vaccine efficacy is also reduced in older adults, who are one of the main target groups for vaccination. The continual threat of pandemic influenza, with the known potential for rapid spread around the world and high mortality rates, has prompted researchers to develop a number of novel approaches to providing immunity to this virus, focusing on target antigens which are highly conserved between different influenza A virus subtypes. Several of these have now been taken into clinical development, and this review discusses the progress that has been made, as well as considering the requirements for licensing these new vaccines and how they might be used in the future. © 2012 John Wiley & Sons Ltd.

López-Gil E, Lorenzo G, Hevia E, Borrego B, Eiden M, Groschup M, Gilbert SC, Brun A. 2013. A single immunization with MVA expressing GnGc glycoproteins promotes epitope-specific CD8+-T cell activation and protects immune-competent mice against a lethal RVFV infection. PLoS Negl Trop Dis, 7 (7), pp. e2309. | Show Abstract | Read more

BACKGROUND: Rift Valley fever virus (RVFV) is a mosquito-borne pathogen causing an important disease in ruminants often transmitted to humans after epizootic outbreaks in African and Arabian countries. To help combat the spread of the disease, prophylactic measures need to be developed and/or improved. METHODOLOGY/PRINCIPAL FINDINGS: In this work, we evaluated the immunogenicity and protective efficacy of recombinant plasmid DNA and modified vaccinia virus Ankara (rMVA) vectored vaccines against Rift Valley fever in mice. These recombinant vaccines encoded either of two components of the Rift Valley fever virus: the viral glycoproteins (Gn/Gc) or the nucleoprotein (N). Following lethal challenge with live RVFV, mice immunized with a single dose of the rMVA-Gn/Gc vaccine showed no viraemia or clinical manifestation of disease, but mounted RVFV neutralizing antibodies and glycoprotein specific CD8+ T-cell responses. Neither DNA-Gn/Gc alone nor a heterologous prime-boost immunization schedule (DNA-Gn/Gc followed by rMVAGn/Gc) was better than the single rMVA-Gn/Gc immunization schedule with regards to protective efficacy. However, the rMVA-Gn/Gc vaccine failed to protect IFNAR(-/-) mice upon lethal RVFV challenge suggesting a role for innate responses in protection against RVFV. Despite induction of high titer antibodies against the RVFV nucleoprotein, the rMVA-N vaccine, whether in homologous or heterologous prime-boost schedules with the corresponding recombinant DNA vaccine, only conferred partial protection to RVFV challenge. CONCLUSIONS/SIGNIFICANCE: Given the excellent safety profile of rMVA based vaccines in humans and animals, our data supports further development of rMVA-Gn/Gc as a vaccine strategy that can be used for the prevention of Rift Valley fever in both humans and livestock.

Draper SJ, Cottingham MG, Gilbert SC. 2013. Utilizing poxviral vectored vaccines for antibody induction-progress and prospects. Vaccine, 31 (39), pp. 4223-4230. | Show Abstract | Read more

Over the last decade, poxviral vectors emerged as a mainstay approach for the induction of T cell-mediated immunity by vaccination, and their suitability for human use has led to widespread clinical testing of candidate vectors against infectious intracellular pathogens and cancer. In contrast, poxviruses have been widely perceived in the vaccine field as a poor choice of vector for the induction of humoral immunity. However, a growing body of data, from both animal models and recent clinical trials, now suggests that these vectors can be successfully utilized to prime and boost B cells and effective antibody responses. Significant progress has been made in the context of heterologous prime-boost immunization regimes, whereby poxviruses are able to boost responses primed by other vectors, leading to the induction of high-titre antigen-specific antibody responses. In other cases, poxviral vectors have been shown to stimulate humoral immunity against both themselves and encoded transgenes, in particular viral surface proteins such as influenza haemagglutinin. In the veterinary field, recombinant poxviral vectors have made a significant impact with numerous vectors licensed for use against a variety of animal viruses. On-going studies continue to explore the potential of poxviral vectors to modulate qualitative aspects of the humoral response, as well as their amenability to adjuvantation seeking to improve quantitative antibody immunogenicity. Nevertheless, the underlying mechanisms of B cell induction by recombinant poxviruses remain poorly defined, and further work is necessary to help guide the rational optimization of future poxviral vaccine candidates aiming to induce antibodies.

Rowland R, O'Hara GA, Hamill M, Poulton ID, Donaldson H, Dinsmore L, James T, Barnes E et al. 2013. Determining the validity of hospital laboratory reference intervals for healthy young adults participating in early clinical trials of candidate vaccines. Hum Vaccin Immunother, 9 (8), pp. 1741-1751. | Show Abstract | Read more

This was a retrospective study to determine the validity of institutional reference intervals for interpreting biochemistry and hematology results in healthy adults in the context of clinical trials of preventive vaccines. An example population of 974 healthy adults participating in clinical trials at the Jenner Institute, Oxford, UK, between 1999 and 2009 was studied. Methods for calculating the central 95% ranges and determining the coefficients of within person variation were demonstrated. Recommendations have been made as to how these data can be usefully applied to the interpretation of blood results in healthy adult subjects for the purposes of clinical trial inclusion decisions and post-vaccination safety monitoring.

Goodman AL, Forbes EK, Williams AR, Douglas AD, de Cassan SC, Bauza K, Biswas S, Dicks MD et al. 2013. The utility of Plasmodium berghei as a rodent model for anti-merozoite malaria vaccine assessment. Sci Rep, 3 pp. 1706. | Show Abstract | Read more

Rodent malaria species Plasmodium yoelii and P. chabaudi have been widely used to validate vaccine approaches targeting blood-stage merozoite antigens. However, increasing data suggest the P. berghei rodent malaria may be able to circumvent vaccine-induced anti-merozoite responses. Here we confirm a failure to protect against P. berghei, despite successful antibody induction against leading merozoite antigens using protein-in-adjuvant or viral vectored vaccine delivery. No subunit vaccine approach showed efficacy in mice following immunization and challenge with the wild-type P. berghei strains ANKA or NK65, or against a chimeric parasite line encoding a merozoite antigen from P. falciparum. Protection was not improved in knockout mice lacking the inhibitory Fc receptor CD32b, nor against a Δsmac P. berghei parasite line with a non-sequestering phenotype. An improved understanding of the mechanisms responsible for protection, or failure of protection, against P. berghei merozoites could guide the development of an efficacious vaccine against P. falciparum.

Mullarkey CE, Boyd A, van Laarhoven A, Lefevre EA, Veronica Carr B, Baratelli M, Molesti E, Temperton NJ et al. 2013. Improved adjuvanting of seasonal influenza vaccines: preclinical studies of MVA-NP+M1 coadministration with inactivated influenza vaccine. Eur J Immunol, 43 (7), pp. 1940-1952. | Show Abstract | Read more

Licensed seasonal influenza vaccines induce antibody (Ab) responses against influenza hemagglutinin (HA) that are limited in their ability to protect against different strains of influenza. Cytotoxic T lymphocytes recognizing the conserved internal nucleoprotein (NP) and matrix protein (M1) are capable of mediating a cross-subtype immune response against influenza. Modified vaccinia Ankara (MVA) virus encoding NP and M1 (MVA-NP+M1) is designed to boost preexisting T-cell responses in adults in order to elicit a cross-protective immune response. We examined the coadministration of HA protein formulations and candidate MVA-NP+M1 influenza vaccines in murine, avian, and swine models. Ab responses postimmunization were measured by ELISA and pseudotype neutralization assays. Here, we demonstrate that MVA-NP+M1 can act as an adjuvant enhancing Ab responses to HA while simultaneously inducing potent T-cell responses to conserved internal Ags. We show that this regimen leads to the induction of cytophilic Ab isotypes that are capable of inhibiting hemagglutination and in the context of H5 exhibit cross-clade neutralization. The simultaneous induction of T cells and Ab responses has the potential to improve seasonal vaccine performance and could be employed in pandemic situations.

Douglas AD, Edwards NJ, Duncan CJ, Thompson FM, Sheehy SH, O'Hara GA, Anagnostou N, Walther M et al. 2013. Comparison of modeling methods to determine liver-to-blood inocula and parasite multiplication rates during controlled human malaria infection. J Infect Dis, 208 (2), pp. 340-345. | Show Abstract | Read more

Controlled human malaria infection is used to measure efficacy of candidate malaria vaccines before field studies are undertaken. Mathematical modeling using data from quantitative polymerase chain reaction (qPCR) parasitemia monitoring can discriminate between vaccine effects on the parasite's liver and blood stages. Uncertainty regarding the most appropriate modeling method hinders interpretation of such trials. We used qPCR data from 267 Plasmodium falciparum infections to compare linear, sine-wave, and normal-cumulative-density-function models. We find that the parameters estimated by these models are closely correlated, and their predictive accuracy for omitted data points was similar. We propose that future studies include the linear model.

Gilbert SC. 2013. Clinical development of Modified Vaccinia virus Ankara vaccines. Vaccine, 31 (39), pp. 4241-4246. | Show Abstract | Read more

The smallpox vaccine Vaccinia was successfully used to eradicate smallpox, but although very effective, it was a very reactogenic vaccine and responsible for the deaths of one or two people per million vaccinated. Modified Vaccinia virus Ankara (MVA) is a replication-deficient and attenuated derivative, also used in the smallpox eradication campaign and now being developed as a recombinant viral vector to produce vaccines against infectious diseases and cancer. Many clinical trials of these new vaccines have been conducted, and the findings of these trials are reviewed here. The safety of MVA is now well documented, immunogenicity is influenced by the dose and vaccination regimen, and information on the efficacy of MVA-vectored vaccines is now beginning to accumulate.

Lambe T, Carey JB, Li Y, Spencer AJ, van Laarhoven A, Mullarkey CE, Vrdoljak A, Moore AC, Gilbert SC. 2013. Immunity against heterosubtypic influenza virus induced by adenovirus and MVA expressing nucleoprotein and matrix protein-1. Sci Rep, 3 pp. 1443. | Show Abstract | Read more

Alternate prime/boost vaccination regimens employing recombinant replication-deficient adenovirus or MVA, expressing Influenza A virus nucleoprotein and matrix protein 1, induced antigen-specific T cell responses in intradermally (ID) vaccinated mice; with the strongest responses resulting from Ad/MVA immunization. In BALB/C mice the immunodominant response was shifted from the previously identified immunodominant epitope to a novel epitope when the antigen was derived from A/Panama/2007/1999 rather than A/PR/8. Alternate immunization routes did not affect the magnitude of antigen-specific systemic IFN-γ response, but higher CD8(+) T-cell IFN-γ immune responses were seen in the bronchoalveolar lavage following intransal (IN) boosting after intramuscular (IM) priming, whilst higher splenic antigen-specific CD8(+) T cell IFN-γ was seen following IM boosting. Partial protection against heterologous influenza virus challenge was achieved following either IM/IM or IM/IN but not ID/ID immunization. These data may be of relevance for the design of optimal immunization regimens for human influenza vaccines, especially for influenza-naïve infants.

Gilbert SC. 2013. No easy route to a pandemic influenza vaccine. Lancet Infect Dis, 13 (3), pp. 188-189. | Read more

Dean G, Whelan A, Clifford D, Salguero FJ, Xing Z, Gilbert S, McShane H, Hewinson RG, Vordermeier M, Villarreal-Ramos B. 2014. Comparison of the immunogenicity and protection against bovine tuberculosis following immunization by BCG-priming and boosting with adenovirus or protein based vaccines. Vaccine, 32 (11), pp. 1304-1310. | Show Abstract | Read more

There is a requirement for vaccines or vaccination strategies that confer better protection against TB than the current live attenuated Mycobacterium bovis Bacillus Calmette-Guerin (BCG) vaccine for use in cattle. Boosting with recombinant viral vectors expressing mycobacterial proteins, such as Ag85A, has shown a degree of promise as a strategy for improving on the protection afforded by BCG. Experiments in small animal models have indicated that broadening the immune response to include mycobacterial antigens other than Ag85A, such as Rv0288, induced by boosting with Ad5 constructs has a direct effect on the protection afforded against TB. Here, we compared the immunogenicity and protection against challenge with M. bovis afforded by boosting BCG-vaccinated cattle with a human type 5 (Ad5)-based vaccine expressing the mycobacterial antigens Ag85A (Ad5-85A); or Ag85A, Rv0251, Rv0287 and Rv0288 (Ad5-TBF); or with protein TBF emulsified in adjuvant (Adj-TBF). Boosting with TBF broaden the immune response. The kinetics of Ad5-TBF and Adj-TBF were shown to be different, with effector T cell responses from the latter developing more slowly but being more durable than those induced by Ad5-TBF. No increase in protection compared to BCG alone was afforded by Ad5-TBF or Adj-TBF by gross pathology or bacteriology. Using histopathology, as a novel parameter of protection, we show that boosting BCG vaccinated cattle with Ad5-85A induced significantly better protection than BCG alone.

Kreijtz JH, Gilbert SC, Sutter G. 2013. Poxvirus vectors. Vaccine, 31 (39), pp. 4217-4219. | Read more

Pearson FE, McNeilly CL, Crichton ML, Primiero CA, Yukiko SR, Fernando GJ, Chen X, Gilbert SC, Hill AV, Kendall MA. 2013. Dry-coated live viral vector vaccines delivered by nanopatch microprojections retain long-term thermostability and induce transgene-specific T cell responses in mice. PLoS One, 8 (7), pp. e67888. | Show Abstract | Read more

The disadvantages of needle-based immunisation motivate the development of simple, low cost, needle-free alternatives. Vaccine delivery to cutaneous environments rich in specialised antigen-presenting cells using microprojection patches has practical and immunological advantages over conventional needle delivery. Additionally, stable coating of vaccine onto microprojections removes logistical obstacles presented by the strict requirement for cold-chain storage and distribution of liquid vaccine, or lyophilised vaccine plus diluent. These attributes make these technologies particularly suitable for delivery of vaccines against diseases such as malaria, which exerts its worst effects in countries with poorly-resourced healthcare systems. Live viral vectors including adenoviruses and poxviruses encoding exogenous antigens have shown significant clinical promise as vaccines, due to their ability to generate high numbers of antigen-specific T cells. Here, the simian adenovirus serotype 63 and the poxvirus modified vaccinia Ankara--two vectors under evaluation for the delivery of malaria antigens to humans--were formulated for coating onto Nanopatch microprojections and applied to murine skin. Co-formulation with the stabilising disaccharides trehalose and sucrose protected virions during the dry-coating process. Transgene-specific CD8(+) T cell responses following Nanopatch delivery of both vectors were similar to intradermal injection controls after a single immunisation (despite a much lower delivered dose), though MVA boosting of pre-primed responses with Nanopatch was found to be less effective than the ID route. Importantly, disaccharide-stabilised ChAd63 could be stored for 10 weeks at 37°C with less than 1 log10 loss of viability, and retained single-dose immunogenicity after storage. These data support the further development of microprojection patches for the deployment of live vaccines in hot climates.

Powell TJ, Peng Y, Berthoud TK, Blais ME, Lillie PJ, Hill AV, Rowland-Jones SL, McMichael AJ, Gilbert SC, Dong T. 2013. Examination of influenza specific T cell responses after influenza virus challenge in individuals vaccinated with MVA-NP+M1 vaccine. PLoS One, 8 (5), pp. e62778. | Show Abstract | Read more

Current influenza vaccines stimulate neutralising antibody to the haemagglutinin antigen but as there is antigenic drift in HA it is difficult to prepare a vaccine in advance against an emergent strain. A potential strategy is to induce CD8(+) and CD4(+) T cells that recognize epitopes within internal proteins that are less subject to antigenic drift. Augmenting humoral responses to HA with T cell responses to more conserved antigens may result in a more broadly protective vaccine. In this study, we evaluate the quality of influenza specific T cell responses in a clinical trial using MVA-NP+M1 vaccination followed by influenza virus challenge. In vaccinated volunteers, the expression of Granzyme A, Perforin and CD57 on influenza HLA A*02 M158-66 antigen specific cells was higher than non-vaccinated volunteers before and after challenge despite a similar frequency of antigen specific cells. BCL2 expression was lower in vaccinated volunteers. These data indicate that antigen specific T cells are a useful additional measure for use in human vaccination or immunization studies.

Ogwang C, Afolabi M, Kimani D, Jagne YJ, Sheehy SH, Bliss CM, Duncan CJ, Collins KA et al. 2013. Safety and immunogenicity of heterologous prime-boost immunisation with Plasmodium falciparum malaria candidate vaccines, ChAd63 ME-TRAP and MVA ME-TRAP, in healthy Gambian and Kenyan adults. PLoS One, 8 (3), pp. e57726. | Show Abstract | Read more

BACKGROUND: Heterologous prime boost immunization with chimpanzee adenovirus 63 (ChAd63) and Modified vaccinia Virus Ankara (MVA) vectored vaccines is a strategy recently shown to be capable of inducing strong cell mediated responses against several antigens from the malaria parasite. ChAd63-MVA expressing the Plasmodium falciparum pre-erythrocytic antigen ME-TRAP (multiple epitope string with thrombospondin-related adhesion protein) is a leading malaria vaccine candidate, capable of inducing sterile protection in malaria naïve adults following controlled human malaria infection (CHMI). METHODOLOGY: We conducted two Phase Ib dose escalation clinical trials assessing the safety and immunogenicity of ChAd63-MVA ME-TRAP in 46 healthy malaria exposed adults in two African countries with similar malaria transmission patterns. RESULTS: ChAd63-MVA ME-TRAP was shown to be safe and immunogenic, inducing high-level T cell responses (median >1300 SFU/million PBMC). CONCLUSIONS: ChAd63-MVA ME-TRAP is a safe and highly immunogenic vaccine regimen in adults with prior exposure to malaria. Further clinical trials to assess safety and immunogenicity in children and infants and protective efficacy in the field are now warranted. TRIAL REGISTRATION: Pactr.org PACTR2010020001771828 Pactr.org PACTR201008000221638 ClinicalTrials.gov NCT01373879 NCT01373879 ClinicalTrials.gov NCT01379430 NCT01379430.

Gilbert SC. 2013. No easy route to a pandemic influenza vaccine The Lancet Infectious Diseases, 13 (3), pp. 188-189. | Read more

Boyd AC, Ruiz-Hernandez R, Peroval MY, Carson C, Balkissoon D, Staines K, Turner AV, Hill AV, Gilbert SC, Butter C. 2013. Towards a universal vaccine for avian influenza: protective efficacy of modified Vaccinia virus Ankara and Adenovirus vaccines expressing conserved influenza antigens in chickens challenged with low pathogenic avian influenza virus. Vaccine, 31 (4), pp. 670-675. | Show Abstract | Read more

Current vaccines targeting surface proteins can drive antigenic variation resulting either in the emergence of more highly pathogenic viruses or of antigenically distinct viruses that escape control by vaccination and thereby persist in the host population. Influenza vaccines typically target the highly mutable surface proteins and do not provide protection against heterologous challenge. Vaccines which induce immune responses against conserved influenza epitopes may confer protection against heterologous challenge. We report here the results of vaccination with recombinant modified Vaccinia virus Ankara (MVA) and Adenovirus (Ad) expressing a fusion construct of nucleoprotein and matrix protein (NP+M1). Prime and boost vaccination regimes were trialled in different ages of chicken and were found to be safe and immunogenic. Interferon-γ (IFN-γ) ELISpot was used to assess the cellular immune response post secondary vaccination. In ovo Ad prime followed by a 4 week post hatch MVA boost was identified as the most immunogenic regime in one outbred and two inbred lines of chicken. Following vaccination, one inbred line (C15I) was challenged with low pathogenic avian influenza (LPAI) H7N7 (A/Turkey/England/1977). Birds receiving a primary vaccination with Ad-NP+M1 and a secondary vaccination with MVA-NP+M1 exhibited reduced cloacal shedding as measured by plaque assay at 7 days post infection compared with birds vaccinated with recombinant viruses containing irrelevant antigen. This preliminary indication of efficacy demonstrates proof of concept in birds; induction of T cell responses in chickens by viral vectors containing internal influenza antigens may be a productive strategy for the development of vaccines to induce heterologous protection against influenza in poultry.

Rowland R, Pathan AA, Satti I, Poulton ID, Matsumiya MM, Whittaker M, Minassian AM, O'Hara GA et al. 2013. Safety and immunogenicity of an FP9-vectored candidate tuberculosis vaccine (FP85A), alone and with candidate vaccine MVA85A in BCG-vaccinated healthy adults: a phase I clinical trial. Hum Vaccin Immunother, 9 (1), pp. 50-62. | Show Abstract | Read more

The safety and immunogenicity of a new candidate tuberculosis (TB) vaccine, FP85A was evaluated alone and in heterologous prime-boost regimes with another candidate TB vaccine, MVA85A. This was an open label, non-controlled, non-randomized Phase I clinical trial. Healthy previously BCG-vaccinated adult subjects were enrolled sequentially into three groups and vaccinated with FP85A alone, or both FP85A and MVA85A, with a four week interval between vaccinations. Passive and active data on adverse events were collected. Immunogenicity was evaluated by Enzyme Linked Immunospot (ELISpot), flow cytometry and Enzyme Linked Immunosorbent assay (ELISA). Most adverse events were mild and there were no vaccine-related serious adverse events. FP85A vaccination did not enhance antigen 85A-specific cellular immunity. When MVA85A vaccination was preceded by FP85A vaccination, cellular immune responses were lower compared with when MVA85A vaccination was the first immunisation. MVA85A vaccination, but not FP85A vaccination, induced anti-MVA IgG antibodies. Both MVA85A and FP85A vaccinations induced anti-FP9 IgG antibodies. In conclusion, FP85A vaccination was well tolerated but did not induce antigen-specific cellular immune responses. We hypothesize that FP85A induced anti-FP9 IgG antibodies with cross-reactivity for MVA85A, which may have mediated inhibition of the immune response to subsequent MVA85A. ClinicalTrials.gov identification number: NCT00653770.

Sheehy SH, Duncan CJ, Elias SC, Choudhary P, Biswas S, Halstead FD, Collins KA, Edwards NJ et al. 2012. ChAd63-MVA-vectored blood-stage malaria vaccines targeting MSP1 and AMA1: assessment of efficacy against mosquito bite challenge in humans. Mol Ther, 20 (12), pp. 2355-2368. | Show Abstract | Read more

The induction of cellular immunity, in conjunction with antibodies, may be essential for vaccines to protect against blood-stage infection with the human malaria parasite Plasmodium falciparum. We have shown that prime-boost delivery of P. falciparum blood-stage antigens by chimpanzee adenovirus 63 (ChAd63) followed by the attenuated orthopoxvirus MVA is safe and immunogenic in healthy adults. Here, we report on vaccine efficacy against controlled human malaria infection delivered by mosquito bites. The blood-stage malaria vaccines were administered alone, or together (MSP1+AMA1), or with a pre-erythrocytic malaria vaccine candidate (MSP1+ME-TRAP). In this first human use of coadministered ChAd63-MVA regimes, we demonstrate immune interference whereby responses against merozoite surface protein 1 (MSP1) are dominant over apical membrane antigen 1 (AMA1) and ME-TRAP. We also show that induction of strong cellular immunity against MSP1 and AMA1 is safe, but does not impact on parasite growth rates in the blood. In a subset of vaccinated volunteers, a delay in time to diagnosis was observed and sterilizing protection was observed in one volunteer coimmunized with MSP1+AMA1-results consistent with vaccine-induced pre-erythrocytic, rather than blood-stage, immunity. These data call into question the utility of T cell-inducing blood-stage malaria vaccines and suggest that the focus should remain on high-titer antibody induction against susceptible antigen targets.

Gilbert SC. 2013. Advances in the development of universal influenza vaccines. Influenza Other Respir Viruses, 7 (5), pp. 750-758. | Show Abstract | Read more

Despite the widespread availability and use of influenza vaccines, influenza still poses a considerable threat to public health. Vaccines against seasonal influenza do not offer protection against pandemic viruses, and vaccine efficacy against seasonal viruses is reduced in seasons when the vaccine composition is not a good match for the predominant circulating viruses. Vaccine efficacy is also reduced in older adults, who are one of the main target groups for vaccination. The continual threat of pandemic influenza, with the known potential for rapid spread around the world and high mortality rates, has prompted researchers to develop a number of novel approaches to providing immunity to this virus, focusing on target antigens which are highly conserved between different influenza A virus subtypes. Several of these have now been taken into clinical development, and this review discusses the progress that has been made, as well as considering the requirements for licensing these new vaccines and how they might be used in the future.

Gilbert SC. 2012. Influenza vaccines and immunopathology. Expert Rev Vaccines, 11 (8), pp. 873-875. | Read more

Biswas S, Spencer AJ, Forbes EK, Gilbert SC, Holder AA, Hill AV, Draper SJ. 2012. Recombinant viral-vectored vaccines expressing Plasmodium chabaudi AS apical membrane antigen 1: mechanisms of vaccine-induced blood-stage protection. J Immunol, 188 (10), pp. 5041-5053. | Show Abstract | Read more

Apical membrane Ag 1 (AMA1) is one of the leading candidate Ags for inclusion in a subunit vaccine against blood-stage malaria. However, the efficacy of Ab-inducing recombinant AMA1 protein vaccines in phase IIa/b clinical trials remains disappointing. In this article, we describe the development of recombinant human adenovirus serotype 5 and modified vaccinia virus Ankara vectors encoding AMA1 from the Plasmodium chabaudi chabaudi strain AS. These vectors, when used in a heterologous prime-boost regimen in BALB/c mice, are capable of inducing strong transgene-specific humoral and cellular immune responses. We show that this vaccination regimen is protective against a nonlethal P. chabaudi chabaudi strain AS blood-stage challenge, resulting in reduced peak parasitemias. The role of vaccine-induced, AMA1-specific Abs and T cells in mediating the antiparasite effect was investigated by in vivo depletion of CD4(+) T cells and adoptive-transfer studies into naive and immunodeficient mice. Depletion of CD4(+) T cells led to a loss of vaccine-induced protection. Adoptive-transfer studies confirmed that efficacy is mediated by both CD4(+) T cells and Abs functioning in the context of an intact immune system. Unlike previous studies, these results confirm that Ag-specific CD4(+) T cells, induced by a clinically relevant vaccine-delivery platform, can make a significant contribution to vaccine blood-stage efficacy in the P. chabaudi model. Given that cell-mediated immunity may also contribute to parasite control in human malaria, these data support the clinical development of viral-vectored vaccines that induce both T cell and Abs against Plasmodium falciparum blood-stage malaria Ags like AMA1.

Lambe T, Spencer AJ, Mullarkey CE, Antrobus RD, Yu LM, de Whalley P, Thompson BA, Jones C et al. 2012. T-cell responses in children to internal influenza antigens, 1 year after immunization with pandemic H1N1 influenza vaccine, and response to revaccination with seasonal trivalent-inactivated influenza vaccine. Pediatr Infect Dis J, 31 (6), pp. e86-e91. | Show Abstract | Read more

BACKGROUND: During seasonal influenza epidemics, 5-15% of the population are affected with an illness having a nontrivial mortality, morbidity and economic burden. Inactivated influenza vaccines are routinely used to prevent influenza infection, primarily by inducing humoral immunity. In addition, trivalent-inactivated influenza vaccines have previously been shown to boost influenza-specific T-cell responses in a small percentage of adults. We investigate here the influenza-specific T-cell response, in children, 1 year after pandemic H1N1 vaccination and the ability to boost the T-cell response with trivalent-inactivated influenza immunization. METHODS: Peripheral blood mononuclear cells (PBMCs) were isolated from children previously vaccinated with pandemic H1N1 vaccine, pre- and postseasonal 2010-2011 trivalent influenza vaccine (TIV) vaccination. Samples were analyzed by interferon-gamma enzyme-linked immunosorbent spot for reactogenicity toward internal influenza antigens (nucleoprotein, matrix protein 1 and nonstructural protein 1). RESULTS: Basal ex vivo T-cell responses to nucleoprotein, matrix protein 1 and nonstructural protein 1 measured by interferon-gamma enzyme-linked immunosorbent spot assay were significantly higher in those children who had previously received an AS03B-adjuvanted split virion pandemic vaccine 12 months earlier rather than a nonadjuvanted whole virion vaccine. Boosting of these responses, 21 days after 2010/2011 seasonal TIV vaccination was observed regardless of age or prior pandemic vaccination regime, although boosting was greater in those groups with the lowest initial response. CONCLUSIONS: We show here that children previously vaccinated with the 2009 pandemic H1N1 vaccine have measurable T-cell responses 1 year after vaccination. The magnitudes of these responses are dependent on both age of vaccine and type of pandemic H1N1 vaccine used. After 2010/2011 seasonal TIV vaccination, these T-cell responses undergo a small but significant boost.

Lillie PJ, Berthoud TK, Powell TJ, Lambe T, Mullarkey C, Spencer AJ, Hamill M, Peng Y et al. 2012. Preliminary assessment of the efficacy of a T-cell-based influenza vaccine, MVA-NP+M1, in humans. Clin Infect Dis, 55 (1), pp. 19-25. | Show Abstract | Read more

BACKGROUND: The novel influenza vaccine MVA-NP+M1 is designed to boost cross-reactive T-cell responses to internal antigens of the influenza A virus that are conserved across all subtypes, providing protection against both influenza disease and virus shedding against all influenza A viruses. Following a phase 1 clinical study that demonstrated vaccine safety and immunogenicity, a phase 2a vaccination and influenza challenge study has been conducted in healthy adult volunteers. METHODS: Volunteers with no measurable serum antibodies to influenza A/Wisconsin/67/2005 received either a single vaccination with MVA-NP+M1 or no vaccination. T-cell responses to the vaccine antigens were measured at enrollment and again prior to virus challenge. All volunteers underwent intranasal administration of influenza A/Wisconsin/67/2005 while in a quarantine unit and were monitored for symptoms of influenza disease and virus shedding. RESULTS: Volunteers had a significantly increased T-cell response to the vaccine antigens following a single dose of the vaccine, with an increase in cytolytic effector molecules. Intranasal influenza challenge was undertaken without safety issues. Two of 11 vaccinees and 5 of 11 control subjects developed laboratory-confirmed influenza (symptoms plus virus shedding). Symptoms of influenza were less pronounced in the vaccinees and there was a significant reduction in the number of days of virus shedding in those vaccinees who developed influenza (mean, 1.09 days in controls, 0.45 days in vaccinees, P = .036). CONCLUSIONS: This study provides the first demonstration of clinical efficacy of a T-cell-based influenza vaccine and indicates that further clinical development should be undertaken. CLINICAL TRIALS REGISTRATION: NCT00993083.

Guzman E, Cubillos-Zapata C, Cottingham MG, Gilbert SC, Prentice H, Charleston B, Hope JC. 2012. Modified vaccinia virus Ankara-based vaccine vectors induce apoptosis in dendritic cells draining from the skin via both the extrinsic and intrinsic caspase pathways, preventing efficient antigen presentation. J Virol, 86 (10), pp. 5452-5466. | Show Abstract | Read more

Dendritic cells (DC) are potent antigen-presenting cells and central to the induction of immune responses following infection or vaccination. The collection of DC migrating from peripheral tissues by cannulation of the afferent lymphatic vessels provides DC which can be used directly ex vivo without extensive in vitro manipulations. We have previously used bovine migrating DC to show that recombinant human adenovirus 5 vectors efficiently transduce afferent lymph migrating DEC-205(+) CD11c(+) CD8(-) DC (ALDC). We have also shown that recombinant modified vaccinia virus Ankara (MVA) infects ALDC in vitro, causing downregulation of costimulatory molecules, apoptosis, and cell death. We now show that in the bovine system, modified vaccinia virus Ankara-induced apoptosis in DC draining from the skin occurs soon after virus binding via the caspase 8 pathway and is not associated with viral gene expression. We also show that after virus entry, the caspase 9 pathway cascade is initiated. The magnitude of T cell responses to mycobacterial antigen 85A (Ag85A) expressed by recombinant MVA-infected ALDC is increased by blocking caspase-induced apoptosis. Apoptotic bodies generated by recombinant MVA (rMVA)-Ag85A-infected ALDC and containing Ag85A were phagocytosed by noninfected migrating ALDC expressing SIRPα via actin-dependent phagocytosis, and these ALDC in turn presented antigen. However, the addition of fresh ALDC to MVA-infected cultures did not improve on the magnitude of the T cell responses; in contrast, these noninfected DC showed downregulation of major histocompatibility complex class II (MHC-II), CD40, CD80, and CD86. We also observed that MVA-infected ALDC promoted migration of DEC-205(+) SIRPα(+) CD21(+) DC as well as CD4(+) and CD8(+) T cells independently of caspase activation. These in vitro studies show that induction of apoptosis in DC by MVA vectors is detrimental to the subsequent induction of T cell responses.

Cottingham MG, Carroll F, Morris SJ, Turner AV, Vaughan AM, Kapulu MC, Colloca S, Siani L, Gilbert SC, Hill AV. 2012. Preventing spontaneous genetic rearrangements in the transgene cassettes of adenovirus vectors. Biotechnol Bioeng, 109 (3), pp. 719-728. | Show Abstract | Read more

First-generation, E1/E3-deleted adenoviral vectors with diverse transgenes are produced routinely in laboratories worldwide for development of novel prophylactics and therapies for a variety of applications, including candidate vaccines against important infectious diseases, such as HIV/AIDS, tuberculosis, and malaria. Here, we show, for two different transgenes (both encoding malarial antigens) inserted at the E1 locus, that rare viruses containing a transgene-inactivating mutation exhibit a selective growth advantage during propagation in E1-complementing HEK293 cells, such that they rapidly become the major or sole species in the viral population. For one of these transgenes, we demonstrate that viral yield and cytopathic effect are enhanced by repression of transgene expression in the producer cell line, using the tetracycline repressor system. In addition to these transgene-inactivating mutations, one of which occurred during propagation of the pre-viral genomic clone in bacteria, and the other after viral reconstitution in HEK293 cells, we describe two other types of mutation, a small deletion and a gross rearranging duplication, in one of the transgenes studied. These were of uncertain origin, and the effects on transgene expression and viral growth were not fully characterized. We demonstrate that, together with minor protocol modifications, repression of transgene expression in HEK293 cells during viral propagation enables production of a genetically stable chimpanzee adenovirus vector expressing a malarial antigen which had previously been impossible to derive. These results have important implications for basic and pre-clinical studies using adenoviral vectors and for derivation of adenoviral vector products destined for large-scale amplification during biomanufacture.

O'Hara GA, Duncan CJ, Ewer KJ, Collins KA, Elias SC, Halstead FD, Goodman AL, Edwards NJ et al. 2012. Clinical assessment of a recombinant simian adenovirus ChAd63: a potent new vaccine vector. J Infect Dis, 205 (5), pp. 772-781. | Show Abstract | Read more

BACKGROUND: Vaccine development in human Plasmodium falciparum malaria has been hampered by the exceptionally high levels of CD8(+) T cells required for efficacy. Use of potently immunogenic human adenoviruses as vaccine vectors could overcome this problem, but these are limited by preexisting immunity to human adenoviruses. METHODS: From 2007 to 2010, we undertook a phase I dose and route finding study of a new malaria vaccine, a replication-incompetent chimpanzee adenovirus 63 (ChAd63) encoding the preerythrocytic insert multiple epitope thrombospondin-related adhesion protein (ME-TRAP; n = 54 vaccinees) administered alone (n = 28) or with a modified vaccinia virus Ankara (MVA) ME-TRAP booster immunization 8 weeks later (n = 26). We observed an excellent safety profile. High levels of TRAP antigen-specific CD8(+) and CD4(+) T cells, as detected by interferon γ enzyme-linked immunospot assay and flow cytometry, were induced by intramuscular ChAd63 ME-TRAP immunization at doses of 5 × 10(10) viral particles and above. Subsequent administration of MVA ME-TRAP boosted responses to exceptionally high levels, and responses were maintained for up to 30 months postvaccination. CONCLUSIONS: The ChAd63 chimpanzee adenovirus vector appears safe and highly immunogenic, providing a viable alternative to human adenoviruses as vaccine vectors for human use. CLINICAL TRIALS REGISTRATION: NCT00890019.

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Sheehy SH, Duncan CJ, Elias SC, Choudhary P, Biswas S, Halstead FD, Collins KA, Edwards NJ et al. 2012. ChAd63-MVA-vectored blood-stage Malaria vaccines targeting MSP1 and AMA1: Assessment of efficacy against mosquito bite challenge in humans Molecular Therapy, 20 (12), pp. 2355-2368. | Show Abstract | Read more

The induction of cellular immunity, in conjunction with antibodies, may be essential for vaccines to protect against blood-stage infection with the human malaria parasite Plasmodium falciparum. We have shown that prime-boost delivery of P. falciparum blood-stage antigens by chimpanzee adenovirus 63 (ChAd63) followed by the attenuated orthopoxvirus MVA is safe and immunogenic in healthy adults. Here, we report on vaccine efficacy against controlled human malaria infection delivered by mosquito bites. The blood-stage malaria vaccines were administered alone, or together (MSP1AMA1), or with a pre-erythrocytic malaria vaccine candidate (MSP1ME-TRAP). In this first human use of coadministered ChAd63-MVA regimes, we demonstrate immune interference whereby responses against merozoite surface protein 1 (MSP1) are dominant over apical membrane antigen 1 (AMA1) and ME-TRAP. We also show that induction of strong cellular immunity against MSP1 and AMA1 is safe, but does not impact on parasite growth rates in the blood. In a subset of vaccinated volunteers, a delay in time to diagnosis was observed and sterilizing protection was observed in one volunteer coimmunized with MSP1AMA1 - results consistent with vaccine-induced pre-erythrocytic, rather than blood-stage, immunity. These data call into question the utility of T cell-inducing blood-stage malaria vaccines and suggest that the focus should remain on high-titer antibody induction against susceptible antigen targets. © The American Society of Gene & Cell Therapy.

Antrobus RD, Lillie PJ, Berthoud TK, Spencer AJ, McLaren JE, Ladell K, Lambe T, Milicic A, Price DA, Hill AV, Gilbert SC. 2012. A T cell-inducing influenza vaccine for the elderly: safety and immunogenicity of MVA-NP+M1 in adults aged over 50 years. PLoS One, 7 (10), pp. e48322. | Show Abstract | Read more

BACKGROUND: Current influenza vaccines have reduced immunogenicity and are of uncertain efficacy in older adults. We assessed the safety and immunogenicity of MVA-NP+M1, a viral-vectored influenza vaccine designed to boost memory T cell responses, in a group of older adults. METHODS: Thirty volunteers (aged 50-85) received a single intramuscular injection of MVA-NP+M1 at a dose of 1·5×10(8) plaque forming units (pfu). Safety and immunogenicity were assessed over a period of one year. The frequency of T cells specific for nucleoprotein (NP) and matrix protein 1 (M1) was determined by interferon-gamma (IFN-γ) ELISpot, and their phenotypic and functional properties were characterized by polychromatic flow cytometry. In a subset of M1-specific CD8(+) T cells, T cell receptor (TCR) gene expression was evaluated using an unbiased molecular approach. RESULTS: Vaccination with MVA-NP+M1 was well tolerated. ELISpot responses were boosted significantly above baseline following vaccination. Increases were detected in both CD4(+) and CD8(+) T cell subsets. Clonality studies indicated that MVA-NP+M1 expanded pre-existing memory CD8(+) T cells, which displayed a predominant CD27(+)CD45RO(+)CD57(-)CCR7(-) phenotype both before and after vaccination. CONCLUSIONS: MVA-NP+M1 is safe and immunogenic in older adults. Unlike seasonal influenza vaccination, the immune responses generated by MVA-NP+M1 are similar between younger and older individuals. A T cell-inducing vaccine such as MVA-NP+M1 may therefore provide a way to circumvent the immunosenescence that impairs routine influenza vaccination. TRIAL REGISTRATION: ClinicalTrials.gov NCT00942071.

Lillie PJ, Duncan CJA, Sheehy SH, Meyer J, O'Hara GA, Gilbert SC, Hill AVS. 2012. Distinguishing malaria and influenza: Early clinical features in controlled human experimental infection studies Travel Medicine and Infectious Disease, 10 (4), pp. 192-196. | Show Abstract | Read more

During the H1N1 influenza pandemic (pH1N1/09) diagnostic algorithms were developed to guide antiviral provision. However febrile illnesses are notoriously difficult to distinguish clinically. Recent evidence highlights the importance of incorporating travel history into diagnostic algorithms to prevent the catastrophic misdiagnosis of life-threatening infections such as malaria. We applied retrospectively the UK pH1N1/09 case definition to a unique cohort of healthy adult volunteers exposed to Plasmodium falciparum malaria or influenza to assess the predictive value of this case definition, and to explore the distinguishing clinical features of early phase infection with these pathogens under experimental conditions. For influenza exposure the positive predictive value of the pH1N1/09 case definition was only 0.38 (95% CI: 0.06-0.60), with a negative predictive value of 0.27 (95% CI: 0.02-0.51). Interestingly, 8/11 symptomatic malaria-infected adults would have been inappropriately classified with influenza by the pH1N1/09 case definition, while 5/8 symptomatic influenza-exposed volunteers would have been classified without influenza (P = 0.18 Fisher's exact). Cough (P = 0.005) and nasal symptoms (P = 0.001) were the only clinical features that distinguished influenza-exposed from malaria-exposed volunteers. An open mind regarding the clinical cause of undifferentiated febrile illness, particularly in the absence of upper respiratory tract symptoms, remains important even during influenza pandemic settings. These data support incorporating travel history into pandemic algorithms. © 2012 Elsevier Ltd. All rights reserved.

Duncan CJ, Rowland R, Lillie PJ, Meyer J, Sheehy SH, O'Hara GA, Hamill M, Donaldson H et al. 2012. Incidental diagnosis in healthy clinical trial subjects. Clin Transl Sci, 5 (4), pp. 348-350. | Show Abstract | Read more

Previously unrecognized medical conditions identified in volunteers for early phase clinical studies have significant clinical and ethical implications for the participant. It is therefore crucial that the potential for unexpected diagnosis is addressed during the informed consent process. But the frequency of incidental diagnosis in healthy volunteers who attend for clinical trial screening remains unclear. To assess this we retrospectively analyzed 1,131 independent screening visits for 990 volunteers at a single academic center over a 10-year period to describe the frequency and nature of new clinical findings. Overall 23 of 990 volunteers (2.3%) were excluded at screening for a newly diagnosed medical abnormality. Some clinically important conditions, such as nephrotic syndrome and familial hypercholesterolemia were identified. The frequency of abnormalities was associated with increasing age in males (p= 0.02 χ(2) for trend) but not females (p= 0.82). These data will assist those planning and conducting phase I/II vaccine trials in healthy volunteers, and importantly should strengthen the informed consent of future trial participants.

Dicks MD, Spencer AJ, Edwards NJ, Wadell G, Bojang K, Gilbert SC, Hill AV, Cottingham MG. 2012. A novel chimpanzee adenovirus vector with low human seroprevalence: improved systems for vector derivation and comparative immunogenicity. PLoS One, 7 (7), pp. e40385. | Show Abstract | Read more

Recombinant adenoviruses are among the most promising tools for vaccine antigen delivery. Recently, the development of new vectors has focused on serotypes to which the human population is less exposed in order to circumvent pre-existing anti vector immunity. This study describes the derivation of a new vaccine vector based on a chimpanzee adenovirus, Y25, together with a comparative assessment of its potential to elicit transgene product specific immune responses in mice. The vector was constructed in a bacterial artificial chromosome to facilitate genetic manipulation of genomic clones. In order to conduct a fair head-to-head immunological comparison of multiple adenoviral vectors, we optimised a method for accurate determination of infectious titre, since this parameter exhibits profound natural variability and can confound immunogenicity studies when doses are based on viral particle estimation. Cellular immunogenicity of recombinant E1 E3-deleted vector ChAdY25 was comparable to that of other species E derived chimpanzee adenovirus vectors including ChAd63, the first simian adenovirus vector to enter clinical trials in humans. Furthermore, the prevalence of virus neutralizing antibodies (titre >1:200) against ChAdY25 in serum samples collected from two human populations in the UK and Gambia was particularly low compared to published data for other chimpanzee adenoviruses. These findings support the continued development of new chimpanzee adenovirus vectors, including ChAdY25, for clinical use.

Orubu T, Alharbi NK, Lambe T, Gilbert SC, Cottingham MG. 2012. Expression and cellular immunogenicity of a transgenic antigen driven by endogenous poxviral early promoters at their authentic loci in MVA. PLoS One, 7 (6), pp. e40167. | Show Abstract | Read more

CD8(+) T cell responses to vaccinia virus are directed almost exclusively against early gene products. The attenuated strain modified vaccinia virus Ankara (MVA) is under evaluation in clinical trials of new vaccines designed to elicit cellular immune responses against pathogens including Plasmodium spp., M. tuberculosis and HIV-1. All of these recombinant MVAs (rMVA) utilize the well-established method of linking the gene of interest to a cloned poxviral promoter prior to insertion into the viral genome at a suitable locus by homologous recombination in infected cells. Using BAC recombineering, we show that potent early promoters that drive expression of non-functional or non-essential MVA open reading frames (ORFs) can be harnessed for immunogenic expression of recombinant antigen. Precise replacement of the MVA orthologs of C11R, F11L, A44L and B8R with a model antigen positioned to use the same translation initiation codon allowed early transgene expression similar to or slightly greater than that achieved by the commonly-used p7.5 or short synthetic promoters. The frequency of antigen-specific CD8(+) T cells induced in mice by single shot or adenovirus-prime, rMVA-boost vaccination were similarly equal or marginally enhanced using endogenous promoters at their authentic genomic loci compared to the traditional constructs. The enhancement in immunogenicity observed using the C11R or F11L promoters compared with p7.5 was similar to that obtained with the mH5 promoter compared with p7.5. Furthermore, the growth rates of the viruses were unimpaired and the insertions were genetically stable. Insertion of a transgenic ORF in place of a viral ORF by BAC recombineering can thus provide not only a potent promoter, but also, concomitantly, a suitable insertion site, potentially facilitating development of MVA vaccines expressing multiple recombinant antigens.

Spencer AJ, Hill F, Honeycutt JD, Cottingham MG, Bregu M, Rollier CS, Furze J, Draper SJ et al. 2012. Fusion of the Mycobacterium tuberculosis antigen 85A to an oligomerization domain enhances its immunogenicity in both mice and non-human primates. PLoS One, 7 (3), pp. e33555. | Show Abstract | Read more

To prevent important infectious diseases such as tuberculosis, malaria and HIV, vaccines inducing greater T cell responses are required. In this study, we investigated whether fusion of the M. tuberculosis antigen 85A to recently described adjuvant IMX313, a hybrid avian C4bp oligomerization domain, could increase T cell responses in pre-clinical vaccine model species. In mice, the fused antigen 85A showed consistent increases in CD4(+) and CD8(+) T cell responses after DNA and MVA vaccination. In rhesus macaques, higher IFN-γ responses were observed in animals vaccinated with MVA-Ag85A IMX313 after both primary and secondary immunizations. In both animal models, fusion to IMX313 induced a quantitative enhancement in the response without altering its quality: multifunctional cytokines were uniformly increased and differentiation into effector and memory T cell subsets was augmented rather than skewed. An extensive in vivo characterization suggests that IMX313 improves the initiation of immune responses as an increase in antigen 85A specific cells was observed as early as day 3 after vaccination. This report demonstrates that antigen multimerization using IMX313 is a simple and effective cross-species method to improve vaccine immunogenicity with potentially broad applicability.

Hope JC, Guzman E, Cubillos-Zapata C, Stephens SA, Gilbert SC, Prentice H, Sopp P, Howard CJ, Charleston B. 2012. Migratory sub-populations of afferent lymphatic dendritic cells differ in their interactions with Mycobacterium bovis Bacille Calmette Guerin. Vaccine, 30 (13), pp. 2357-2367. | Show Abstract | Read more

Understanding how pathogens or vaccine antigens are targeted to dendritic cell (DC) subsets is important for disease pathogenesis studies and vaccine design. We characterised the sub-populations of migrating bovine DC with functional and phenotypic diversity present in pseudoafferent lymph draining the skin. These skin draining DC exist as a series of maturation dependent subsets with differential capacities for antigen uptake and cytokine expression, and include both Langerhans' cells (LC) and dermal derived cells. Furthermore, Mycobacterium bovis Bacille Calmette Guerin, a vaccine which is administered by the intradermal route, was only taken up by a small number of the migrating DC, which were SIRPα(+) and expressed the mannose receptor and CD1b. This was evident following in vitro infection and also in vivo following inoculation of green fluorescent BCG over the lymphatic cannulation site. Only the SIRPα(+) DC were able to present antigen to T cells isolated from BCG vaccinated calves. Furthermore, presentation of BCG antigens by DC to T lymphocytes was ineffective compared to mycobacterial proteins. However, mycobacterial antigen 85 was delivered more effectively to DC via an adenoviral vector and the magnitude of the subsequent antigen-specific T cell response was significantly increased. This study further extends our understanding of the biology of migrating DC, identifies potential explanations for the modest success of BCG vaccination and demonstrates that targeted delivery of antigens via adenoviruses to DC can improve antigen presentation.

Sheehy SH, Duncan CJ, Elias SC, Biswas S, Collins KA, O'Hara GA, Halstead FD, Ewer KJ et al. 2012. Phase Ia clinical evaluation of the safety and immunogenicity of the Plasmodium falciparum blood-stage antigen AMA1 in ChAd63 and MVA vaccine vectors. PLoS One, 7 (2), pp. e31208. | Show Abstract | Read more

BACKGROUND: Traditionally, vaccine development against the blood-stage of Plasmodium falciparum infection has focused on recombinant protein-adjuvant formulations in order to induce high-titer growth-inhibitory antibody responses. However, to date no such vaccine encoding a blood-stage antigen(s) alone has induced significant protective efficacy against erythrocytic-stage infection in a pre-specified primary endpoint of a Phase IIa/b clinical trial designed to assess vaccine efficacy. Cell-mediated responses, acting in conjunction with functional antibodies, may be necessary for immunity against blood-stage P. falciparum. The development of a vaccine that could induce both cell-mediated and humoral immune responses would enable important proof-of-concept efficacy studies to be undertaken to address this question. METHODOLOGY: We conducted a Phase Ia, non-randomized clinical trial in 16 healthy, malaria-naïve adults of the chimpanzee adenovirus 63 (ChAd63) and modified vaccinia virus Ankara (MVA) replication-deficient viral vectored vaccines encoding two alleles (3D7 and FVO) of the P. falciparum blood-stage malaria antigen; apical membrane antigen 1 (AMA1). ChAd63-MVA AMA1 administered in a heterologous prime-boost regime was shown to be safe and immunogenic, inducing high-level T cell responses to both alleles 3D7 (median 2036 SFU/million PBMC) and FVO (median 1539 SFU/million PBMC), with a mixed CD4(+)/CD8(+) phenotype, as well as substantial AMA1-specific serum IgG responses (medians of 49 µg/mL and 41 µg/mL for 3D7 and FVO AMA1 respectively) that demonstrated growth inhibitory activity in vitro. CONCLUSIONS: ChAd63-MVA is a safe and highly immunogenic delivery platform for both alleles of the AMA1 antigen in humans which warrants further efficacy testing. ChAd63-MVA is a promising heterologous prime-boost vaccine strategy that could be applied to numerous other diseases where strong cellular and humoral immune responses are required for protection. TRIAL REGISTRATION: ClinicalTrials.gov NCT01095055.

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Gilbert SC. 2012. T-cell-inducing vaccines - what's the future Immunology, 135 (1), pp. 19-26. | Show Abstract | Read more

In the twentieth century vaccine development has moved from the use of attenuated or killed micro-organisms to protein sub-unit vaccines, with vaccine immunogenicity assessed by measuring antibodies induced by vaccination. However, for many infectious diseases T cells are an important part of naturally acquired protective immune responses, and inducing these by vaccination has been the aim of much research. The progress that has been made in developing effective T-cell-inducing vaccines against viral and parasitic diseases such as HIV and malaria is discussed, along with recent developments in therapeutic vaccine development for chronic viral infections and cancer. Although many ways of inducing T cells by vaccination have been assessed, the majority result in low level, non-protective responses. Sufficient clinical research has now been conducted to establish that replication-deficient viral vectored vaccines lead the field in inducing strong and broad responses, and efficacy studies of T-cell-inducing vaccines against a number of diseases are finally demonstrating that this is a valid approach to filling the gaps in our defence against not only infectious disease, but some forms of cancer. © 2011 The Author. Immunology © 2011 Blackwell Publishing Ltd.

Busquets N, Lopez E, Lorenzo G, Abad FX, Galindo I, Rivas R, Solanes D, Bensaid A et al. 2011. Evaluation of the protective potential of a recombinant modified vaccinia virus Ankara (MVA) encoding rift valley fever virus (RVFV) glycoproteins in lambs TROPICAL MEDICINE & INTERNATIONAL HEALTH, 16 pp. 239-239.

Dicks MDJ, Spencer A, Edwards N, Gilbert S, Hill S, Cottingham MG. 2011. Improved systems for generating and evaluating adenovirus vaccine vectors reveal differences in immunogenicity between vectors of different adenoviral species HUMAN GENE THERAPY, 22 (10), pp. A35-A35.

Gharbi M, Darghouth MA, Weir W, Katzer F, Boulter N, Adamson R, Gilbert SC, Jongejan F et al. 2011. Prime-boost immunisation against tropical theileriosis with two parasite surface antigens: evidence for protection and antigen synergy. Vaccine, 29 (38), pp. 6620-6628. | Show Abstract | Read more

Current methods for control of tropical theileriosis in cattle suffer from several disadvantages that could be circumvented by development of an effective sub-unit vaccine. Previous work has utilised two major surface antigens (SPAG-1 and Tams1) and conventional adjuvants to provide partial protection against parasite challenge. In this study we have delivered these antigens using the prime-boost system and analysed whether a combination regime can enhance protection against lethal challenge. Delivery of the boost as recombinant protein or expressed from a recombinant MVA vector was also assessed. The results confirmed that immunisation with Tams1 alone could reduce the severity of several disease parameters compared to non-immunised controls and these effects were more marked when recombinant protein was used for boosting compared to MVA delivery. A similar outcome was obtained by immunisation with SPAG-1 alone. Significantly, delivery of SPAG-1 and Tams1 as a cocktail showed enhanced protection. This was manifest by significant improvement in a large range of clinical and parasitological parameters and, most dramatically, by the survival and recovery of 50% of the immunised animals compared to 0% of the controls. Analysis of the antibody response post-challenge showed that while there was a strong response to Tams1, no response to SPAG-1 was detected. In contrast, lymphoproliferation assays showed a significant enhancement of response at day 7 post-challenge in calves of the SPAG-1 group but a dramatic decrease of the proliferation activity in all three groups receiving Tams1. We conclude that immunisation with a cocktail of SPAG-1 and Tams1 generates a synergistic protective response that significantly improves the efficacy of recombinant vaccination against tropical theileriosis. Potential effector mechanisms that could mediate this response are discussed.

Forbes EK, Biswas S, Collins KA, Gilbert SC, Hill AV, Draper SJ. 2011. Combining liver- and blood-stage malaria viral-vectored vaccines: investigating mechanisms of CD8+ T cell interference. J Immunol, 187 (7), pp. 3738-3750. | Show Abstract | Read more

Replication-deficient adenovirus and modified vaccinia virus Ankara (MVA) vectors expressing single pre-erythrocytic or blood-stage Plasmodium falciparum Ags have entered clinical testing using a heterologous prime-boost immunization approach. In this study, we investigated the utility of the same immunization regimen when combining viral vectored vaccines expressing the 42-kDa C terminus of the blood-stage Ag merozoite surface protein 1 and the pre-erythrocytic Ag circumsporozoite protein in the Plasmodium yoelii mouse model. We find that vaccine coadministration leads to maintained Ab responses and efficacy against blood-stage infection, but reduced secondary CD8(+) T cell responses against both Ags and efficacy against liver-stage infection. CD8(+) T cell interference can be minimized by coadministering the MVA vaccines at separate sites, resulting in enhanced liver-stage efficacy in mice immunized against both Ags compared with just one. CD8(+) T cell interference (following MVA coadministration as a mixture) may be caused partly by a lack of physiologic space for high-magnitude responses against multiple Ags, but is not caused by competition for presentation of Ag on MHC class I molecules, nor is it due to restricted T cell access to APCs presenting both Ags. Instead, enhanced killing of peptide-pulsed cells is observed in mice possessing pre-existing T cells against two Ags compared with just one, suggesting that priming against multiple Ags may in part reduce the potency of multiantigen MVA vectors to stimulate secondary CD8(+) T cell responses. These data have important implications for the development of a multistage or multicomponent viral vectored malaria vaccine for use in humans.

Sheehy SH, Duncan CJ, Elias SC, Collins KA, Ewer KJ, Spencer AJ, Williams AR, Halstead FD et al. 2011. Phase Ia clinical evaluation of the Plasmodium falciparum blood-stage antigen MSP1 in ChAd63 and MVA vaccine vectors. Mol Ther, 19 (12), pp. 2269-2276. | Show Abstract | Read more

Efficacy trials of antibody-inducing protein-in-adjuvant vaccines targeting the blood-stage Plasmodium falciparum malaria parasite have so far shown disappointing results. The induction of cell-mediated responses in conjunction with antibody responses is thought to be one alternative strategy that could achieve protective efficacy in humans. Here, we prepared chimpanzee adenovirus 63 (ChAd63) and modified vaccinia virus Ankara (MVA) replication-deficient vectors encoding the well-studied P. falciparum blood-stage malaria antigen merozoite surface protein 1 (MSP1). A phase Ia clinical trial was conducted in healthy adults of a ChAd63-MVA MSP1 heterologous prime-boost immunization regime. The vaccine was safe and generally well tolerated. Fewer systemic adverse events (AEs) were observed following ChAd63 MSP1 than MVA MSP1 administration. Exceptionally strong T-cell responses were induced, and these displayed a mixed of CD4(+) and CD8(+) phenotype. Substantial MSP1-specific serum immunoglobulin G (IgG) antibody responses were also induced, which were capable of recognizing native parasite antigen, but these did not reach titers sufficient to neutralize P. falciparum parasites in vitro. This viral vectored vaccine regime is thus a leading approach for the induction of strong cellular and humoral immunogenicity against difficult disease targets in humans. Further studies are required to assess whether this strategy can achieve protective efficacy against blood-stage malaria infection.

de Cassan SC, Forbes EK, Douglas AD, Milicic A, Singh B, Gupta P, Chauhan VS, Chitnis CE, Gilbert SC, Hill AV, Draper SJ. 2011. The requirement for potent adjuvants to enhance the immunogenicity and protective efficacy of protein vaccines can be overcome by prior immunization with a recombinant adenovirus. J Immunol, 187 (5), pp. 2602-2616. | Show Abstract | Read more

A central goal in vaccinology is the induction of high and sustained Ab responses. Protein-in-adjuvant formulations are commonly used to achieve such responses. However, their clinical development can be limited by the reactogenicity of some of the most potent preclinical adjuvants and the cost and complexity of licensing new adjuvants for human use. Also, few adjuvants induce strong cellular immunity, which is important for protection against many diseases, such as malaria. We compared classical adjuvants such as aluminum hydroxide to new preclinical adjuvants and adjuvants in clinical development, such as Abisco 100, CoVaccine HT, Montanide ISA720, and stable emulsion-glucopyranosyl lipid A, for their ability to induce high and sustained Ab responses and T cell responses. These adjuvants induced a broad range of Ab responses when used in a three-shot protein-in-adjuvant regimen using the model Ag OVA and leading blood-stage malaria vaccine candidate Ags. Surprisingly, this range of Ab immunogenicity was greatly reduced when a protein-in-adjuvant vaccine was used to boost Ab responses primed by a human adenovirus serotype 5 vaccine recombinant for the same Ag. This human adenovirus serotype 5-protein regimen also induced a more cytophilic Ab response and demonstrated improved efficacy of merozoite surface protein-1 protein vaccines against a Plasmodium yoelii blood-stage challenge. This indicates that the differential immunogenicity of protein vaccine adjuvants may be largely overcome by prior immunization with recombinant adenovirus, especially for adjuvants that are traditionally considered poorly immunogenic in the context of subunit vaccination and may circumvent the need for more potent chemical adjuvants.

Douglas AD, Andrews L, Draper SJ, Bojang K, Milligan P, Gilbert SC, Imoukhuede EB, Hill AV. 2011. Substantially reduced pre-patent parasite multiplication rates are associated with naturally acquired immunity to Plasmodium falciparum. J Infect Dis, 203 (9), pp. 1337-1340. | Show Abstract | Read more

Naturally acquired immunity to Plasmodium falciparum's asexual blood stage reduces parasite multiplication at microscopically detectable densities. The effect of natural immunity on initial prepatent parasite multiplication during the period following a new infection has been uncertain, contributing to doubt regarding the utility of experimental challenge models for blood-stage vaccine trials. Here we present data revealing that parasite multiplication rates during the initial prepatent period in semi-immune Gambian adults are substantially lower than in malaria-naive participants. This supports the view that a blood-stage vaccine capable of emulating the disease-reducing effect of natural immunity could achieve a detectable effect during the prepatent period.

Vordermeier HM, Villarreal-Ramos B, Cockle PJ, McAulay M, Rhodes SG, Thacker T, Gilbert SC, McShane H, Hill AVS, Xing Z, Hewinson RG. 2011. Viral Booster Vaccines Improve Mycobacterium bovis BCG-Induced Protection against Bovine Tuberculosis, (vol 77, pg 3371, 2009) INFECTION AND IMMUNITY, 79 (5), pp. 2134-2134. | Read more

Porter DW, Thompson FM, Berthoud TK, Hutchings CL, Andrews L, Biswas S, Poulton I, Prieur E et al. 2011. A human Phase I/IIa malaria challenge trial of a polyprotein malaria vaccine. Vaccine, 29 (43), pp. 7514-7522. | Show Abstract | Read more

We examined the safety, immunogenicity and efficacy of a prime-boost vaccination regime involving two poxvirus malaria subunit vaccines, FP9-PP and MVA-PP, expressing the same polyprotein consisting of six pre-erythrocytic antigens from Plasmodium falciparum. Following safety assessment of single doses, 15 volunteers received a heterologous prime-boost vaccination regime and underwent malaria sporozoite challenge. The vaccines were safe but interferon-γ ELISPOT responses were low compared to other poxvirus vectors, despite targeting multiple antigens. There was no vaccine efficacy as measured by delay in time to parasitaemia. A number of possible explanations are discussed, including the very large insert size of the polyprotein transgene.

Duncan CJ, Sheehy SH, Ewer KJ, Douglas AD, Collins KA, Halstead FD, Elias SC, Lillie PJ et al. 2011. Impact on malaria parasite multiplication rates in infected volunteers of the protein-in-adjuvant vaccine AMA1-C1/Alhydrogel+CPG 7909. PLoS One, 6 (7), pp. e22271. | Show Abstract | Read more

BACKGROUND: Inhibition of parasite growth is a major objective of blood-stage malaria vaccines. The in vitro assay of parasite growth inhibitory activity (GIA) is widely used as a surrogate marker for malaria vaccine efficacy in the down-selection of candidate blood-stage vaccines. Here we report the first study to examine the relationship between in vivo Plasmodium falciparum growth rates and in vitro GIA in humans experimentally infected with blood-stage malaria. METHODS: In this phase I/IIa open-label clinical trial five healthy malaria-naive volunteers were immunised with AMA1/C1-Alhydrogel+CPG 7909, and together with three unvaccinated controls were challenged by intravenous inoculation of P. falciparum infected erythrocytes. RESULTS: A significant correlation was observed between parasite multiplication rate in 48 hours (PMR) and both vaccine-induced growth-inhibitory activity (Pearson r = -0.93 [95% CI: -1.0, -0.27] P = 0.02) and AMA1 antibody titres in the vaccine group (Pearson r = -0.93 [95% CI: -0.99, -0.25] P = 0.02). However immunisation failed to reduce overall mean PMR in the vaccine group in comparison to the controls (vaccinee 16 fold [95% CI: 12, 22], control 17 fold [CI: 0, 65] P = 0.70). Therefore no impact on pre-patent period was observed (vaccine group median 8.5 days [range 7.5-9], control group median 9 days [range 7-9]). CONCLUSIONS: Despite the first observation in human experimental malaria infection of a significant association between vaccine-induced in vitro growth inhibitory activity and in vivo parasite multiplication rate, this did not translate into any observable clinically relevant vaccine effect in this small group of volunteers. TRIAL REGISTRATION: ClinicalTrials.gov [NCT00984763].

Berthoud TK, Hamill M, Lillie PJ, Hwenda L, Collins KA, Ewer KJ, Milicic A, Poyntz HC et al. 2011. Potent CD8+ T-cell immunogenicity in humans of a novel heterosubtypic influenza A vaccine, MVA-NP+M1. Clin Infect Dis, 52 (1), pp. 1-7. | Show Abstract | Read more

BACKGROUND: Influenza A viruses cause occasional pandemics and frequent epidemics. Licensed influenza vaccines that induce high antibody titers to the highly polymorphic viral surface antigen hemagglutinin must be re-formulated and readministered annually. A vaccine providing protective immunity to the highly conserved internal antigens could provide longer-lasting protection against multiple influenza subtypes. METHODS: We prepared a Modified Vaccinia virus Ankara (MVA) vector encoding nucleoprotein and matrix protein 1 (MVA-NP+M1) and conducted a phase I clinical trial in healthy adults. RESULTS: The vaccine was generally safe and well tolerated, with significantly fewer local side effects after intramuscular rather than intradermal administration. Systemic side effects increased at the higher dose in both frequency and severity, with 5 out of 8 volunteers experiencing severe nausea/vomiting, malaise, or rigors. Ex vivo T-cell responses to NP and M1 measured by IFN-γ ELISPOT assay were significantly increased after vaccination (prevaccination median of 123 spot-forming units/million peripheral blood mononuclear cells, postvaccination peak response median 339, 443, and 1443 in low-dose intradermal, low-dose intramuscular, and high-dose intramuscular groups, respectively), and the majority of the antigen-specific T cells were CD8(+). CONCLUSIONS: We conclude that the vaccine was both safe and remarkably immunogenic, leading to frequencies of responding T cells that appear to be much higher than those induced by any other influenza vaccination approach. Further studies will be required to find the optimum dose and to assess whether the increased T-cell response to conserved influenza proteins results in protection from influenza disease.

Biswas S, Dicks MD, Long CA, Remarque EJ, Siani L, Colloca S, Cottingham MG, Holder AA, Gilbert SC, Hill AV, Draper SJ. 2011. Transgene optimization, immunogenicity and in vitro efficacy of viral vectored vaccines expressing two alleles of Plasmodium falciparum AMA1. PLoS One, 6 (6), pp. e20977. | Show Abstract | Read more

BACKGROUND: Apical membrane antigen 1 (AMA1) is a leading candidate vaccine antigen against blood-stage malaria, although to date numerous clinical trials using mainly protein-in-adjuvant vaccines have shown limited success. Here we describe the pre-clinical development and optimization of recombinant human and simian adenoviral (AdHu5 and ChAd63) and orthopoxviral (MVA) vectors encoding transgene inserts for Plasmodium falciparum AMA1 (PfAMA1). METHODOLOGY/PRINCIPAL FINDINGS: AdHu5-MVA prime-boost vaccination in mice and rabbits using these vectors encoding the 3D7 allele of PfAMA1 induced cellular immune responses as well as high-titer antibodies that showed growth inhibitory activity (GIA) against the homologous but not heterologous parasite strains. In an effort to overcome the issues of PfAMA1 antigenic polymorphism and pre-existing immunity to AdHu5, a simian adenoviral (ChAd63) vector and MVA encoding two alleles of PfAMA1 were developed. This antigen, composed of the 3D7 and FVO alleles of PfAMA1 fused in tandem and with expression driven by a single promoter, was optimized for antigen secretion and transmembrane expression. These bi-allelic PfAMA1 vaccines, when administered to mice and rabbits, demonstrated comparable immunogenicity to the mono-allelic vaccines and purified serum IgG now showed GIA against the two divergent strains of P. falciparum encoded in the vaccine. CD8(+) and CD4(+) T cell responses against epitopes that were both common and unique to the two alleles of PfAMA1 were also measured in mice. CONCLUSIONS/SIGNIFICANCE: Optimized transgene inserts encoding two divergent alleles of the same antigen can be successfully inserted into adeno- and pox-viral vaccine vectors. Adenovirus-MVA immunization leads to the induction of T cell responses common to both alleles, as well as functional antibody responses that are effective against both of the encoded strains of P. falciparum in vitro. These data support the further clinical development of these vaccine candidates in Phase I/IIa clinical trials.

Cited:

86

Scopus

Sheehy SH, Duncan CJA, Elias SC, Collins KA, Ewer KJ, Spencer AJ, Williams AR, Halstead FD et al. 2011. Phase Ia clinical evaluation of the plasmodium falciparum blood-stage antigen MSP1 in ChAd63 and MVA vaccine vectors Molecular Therapy, 19 (12), pp. 2269-2276. | Show Abstract | Read more

Efficacy trials of antibody-inducing protein-in-adjuvant vaccines targeting the blood-stage Plasmodium falciparum malaria parasite have so far shown disappointing results. The induction of cell-mediated responses in conjunction with antibody responses is thought to be one alternative strategy that could achieve protective efficacy in humans. Here, we prepared chimpanzee adenovirus 63 (ChAd63) and modified vaccinia virus Ankara (MVA) replication-deficient vectors encoding the well-studied P. falciparum blood-stage malaria antigen merozoite surface protein 1 (MSP1). A phase Ia clinical trial was conducted in healthy adults of a ChAd63-MVA MSP1 heterologous prime-boost immunization regime. The vaccine was safe and generally well tolerated. Fewer systemic adverse events (AEs) were observed following ChAd63 MSP1 than MVA MSP1 administration. Exceptionally strong T-cell responses were induced, and these displayed a mixed of CD4 and CD8 phenotype. Substantial MSP1-specific serum immunoglobulin G (IgG) antibody responses were also induced, which were capable of recognizing native parasite antigen, but these did not reach titers sufficient to neutralize P. falciparum parasites in vitro. This viral vectored vaccine regime is thus a leading approach for the induction of strong cellular and humoral immunogenicity against difficult disease targets in humans. Further studies are required to assess whether this strategy can achieve protective efficacy against blood-stage malaria infection. © The American Society of Gene & Cell Therapy.

Cubillos-Zapata C, Guzman E, Turner A, Gilbert SC, Prentice H, Hope JC, Charleston B. 2011. Differential effects of viral vectors on migratory afferent lymph dendritic cells in vitro predict enhanced immunogenicity in vivo Journal of Virology, 85 (18), pp. 9385-9394. | Show Abstract | Read more

Targeting dendritic cells (DC) is key to driving effective immune responses. Lymphatic cannulation provides access to the heterogeneous populations of DC draining peripheral sites in rodents and ruminants. Afferent lymph DEC-205 + CD11c ( SIRPα + DC were preferentially infected ex vivo with three vaccine viral vectors: recombinant human replication-defective human adenovirus 5 (rhuAdV5), recombinant modified vaccinia virus Ankara (rMVA), and recombinant fowlpox virus (rFPV), all expressing green fluorescent protein (GFP). The rhuAdV5-infected cells remained viable, and peak GFP expression was observed 16 to 24 h posttransduction. Increasing the incubation period of DC with rhuAdV5 enhanced GFP expression. In contrast, DC infected with rMVA-GFP or rFPV-GFP became rapidly apoptotic and GFP expression peaked at 6 h postinfection. Delivery of foot-and-mouth disease virus (FMDV) A 22 antigen to DC by rhuAdV5-FMDV-A 22 ex vivo resulted in significantly greater CD4+ T cell proliferation than did delivery by rFPV-FMDV-A 22. Delivery of rhuAdV5-GFP in oil adjuvant in vivo, to enhance DC-vector contact, resulted in increased GFP expression in migrating DC compared to that with vector alone. Similarly, CD4 + T cell responses were significantly enhanced when using rhuAdV5-FMDV-A 22 in adjuvant. Therefore, the interaction between viral vectors and afferent lymph DC ex vivo can predict the outcome of in vivo immunization and provide a means of rapidly assessing the effects of vector modification. © 2011, American Society for Microbiology.

Hopkins R, Bridgeman A, Joseph J, Gilbert SC, McShane H, Hanke T. 2011. Dual neonate vaccine platform against HIV-1 and M. tuberculosis. PLoS One, 6 (5), pp. e20067. | Show Abstract | Read more

Acquired immunodeficiency syndrome and tuberculosis (TB) are two of the world's most devastating diseases. The first vaccine the majority of infants born in Africa receive is Mycobacterium bovis bacillus Calmette-Guérin (BCG) as a prevention against TB. BCG protects against disseminated disease in the first 10 years of life, but provides a variable protection against pulmonary TB and enhancing boost delivered by recombinant modified vaccinia virus Ankara (rMVA) expressing antigen 85A (Ag85A) of M. tuberculosis is currently in phase IIb evaluation in African neonates. If the newborn's mother is positive for human immunodeficiency virus type 1 (HIV-1), the baby is at high risk of acquiring HIV-1 through breastfeeding. We suggested that a vaccination consisting of recombinant BCG expressing HIV-1 immunogen administered at birth followed by a boost with rMVA sharing the same immunogen could serve as a strategy for prevention of mother-to-child transmission of HIV-1 and rMVA expressing an African HIV-1-derived immunogen HIVA is currently in phase I trials in African neonates. Here, we aim to develop a dual neonate vaccine platform against HIV-1 and TB consisting of BCG.HIVA administered at birth followed by a boost with MVA.HIVA.85A. Thus, mMVA.HIVA.85A and sMVA.HIVA.85A vaccines were constructed, in which the transgene transcription is driven by either modified H5 or short synthetic promoters, respectively, and tested for immunogenicity alone and in combination with BCG.HIVA(222). mMVA.HIVA.85A was produced markerless and thus suitable for clinical manufacture. While sMVA.HIVA.85A expressed higher levels of the immunogens, it was less immunogenic than mMVA.HIVA.85A in BALB/c mice. A BCG.HIVA(222)-mMVA.HIVA.85A prime-boost regimen induced robust T cell responses to both HIV-1 and M. tuberculosis. Therefore, proof-of-principle for a dual anti-HIV-1/M. tuberculosis infant vaccine platform is established. Induction of immune responses against these pathogens soon after birth is highly desirable and may provide a basis for lifetime protection maintained by boosts later in life.

Draper SJ, Biswas S, Spencer AJ, Remarque EJ, Capone S, Naddeo M, Dicks MD, Faber BW et al. 2010. Enhancing blood-stage malaria subunit vaccine immunogenicity in rhesus macaques by combining adenovirus, poxvirus, and protein-in-adjuvant vaccines. J Immunol, 185 (12), pp. 7583-7595. | Show Abstract | Read more

Protein-in-adjuvant formulations and viral-vectored vaccines encoding blood-stage malaria Ags have shown efficacy in rodent malaria models and in vitro assays against Plasmodium falciparum. Abs and CD4(+) T cell responses are associated with protective efficacy against blood-stage malaria, whereas CD8(+) T cells against some classical blood-stage Ags can also have a protective effect against liver-stage parasites. No subunit vaccine strategy alone has generated demonstrable high-level efficacy against blood-stage infection in clinical trials. The induction of high-level Ab responses, as well as potent T and B cell effector and memory populations, is likely to be essential to achieve immediate and sustained protective efficacy in humans. This study describes in detail the immunogenicity of vaccines against P. falciparum apical membrane Ag 1 in rhesus macaques (Macaca mulatta), including the chimpanzee adenovirus 63 (AdCh63), the poxvirus modified vaccinia virus Ankara (MVA), and protein vaccines formulated in Alhydrogel or CoVaccine HT adjuvants. AdCh63-MVA heterologous prime-boost immunization induces strong and long-lasting multifunctional CD8(+) and CD4(+) T cell responses that exhibit a central memory-like phenotype. Three-shot (AdCh63-MVA-protein) or two-shot (AdCh63-protein) regimens induce memory B cells and high-titer functional IgG responses that inhibit the growth of two divergent strains of P. falciparum in vitro. Prior immunization with adenoviral vectors of alternative human or simian serotype does not affect the immunogenicity of the AdCh63 apical membrane Ag 1 vaccine. These data encourage the further clinical development and coadministration of protein and viral vector vaccine platforms in an attempt to induce broad cellular and humoral immune responses against blood-stage malaria Ags in humans.

Douglas AD, de Cassan SC, Dicks MD, Gilbert SC, Hill AV, Draper SJ. 2010. Tailoring subunit vaccine immunogenicity: maximizing antibody and T cell responses by using combinations of adenovirus, poxvirus and protein-adjuvant vaccines against Plasmodium falciparum MSP1. Vaccine, 28 (44), pp. 7167-7178. | Show Abstract | Read more

Subunit vaccination modalities tend to induce particular immune effector responses. Viral vectors are well known for their ability to induce strong T cell responses, while protein-adjuvant vaccines have been used primarily for induction of antibody responses. Here, we demonstrate in mice using a Plasmodium falciparum merozoite surface protein 1 (PfMSP1) antigen that novel regimes combining adenovirus and poxvirus vectored vaccines with protein antigen in Montanide ISA720 adjuvant can achieve simultaneous antibody and T cell responses which equal, or in some cases surpass, the best immune responses achieved by either the viral vectors or the protein vaccine alone. Such broad responses can be achieved either using three-stage vaccination protocols, or with an equally effective two-stage protocol in which viral vectors are admixed with protein and adjuvant, and were apparent despite the use of a protein antigen that represented only a portion of the viral vector antigen. We describe further possible advantages of viral vectors in achieving consistent antibody priming, enhanced antibody avidity, and cytophilic isotype skew. These data strengthen the evidence that tailored combinations of vaccine platforms can achieve desired combinations of immune responses, and further encourage the co-administration of antibody-inducing recombinant protein vaccines with T cell- and antibody-inducing recombinant viral vectors as one strategy that may achieve protective blood-stage malaria immunity in humans.

Goodman AL, Epp C, Moss D, Holder AA, Wilson JM, Gao GP, Long CA, Remarque EJ et al. 2010. New candidate vaccines against blood-stage Plasmodium falciparum malaria: prime-boost immunization regimens incorporating human and simian adenoviral vectors and poxviral vectors expressing an optimized antigen based on merozoite surface protein 1. Infect Immun, 78 (11), pp. 4601-4612. | Show Abstract | Read more

Although merozoite surface protein 1 (MSP-1) is a leading candidate vaccine antigen for blood-stage malaria, its efficacy in clinical trials has been limited in part by antigenic polymorphism and potentially by the inability of protein-in-adjuvant vaccines to induce strong cellular immunity. Here we report the design of novel vectored Plasmodium falciparum vaccines capable of overcoming such limitations. We optimized an antigenic insert comprising the four conserved blocks of MSP-1 fused to tandemly arranged sequences that represent both allelic forms of the dimorphic 42-kDa C-terminal region. Inserts were expressed by adenoviral and poxviral vectors and employed in heterologous prime-boost regimens. Simian adenoviral vectors were used in an effort to circumvent preexisting immunity to human adenoviruses. In preclinical studies these vaccines induced potent cellular immune responses and high-titer antibodies directed against MSP-1. The antibodies induced were found to have growth-inhibitory activity against dimorphic allelic families of P. falciparum. These vectored vaccines should allow assessment in humans of the safety and efficacy of inducing strong cellular as well as cross-strain humoral immunity to P. falciparum MSP-1.

Young SL, Slobbe LJ, Peacey M, Gilbert SC, Buddle BM, de Lisle GW, Buchan GS. 2010. Immunogenicity and protective efficacy of mycobacterial DNA vaccines incorporating plasmid-encoded cytokines against Mycobacterium bovis. Immunol Cell Biol, 88 (6), pp. 651-657. | Show Abstract | Read more

DNA-based vaccines, alone or in combination with other sub-unit vaccination regimes, represent an alternative to live mycobacterial vaccines for protective immunization against tuberculosis. Here, we have used a murine immunization or Mycobacterium bovis aerosol challenge model to assess the immunogenicity and protective efficacy of mycobacterial DNA vaccines. Mice that received immunization with DNA constructs encoding M. bovis antigen 85A (Ag85-A) and arget(ESAT-6) produced measurable interferon-gamma (IFN-gamma) responses to CD4(+) T-cell epitope-peptide recall antigens in vitro. The magnitude of these responses was enhanced by co-delivery of a construct encoding murine cytokines (macrophage inhibitory protein (MIP)-1 alpha or interleukin(IL)-7), although they did not the match responses observed in mice that received Bacille Calmette-Guerin(BCG) immunisation. In contrast, DNA priming followed by boosting with modified vaccinia Ankara (MVA) vaccine (expressing M. tuberculosis Ag85-A) invoked higher IFN-gamma levels, with the most immunogenic regime of Ag85 or ESAT or IL-7 prime followed by MVA boost being of commensurate immunogenicity to BCG. Despite this, neither DNA alone nor DNA-prime or MVA boost regimes conferred measurable protection against aerosol challenge with virulent M. bovis. These data highlight both the promise and the shortcomings of new generation subunit tuberculosis vaccines, with particular emphasis on their potential as vaccines against M. bovis.

Cottingham MG, Gilbert SC. 2010. Rapid generation of markerless recombinant MVA vaccines by en passant recombineering of a self-excising bacterial artificial chromosome. J Virol Methods, 168 (1-2), pp. 233-236. | Show Abstract | Read more

The non-replicating poxviral vector modified vaccinia virus Ankara (MVA) is currently a leading candidate for development of novel recombinant vaccines against globally important diseases. The 1980s technology for making recombinant MVA (and other poxviruses) is powerful and robust, but relies on rare recombination events in poxviral-infected cells. In the 21st century, it has become possible to apply bacterial artificial chromosome (BAC) technology to poxviruses, as first demonstrated by B. Moss' lab in 2002 for vaccinia virus. A similar BAC clone of MVA was subsequently derived, but while recombination-mediated genetic engineering for rapid production was used of deletion mutants, an alternative method was required for efficient insertion of transgenes. Furthermore "markerless" viruses, which carry no trace of the selectable marker used for their isolation, are increasingly required for clinical trials, and the viruses derived via the new method contained the BAC sequence in their genomic DNA. Two methods are adapted to MVA-BAC to provide more rapid generation of markerless recombinants in weeks rather than months. "En passant" recombineering is applied to the insertion of a transgene expression cassette and the removal of the selectable marker in bacteria; and a self-excising variant of MVA-BAC is constructed, in which the BAC cassette region is rapidly and efficiently lost from the viral genome following rescue of the BAC into infectious virus. These methods greatly facilitate and accelerate production of recombinant MVA, including markerless constructs.

Alcock R, Cottingham MG, Rollier CS, Furze J, De Costa SD, Hanlon M, Spencer AJ, Honeycutt JD et al. 2010. Long-term thermostabilization of live poxviral and adenoviral vaccine vectors at supraphysiological temperatures in carbohydrate glass. Sci Transl Med, 2 (19), pp. 19ra12. | Show Abstract | Read more

Live recombinant viral vectors based on adenoviruses and poxviruses are among the most promising platforms for development of new vaccines against diseases such as malaria, tuberculosis, and HIV-AIDS. Vaccines based on live viruses must remain infectious to be effective, so therefore need continuous refrigeration to maintain stability and viability, a requirement that can be costly and difficult, especially in developing countries. The sugars sucrose and trehalose are commonly used as stabilizing agents and cryoprotectants for biological products. Here, we have exploited the ability of these sugars to vitrify on desiccation to develop a thermostabilization technique for live viral vaccine vectors. By slowly drying vaccines suspended in solutions of these disaccharide stabilizers onto a filter-like support membrane at ambient temperature, an ultrathin glass is deposited on the fibers of the inert matrix. Immobilization of two recombinant vaccine vectors-E1/E3-deleted human adenovirus type 5 and modified vaccinia virus Ankara-in this glass on the membranes enabled complete recovery of viral titer and immunogenicity after storage at up to 45 degrees C for 6 months and even longer with minimal losses. Furthermore, the membrane carrying the stabilized vaccine can be incorporated into a holder attached to a syringe for almost simultaneous reconstitution and injection at point of use. The technology may potentially be developed for the deployment of viral vector-based biopharmaceuticals in resource-poor settings.

Hill AV, Reyes-Sandoval A, O'Hara G, Ewer K, Lawrie A, Goodman A, Nicosia A, Folgori A et al. 2010. Prime-boost vectored malaria vaccines: progress and prospects. Hum Vaccin, 6 (1), pp. 78-83. | Show Abstract | Read more

The difficulty of inducing protective immunity through antibodies against sporozoites led to efforts to assess vectored vaccines as a means of inducing protective T-cell immunity against the malaria liver-stage parasite. Although DNA vectored vaccines used alone were poorly immunogenic and not protective, high levels of parasite clearance in the liver has been achieved with viral vectored vaccines used in heterologous prime-boost regimes. Such vectored vaccination regimes represent one of only two approaches that have induced repeatable partial efficacy in human P. falciparum subunit vaccine trials. Interestingly, vectors expressing the TRAP antigen have been consistently been more immunogenic and protective than vectors expressing the circumsporozoite protein in human trials. However, sterile protection requires induction of very potent T-cell responses that are currently only achievable with heterologous prime-boost regimes. Recently, simian adenoviruses have been assessed as priming agents in Adenovirus-MVA regimes in both phase I and phase IIa trials in the UK, based on very promising pre-clinical results showing better immunogenicity and efficacy than previous prime-boost regimes. The same vectors are also being assessed clinically expressing blood-stage antigens, attempting to induce both protective antibodies and T cells as recently demonstrated in murine efficacy studies. These viral vectors now provide a major option for inclusion in a high efficacy multi-stage malaria vaccine that should achieve deployable levels of efficacy in endemic settings.

Reyes-Sandoval A, Berthoud T, Alder N, Siani L, Gilbert SC, Nicosia A, Colloca S, Cortese R, Hill AV. 2010. Prime-boost immunization with adenoviral and modified vaccinia virus Ankara vectors enhances the durability and polyfunctionality of protective malaria CD8+ T-cell responses. Infect Immun, 78 (1), pp. 145-153. | Show Abstract | Read more

Protection against liver-stage malaria relies on the induction of high frequencies of antigen-specific CD8+ T cells. We have previously reported high protective levels against mouse malaria, albeit short-lived, by a single vaccination with adenoviral vectors coding for a liver-stage antigen (ME.TRAP). Here, we report that prime-boost regimens using modified vaccinia virus Ankara (MVA) and adenoviral vectors encoding ME.TRAP can enhance both short- and long-term sterile protection against malaria. Protection persisted for at least 6 months when simian adenoviruses AdCh63 and AdC9 were used as priming vectors. Kinetic analysis showed that the MVA boost made the adenoviral-primed T cells markedly more polyfunctional, with the number of gamma interferon (INF-gamma), tumor necrosis factor alpha (TNF-alpha), and interleukin-2 (IL-2) triple-positive and INF-gamma and TNF-alpha double-positive cells increasing over time, while INF-gamma single-positive cells declined with time. However, IFN-gamma production prevailed as the main immune correlate of protection, while neither an increase of polyfunctionality nor a high integrated mean fluorescence intensity (iMFI) correlated with protection. These data highlight the ability of optimized viral vector prime-boost regimens to generate more protective and sustained CD8+ T-cell responses, and our results encourage a more nuanced assessment of the importance of inducing polyfunctional CD8(+) T cells by vaccination.

Larsen KC, Spencer AJ, Goodman AL, Gilchrist A, Furze J, Rollier CS, Kiss-Toth E, Gilbert SC et al. 2009. Expression of tak1 and tram induces synergistic pro-inflammatory signalling and adjuvants DNA vaccines. Vaccine, 27 (41), pp. 5589-5598. | Show Abstract | Read more

Improving vaccine immunogenicity remains a major challenge in the fight against developing country diseases like malaria and AIDS. We describe a novel strategy to identify new DNA vaccine adjuvants. We have screened components of the Toll-like receptor signalling pathways for their ability to activate pro-inflammatory target genes in transient transfection assays and assessed in vivo adjuvant activity by expressing the activators from the DNA backbone of vaccines. We find that a robust increase in the immune response necessitates co-expression of two activators. Accordingly, the combination of tak1 and tram elicits synergistic reporter activation in transient transfection assays. In a mouse model this combination, but not the individual molecules, induced approximately twofold increases in CD8+ T-cell immune responses. These results indicate that optimal immunogenicity may require activation of distinct innate immune signalling pathways. Thus this strategy offers a novel route to the discovery of a new generation of adjuvants.

Vordermeier HM, Villarreal-Ramos B, Cockle PJ, McAulay M, Rhodes SG, Thacker T, Gilbert SC, McShane H, Hill AV, Xing Z, Hewinson RG. 2009. Viral booster vaccines improve Mycobacterium bovis BCG-induced protection against bovine tuberculosis. Infect Immun, 77 (8), pp. 3364-3373. | Show Abstract | Read more

Previous work with small-animal laboratory models of tuberculosis has shown that vaccination strategies based on heterologous prime-boost protocols using Mycobacterium bovis bacillus Calmette-Guérin (BCG) to prime and modified vaccinia virus Ankara strain (MVA85A) or recombinant attenuated adenoviruses (Ad85A) expressing the mycobacterial antigen Ag85A to boost may increase the protective efficacy of BCG. Here we report the first efficacy data on using these vaccines in cattle, a natural target species of tuberculous infection. Protection was determined by measuring development of disease as an end point after M. bovis challenge. Either Ad85A or MVA85A boosting resulted in protection superior to that given by BCG alone: boosting BCG with MVA85A or Ad85A induced significant reduction in pathology in four/eight parameters assessed, while BCG vaccination alone did so in only one parameter studied. Protection was particularly evident in the lungs of vaccinated animals (median lung scores for naïve and BCG-, BCG/MVA85A-, and BCG/Ad85A-vaccinated animals were 10.5, 5, 2.5, and 0, respectively). The bacterial loads in lymph node tissues were also reduced after viral boosting of BCG-vaccinated calves compared to those in BCG-only-vaccinated animals. Analysis of vaccine-induced immunity identified memory responses measured by cultured enzyme-linked immunospot assay as well as in vitro interleukin-17 production as predictors of vaccination success, as both responses, measured before challenge, correlated positively with the degree of protection. Therefore, this study provides evidence of improved protection against tuberculosis by viral booster vaccination in a natural target species and has prioritized potential correlates of vaccine efficacy for further evaluation. These findings also have implications for human tuberculosis vaccine development.

Dudareva M, Andrews L, Gilbert SC, Bejon P, Marsh K, Mwacharo J, Kai O, Nicosia A, Hill AV. 2009. Prevalence of serum neutralizing antibodies against chimpanzee adenovirus 63 and human adenovirus 5 in Kenyan children, in the context of vaccine vector efficacy. Vaccine, 27 (27), pp. 3501-3504. | Show Abstract | Read more

Vaccination against Plasmodium falciparum malaria could reduce the worldwide burden of this disease, and decrease its high mortality in children. Replication-defective recombinant adenovirus vectors carrying P. falciparum epitopes may be useful as part of a vaccine that raises cellular immunity to the pre-erythrocytic stage of malaria infection. However, existing immunity to the adenovirus vector results in antibody-mediated neutralization of the vaccine vector, and reduced vaccine immunogenicity. Our aim was to examine a population of children who are at risk from P. falciparum malaria for neutralizing immunity to replication-deficient recombinant chimpanzee adenovirus 63 vector (AdC63), compared to human adenovirus 5 vector (AdHu5). We measured 50% and 90% vector neutralization titers in 200 individual sera, taken from a cohort of children from Kenya, using a secreted alkaline phosphatase neutralization assay. We found that 23% of the children (aged 1-6 years) had high-titer neutralizing antibodies to AdHu5, and 4% had high-titer neutralizing antibodies to AdC63. Immunity to both vectors was age-dependent. Low-level neutralization of AdC63 was significantly less frequent than AdHu5 neutralization at the 90% neutralization level. We conclude that AdC63 may be a useful vector as part of a prime-boost malaria vaccine in children.

Draper SJ, Goodman AL, Biswas S, Forbes EK, Moore AC, Gilbert SC, Hill AV. 2009. Recombinant viral vaccines expressing merozoite surface protein-1 induce antibody- and T cell-mediated multistage protection against malaria. Cell Host Microbe, 5 (1), pp. 95-105. | Show Abstract | Read more

Protecting against both liver and blood stages of infection is a long-sought goal of malaria vaccine design. Recently, we described the use of replication-defective viral vaccine vectors expressing the malaria antigen merozoite surface protein-1 (MSP-1) as an antimalarial vaccine strategy that elicits potent and protective antibody responses against blood-stage parasites. Here, we show that vaccine-induced MSP-1-specific CD4(+) T cells provide essential help for protective B cell responses, and CD8(+) T cells mediate significant antiparasitic activity against liver-stage parasites. Enhanced survival is subsequently seen in immunized mice following challenge with sporozoites, which mimics the natural route of infection more closely than when using infected red blood cells. This effect is evident both in the presence and absence of protective antibodies and is associated with decreased parasite burden in the liver followed by enhanced induction of the cytokine IFN-gamma in the serum. Multistage immunity against malaria can thus be achieved by using viral vectors recombinant for MSP-1.

Verreck FA, Vervenne RA, Kondova I, van Kralingen KW, Remarque EJ, Braskamp G, van der Werff NM, Kersbergen A et al. 2009. MVA.85A boosting of BCG and an attenuated, phoP deficient M. tuberculosis vaccine both show protective efficacy against tuberculosis in rhesus macaques. PLoS One, 4 (4), pp. e5264. | Show Abstract | Read more

BACKGROUND: Continuous high global tuberculosis (TB) mortality rates and variable vaccine efficacy of Mycobacterium bovis Bacille Calmette-Guérin (BCG) motivate the search for better vaccine regimes. Relevant models are required to downselect the most promising vaccines entering clinical efficacy testing and to identify correlates of protection. METHODS AND FINDINGS: Here, we evaluated immunogenicity and protection against Mycobacterium tuberculosis in rhesus monkeys with two novel strategies: BCG boosted by modified vaccinia virus Ankara expressing antigen 85A (MVA.85A), and attenuated M. tuberculosis with a disrupted phoP gene (SO2) as a single-dose vaccine. Both strategies were well tolerated, and immunogenic as evidenced by induction of specific IFNgamma responses. Antigen 85A-specific IFNgamma secretion was specifically increased by MVA.85A boosting. Importantly, both MVA.85A and SO2 treatment significantly reduced pathology and chest X-ray scores upon infectious challenge with M. tuberculosis Erdman strain. MVA.85A and SO2 treatment also showed reduced average lung bacterial counts (1.0 and 1.2 log respectively, compared with 0.4 log for BCG) and significant protective effect by reduction in C-reactive protein levels, body weight loss, and decrease of erythrocyte-associated hematologic parameters (MCV, MCH, Hb, Ht) as markers of inflammatory infection, all relative to non-vaccinated controls. Lymphocyte stimulation revealed Ag85A-induced IFNgamma levels post-infection as the strongest immunocorrelate for protection (spearman's rho: -0.60). CONCLUSIONS: Both the BCG/MVA.85A prime-boost regime and the novel live attenuated, phoP deficient TB vaccine candidate SO2 showed significant protective efficacy by various parameters in rhesus macaques. Considering the phylogenetic relationship between macaque and man and the similarity in manifestations of TB disease, these data support further development of these primary and combination TB vaccine candidates.

Schmidt NW, Podyminogin RL, Butler NS, Badovinac VP, Tucker BJ, Bahjat KS, Lauer P, Reyes-Sandoval A et al. 2008. Memory CD8 T cell responses exceeding a large but definable threshold provide long-term immunity to malaria. Proc Natl Acad Sci U S A, 105 (37), pp. 14017-14022. | Show Abstract | Read more

Infection of mice with sporozoites of Plasmodium berghei or Plasmodium yoelii has been used extensively to evaluate liver-stage protection by candidate preerythrocytic malaria vaccines. Unfortunately, repeated success of such vaccines in mice has not translated readily to effective malaria vaccines in humans. Thus, mice may be used better as models to dissect basic parameters required for immunity to Plasmodium-infection than as preclinical vaccine models. In turn, this basic information may aid in the rational design of malaria vaccines. Here, we describe a model of circumsporozoite-specific memory CD8 T cell generation that protects mice against multiple P. berghei sporozoite challenges for at least 19 months. Using this model we defined a threshold frequency of memory CD8 T cells in the blood that predicts long-term sterilizing immunity against liver-stage infection. Importantly, the number of Plasmodium-specific memory CD8 T cells required for immunity greatly exceeds the number required for resistance to other pathogens. In addition, this model allowed us to identify readily individual immunized mice that exceed or fall below the protective threshold before infection, information that should greatly facilitate studies to dissect basic mechanisms of protective CD8 T cell memory against liver-stage Plasmodium infection. Furthermore, the extremely large threshold in memory CD8 T cell frequencies required for long-term protection in mice may have important implications for development of effective malaria vaccines.

Draper SJ, Moore AC, Goodman AL, Long CA, Holder AA, Gilbert SC, Hill F, Hill AV. 2008. Effective induction of high-titer antibodies by viral vector vaccines. Nat Med, 14 (8), pp. 819-821. | Show Abstract | Read more

Protein-in-adjuvant vaccines have shown limited success against difficult diseases such as blood-stage malaria. Here we show that a recombinant adenovirus-poxvirus prime-boost immunization regime (known to induce strong T cell immunogenicity) can also induce very strong antigen-specific antibody responses, and we identify a simple complement-based adjuvant to further enhance immunogenicity. Antibodies induced against a blood-stage malaria antigen by this viral vector platform are highly effective against Plasmodium yoelii parasites in mice and against Plasmodium falciparum in vitro.

Sridhar S, Reyes-Sandoval A, Draper SJ, Moore AC, Gilbert SC, Gao GP, Wilson JM, Hill AV. 2008. Single-dose protection against Plasmodium berghei by a simian adenovirus vector using a human cytomegalovirus promoter containing intron A. J Virol, 82 (8), pp. 3822-3833. | Show Abstract | Read more

Human adenovirus serotype 5 (AdH5) vector vaccines elicit strong immune responses to the encoded antigen and have been used in various disease models. We designed AdH5 vectors expressing antigen under the control of a human cytomegalovirus (HCMV) immediate-early promoter containing its intron A sequence. The transcriptional levels of antigen and immune responses to antigen for vectors with the HCMV promoter with the intron A sequence (LP) were greater than those for AdH5 vectors using the HCMV promoter sequence without intron A (SP). We compared an E1E3-deleted AdH5 adenoviral vector, which affords more space for insertion of foreign sequences, and showed it to be as immunogenic as an E1-deleted AdH5 vector. Neutralizing antibodies to AdH5 limit the efficacy of vaccines based on the AdH5 serotype, and simian adenoviral vectors offer an attractive option to overcome this problem. We constructed E1E3-deleted human and simian adenoviral vectors encoding the pre-erythrocytic-stage malarial antigen Plasmodium berghei circumsporozoite protein. We compared the immunogenicity and efficacy of AdC6, a recombinant simian adenovirus serotype 6 vector, in a murine malaria model to those of AdH5 and the poxviral vectors MVA and FP9. AdC6 induced sterile protection from a single dose in 90% of mice, in contrast to AdH5 (25%) and poxviral vectors MVA and FP9 (0%). Adenoviral vectors maintained potent CD8(+) T-cell responses for a longer period after immunization than did poxviral vectors and mainly induced an effector memory phenotype of cells. Significantly, AdC6 was able to maintain protection in the presence of preexisting immunity to AdH5.

Reyes-Sandoval A, Sridhar S, Berthoud T, Moore AC, Harty JT, Gilbert SC, Gao G, Ertl HC, Wilson JC, Hill AV. 2008. Single-dose immunogenicity and protective efficacy of simian adenoviral vectors against Plasmodium berghei. Eur J Immunol, 38 (3), pp. 732-741. | Show Abstract | Read more

Simian adenoviral vectors (SAd) offer an attractive alternative to standard human adenovirus serotype 5 (AdH5) subunit vaccination, due to pre-existing immunity affecting vaccine performance. We have used a mouse model of liver-stage malaria to test the efficiency of three chimpanzee-origin adenoviral vectors, AdC6, AdC7 and AdC9 containing ME.TRAP as an insert. AdC7 and AdC9 elicited strong immunogenicity ( approximately 20% of CD8(+) T cells in spleen), equivalent to or outperforming AdH5 and inducing sterile protection in 92% (C9), 83% (H5 and C7) and 67% (C6) of the mice, providing the first evidence of single-dose protection to Plasmodium berghei. Protection was afforded by the SAd despite high levels of pre-existing immunity to AdH5. Phenotypic analysis showed that all adenoviral vectors (Ad) elicited CD8(+) T cell responses with an effector memory T cell (T(EM)) phenotype. By contrast, vaccination with poxviral vectors did not confer protection to P. berghei and induced a predominantly CD8(+) central memory T cell (T(CM)) response. Multifunctional CD8(+) T cell responses (co-expressing IFN-gamma, TNF-alpha and IL-2) were also induced by the Ad in higher percentages than the poxviral vectors. Our data suggest that T(EM) cells are important as a first line of defense against fast-replicating pathogens such as murine Plasmodium and demonstrate the potential of replication-defective SAd as future malaria vaccines for humans.

Graham SP, Pellé R, Yamage M, Mwangi DM, Honda Y, Mwakubambanya RS, de Villiers EP, Abuya E et al. 2008. Characterization of the fine specificity of bovine CD8 T-cell responses to defined antigens from the protozoan parasite Theileria parva. Infect Immun, 76 (2), pp. 685-694. | Show Abstract | Read more

Immunity against the bovine intracellular protozoan parasite Theileria parva has been shown to be mediated by CD8 T cells. Six antigens targeted by CD8 T cells from T. parva-immune cattle of different major histocompatibility complex (MHC) genotypes have been identified, raising the prospect of developing a subunit vaccine. To facilitate further dissection of the specificity of protective CD8 T-cell responses and to assist in the assessment of responses to vaccination, we set out to identify the epitopes recognized in these T. parva antigens and their MHC restriction elements. Nine epitopes in six T. parva antigens, together with their respective MHC restriction elements, were successfully identified. Five of the cytotoxic-T-lymphocyte epitopes were found to be restricted by products of previously described alleles, and four were restricted by four novel restriction elements. Analyses of CD8 T-cell responses to five of the epitopes in groups of cattle carrying the defined restriction elements and immunized with live parasites demonstrated that, with one exception, the epitopes were consistently recognized by animals of the respective genotypes. The analysis of responses was extended to animals immunized with multiple antigens delivered in separate vaccine constructs. Specific CD8 T-cell responses were detected in 19 of 24 immunized cattle. All responder cattle mounted responses specific for antigens for which they carried an identified restriction element. By contrast, only 8 of 19 responder cattle displayed a response to antigens for which they did not carry an identified restriction element. These data demonstrate that the identified antigens are inherently dominant in animals with the corresponding MHC genotypes.

Thompson FM, Porter DW, Okitsu SL, Westerfeld N, Vogel D, Todryk S, Poulton I, Correa S et al. 2008. Evidence of blood stage efficacy with a virosomal malaria vaccine in a phase IIa clinical trial. PLoS One, 3 (1), pp. e1493. | Show Abstract | Read more

BACKGROUND: Previous research indicates that a combination vaccine targeting different stages of the malaria life cycle is likely to provide the most effective malaria vaccine. This trial was the first to combine two existing vaccination strategies to produce a vaccine that induces immune responses to both the pre-erythrocytic and blood stages of the P. falciparum life cycle. METHODS: This was a Phase I/IIa study of a new combination malaria vaccine FFM ME-TRAP+PEV3A. PEV3A includes peptides from both the pre-erythrocytic circumsporozoite protein and the blood-stage antigen AMA-1. This study was conducted at the Centre for Clinical Vaccinology and Tropical Medicine, University of Oxford, Oxford, UK. The participants were healthy, malaria naïve volunteers, from Oxford. The interventions were vaccination with PEV3A alone, or PEV3A+FFM ME-TRAP. The main outcome measure was protection from malaria in a sporozoite challenge model. Other outcomes included measures of parasite specific immune responses induced by either vaccine; and safety, assessed by collection of adverse event data. RESULTS: We observed evidence of blood stage immunity in PEV3A vaccinated volunteers, but no volunteers were completely protected from malaria. PEV3A induced high antibody titres, and antibodies bound parasites in immunofluorescence assays. Moreover, we observed boosting of the vaccine-induced immune response by sporozoite challenge. Immune responses induced by FFM ME-TRAP were unexpectedly low. The vaccines were safe, with comparable side effect profiles to previous trials. Although there was no sterile protection two major observations support an effect of the vaccine-induced response on blood stage parasites: (i) Lower rates of parasite growth were observed in volunteers vaccinated with PEV3A compared to unvaccinated controls (p = 0.012), and this was reflected in the PCR results from PEV3A vaccinated volunteers. These showed early control of parasitaemia by some volunteers in this group. One volunteer, who received PEV3A alone, was diagnosed very late, on day 20 compared to an average of 11.8 days in unvaccinated controls. (ii). Morphologically abnormal parasites were present in the blood of all (n = 24) PEV3A vaccinated volunteers, and in only 2/6 controls (p = 0.001). We describe evidence of vaccine-induced blood stage efficacy for the first time in a sporozoite challenge study.

Cottingham MG, Andersen RF, Spencer AJ, Saurya S, Furze J, Hill AV, Gilbert SC. 2008. Recombination-mediated genetic engineering of a bacterial artificial chromosome clone of modified vaccinia virus Ankara (MVA). PLoS One, 3 (2), pp. e1638. | Show Abstract | Read more

The production, manipulation and rescue of a bacterial artificial chromosome clone of Vaccinia virus (VAC-BAC) in order to expedite construction of expression vectors and mutagenesis of the genome has been described (Domi & Moss, 2002, PNAS99 12415-20). The genomic BAC clone was 'rescued' back to infectious virus using a Fowlpox virus helper to supply transcriptional machinery. We apply here a similar approach to the attenuated strain Modified Vaccinia virus Ankara (MVA), now widely used as a safe non-replicating recombinant vaccine vector in mammals, including humans. Four apparently full-length, rescuable clones were obtained, which had indistinguishable immunogenicity in mice. One clone was shotgun sequenced and found to be identical to the parent. We employed GalK recombination-mediated genetic engineering (recombineering) of MVA-BAC to delete five selected viral genes. Deletion of C12L, A44L, A46R or B7R did not significantly affect CD8(+) T cell immunogenicity in BALB/c mice, but deletion of B15R enhanced specific CD8(+) T cell responses to one of two endogenous viral epitopes (from the E2 and F2 proteins), in accordance with published work (Staib et al., 2005, J. Gen. Virol.86, 1997-2006). In addition, we found a higher frequency of triple-positive IFN-gamma, TNF-alpha and IL-2 secreting E3-specific CD8+ T-cells 8 weeks after vaccination with MVA lacking B15R. Furthermore, a recombinant vaccine capable of inducing CD8(+) T cells against an epitope from Plasmodium berghei was created using GalK counterselection to insert an antigen expression cassette lacking a tandem marker gene into the traditional thymidine kinase locus of MVA-BAC. MVA continues to feature prominently in clinical trials of recombinant vaccines against diseases such as HIV-AIDS, malaria and tuberculosis. Here we demonstrate in proof-of-concept experiments that MVA-BAC recombineering is a viable route to more rapid and efficient generation of new candidate mutant and recombinant vaccines based on a clinically deployable viral vector.

Sinden RE, Dawes EJ, Alavi Y, Waldock J, Finney O, Mendoza J, Butcher GA, Andrews L, Hill AV, Gilbert SC, Basáñez MG. 2007. Progression of Plasmodium berghei through Anopheles stephensi is density-dependent. PLoS Pathog, 3 (12), pp. e195. | Show Abstract | Read more

It is well documented that the density of Plasmodium in its vertebrate host modulates the physiological response induced; this in turn regulates parasite survival and transmission. It is less clear that parasite density in the mosquito regulates survival and transmission of this important pathogen. Numerous studies have described conversion rates of Plasmodium from one life stage to the next within the mosquito, yet few have considered that these rates might vary with parasite density. Here we establish infections with defined numbers of the rodent malaria parasite Plasmodium berghei to examine how parasite density at each stage of development (gametocytes; ookinetes; oocysts and sporozoites) influences development to the ensuing stage in Anopheles stephensi, and thus the delivery of infectious sporozoites to the vertebrate host. We show that every developmental transition exhibits strong density dependence, with numbers of the ensuing stages saturating at high density. We further show that when fed ookinetes at very low densities, oocyst development is facilitated by increasing ookinete number (i.e., the efficiency of ookinete-oocyst transformation follows a sigmoid relationship). We discuss how observations on this model system generate important hypotheses for the understanding of malaria biology, and how these might guide the rational analysis of interventions against the transmission of the malaria parasites of humans by their diverse vector species.

Bull TJ, Gilbert SC, Sridhar S, Linedale R, Dierkes N, Sidi-Boumedine K, Hermon-Taylor J. 2007. A novel multi-antigen virally vectored vaccine against Mycobacterium avium subspecies paratuberculosis. PLoS One, 2 (11), pp. e1229. | Show Abstract | Read more

BACKGROUND: Mycobacterium avium subspecies paratuberculosis causes systemic infection and chronic intestinal inflammation in many species including primates. Humans are exposed through milk and from sources of environmental contamination. Hitherto, the only vaccines available against Mycobacterium avium subspecies paratuberculosis have been limited to veterinary use and comprised attenuated or killed organisms. METHODS: We developed a vaccine comprising a fusion construct designated HAV, containing components of two secreted and two cell surface Mycobacterium avium subspecies paratuberculosis proteins. HAV was transformed into DNA, human Adenovirus 5 (Ad5) and Modified Vaccinia Ankara (MVA) delivery vectors. Full length expression of the predicted 95 kDa fusion protein was confirmed. PRINCIPAL FINDINGS: Vaccination of naïve and Mycobacterium avium subspecies paratuberculosis infected C57BL/6 mice using DNA-prime/MVA-boost or Ad5-prime/MVA-boost protocols was highly immunogenic resulting in significant IFN-gamma ELISPOT responses by splenocytes against recombinant vaccine antigens and a range of HAV specific peptides. This included strong recognition of a T-cell epitope GFAEINPIA located near the C-terminus of the fusion protein. Antibody responses to recombinant vaccine antigens and HAV specific peptides but not GFAEINPIA, also occurred. No immune recognition of vaccine antigens occurred in any sham vaccinated Mycobacterium avium subspecies paratuberculosis infected mice. Vaccination using either protocol significantly attenuated pre-existing Mycobacterium avium subspecies paratuberculosis infection measured by qPCR in spleen and liver and the Ad5-prime/MVA-boost protocol also conferred some protection against subsequent challenge. No adverse effects of vaccination occurred in any of the mice. CONCLUSIONS/SIGNIFICANCE: A range of modern veterinary and clinical vaccines for the treatment and prevention of disease caused by Mycobacterium avium subspecies paratuberculosis are needed. The present vaccine proved to be highly immunogenic without adverse effect in mice and both attenuated pre-existing Mycobacterium avium subspecies paratuberculosis infection and conferred protection against subsequent challenge. Further studies of the present vaccine in naturally infected animals and humans are indicated.

Bejon P, Mwacharo J, Kai O, Todryk S, Keating S, Lowe B, Lang T, Mwangi TW et al. 2007. The induction and persistence of T cell IFN-gamma responses after vaccination or natural exposure is suppressed by Plasmodium falciparum. J Immunol, 179 (6), pp. 4193-4201. | Show Abstract

Epidemiological observations suggest that T cell immunity may be suppressed in malaria-endemic areas. In vitro studies, animal models, and limited data in humans link immunosuppression with malaria, malnutrition, and other parasitic infections. However, there are no data to determine whether malaria-induced immunosuppression is significant in the long-term, or relative data comparing it with other factors in malaria-endemic areas, so as to measure the impact of malaria, other parasitic disease, nutritional status, age. and location on the acquisition and longevity of IFN-gamma responses in children in Kenya. We studied these factors in two cohorts of 1- to 6-year-old children in a malaria-endemic area. T cell responses were induced by vaccination in one cohort, and acquired as a result of natural exposure in a second cohort. Serial ELISPOT assays conducted over a 1-year period measured the induction and kinetics of IFN-gamma production in response to the malaria Ag thrombospondin-related adhesion protein. Induced responses in both cohorts and the longevity of response in the vaccinated cohort were fitted to potential explanatory variables. Parasitemia was prospectively associated with reduced IFN-gamma-producing T cells in both cohorts (by 15-25%), and both parasitemia and episodes of febrile malaria were associated with 19 and 31% greater attrition of T cell responses, respectively. Malaria may reduce the efficacy vaccinations such as bacillus Calmette-Guérin and investigational T cell-inducing vaccines, and may delay the acquisition of immunity following natural exposure to malaria and other pathogens.

Bejon P, Ogada E, Mwangi T, Milligan P, Lang T, Fegan G, Gilbert SC, Peshu N, Marsh K, Hill AV. 2007. Extended follow-up following a phase 2b randomized trial of the candidate malaria vaccines FP9 ME-TRAP and MVA ME-TRAP among children in Kenya. PLoS One, 2 (8), pp. e707. | Show Abstract | Read more

BACKGROUND: "FFM ME-TRAP" is sequential immunisation with two attenuated poxvirus vectors (FP9 and modified vaccinia virus Ankara) delivering the pre-erythrocytic malaria antigen ME-TRAP. Over nine months follow-up in our original study, there was no evidence that FFM ME-TRAP provided protection against malaria. The incidence of malaria was slightly higher in children who received FFM ME-TRAP, but this was not statistically significant (hazard ratio 1.5, 95% CI 1.0-2.3). Although the study was unblinded, another nine months follow-up was planned to monitor the incidence of malaria and other serious adverse events. METHODS AND FINDINGS: 405 children aged 1-6 yrs were initially randomized to vaccination with either FFM ME-TRAP or control (rabies vaccine). 380 children were still available for follow-up after the first nine months. Children were seen weekly and whenever they were unwell for nine months monitoring. The axillary temperature was measured, and blood films taken when febrile. The primary analysis was time to parasitaemia >2,500/microl. During the second nine months monitoring, 49 events met the primary endpoint (febrile malaria with parasites >2,500/microl) in the Intention To Treat (ITT) group. 23 events occurred among the 189 children in the FFM ME-TRAP group, and 26 among the 194 children in the control group. In the full 18 months of monitoring, there were 63 events in the FFM ME-TRAP group and 60 in the control group (HR = 1.2, CI 0.84-1.73, p = 0.35). There was no evidence that the HR changed over the 18 months (test for interaction between time and vaccination p = 0.11). CONCLUSIONS: Vaccination with FFM ME-TRAP was not protective against malaria in this study. Malaria incidence during 18 months of surveillance was similar in both vaccine groups. TRIAL REGISTRATION: Controlled-Trials.com ISRCTN88335123.

Imoukhuede EB, Andrews L, Milligan P, Berthoud T, Bojang K, Nwakanma D, Ismaili J, Buckee C et al. 2007. Low-level malaria infections detected by a sensitive polymerase chain reaction assay and use of this technique in the evaluation of malaria vaccines in an endemic area. Am J Trop Med Hyg, 76 (3), pp. 486-493. | Show Abstract

The feasibility of using a sensitive polymerase chain reaction (PCR) to evaluate malaria vaccines in small group sizes was tested in 102 adult Gambian volunteers who received either the malaria vaccine regimen FP9 ME-TRAP/MVA ME-TRAP or rabies vaccine. All volunteers received the antimalarial drugs primaquine and Lapdap plus artesunate to eliminate malaria parasites. Volunteers in a further group received an additional single treatment with sulfadoxine-pyrimethamine (SP) to prevent new infections. There was substantially lower T-cell immunogenicity than in previous trials with this vaccine regimen and no protection against infection in the malaria vaccine group. Using the primary endpoint of 20 parasites per mL, no difference was found in the prevalence of low-level infections in volunteers who received SP compared with those who did not, indicating that SP did not reduce the incidence of very low-density infection. However, SP markedly reduced the incidence of higher density infections. These findings support the feasibility and potential of this approach to screen pre-erythrocytic vaccines for efficacy against infection in small numbers of vaccinees in endemic areas.

Bejon P, Keating S, Mwacharo J, Kai OK, Dunachie S, Walther M, Berthoud T, Lang T et al. 2006. Early gamma interferon and interleukin-2 responses to vaccination predict the late resting memory in malaria-naïve and malaria-exposed individuals. Infect Immun, 74 (11), pp. 6331-6338. | Show Abstract | Read more

Two different cell populations respond to potent T-cell-inducing vaccinations. The induction and loss of effector cells can be seen using an ex vivo enzyme-linked immunospot (ELISPOT) assay, but the more durable resting memory response is demonstrable by a cultured ELISPOT assay. The relationship of the early effector response to durable resting memory is incompletely understood. Effector phenotype is usually identified by gamma interferon (IFN-gamma) production, but interleukin-2 (IL-2) has been specifically linked to the differentiation of memory cells. Here, IFN-gamma- and IL-2-secreting effector cells were identified by an ex vivo ELISPOT assay 1 week after vaccination and compared with the resting memory responses detected by a cultured ELISPOT assay 3 months later. The different kinetics and induction of IL-2 by different vaccines and natural exposure are described. Furthermore, both early IFN-gamma and IL-2 production independently predicted subsequent memory responses at 3 months in malaria-naïve volunteers, but only IFN-gamma predicted memory in malaria-exposed volunteers. However, dual ELISPOT assays were also performed on malaria-exposed volunteers to identify cells producing both cytokines simultaneously. This demonstrated that double-cytokine-producing cells were highly predictive of memory. This assay may be useful in predicting vaccinations most likely to generate stable, long-term memory responses.

Imoukhuede EB, Berthoud T, Milligan P, Bojang K, Ismaili J, Keating S, Nwakanma D, Keita S et al. 2006. Safety and immunogenicity of the malaria candidate vaccines FP9 CS and MVA CS in adult Gambian men. Vaccine, 24 (42-43), pp. 6526-6533. | Show Abstract | Read more

We assessed the safety and immunogenicity of prime-boost vectors encoding the Plasmodium falciparum circumsporozoite (CS) protein expressed either in the attenuated fowl-pox virus (FP9) or modified vaccinia virus Ankara (MVA). Thirty-two adult Gambians in groups of four to eight received one, two or three doses of FP9 CS and/or MVA CS. No serious adverse event was observed following vaccination. The most immunogenic regimen was two doses of FP9 followed by a single dose of MVA 4 weeks later (an average of 1000 IFN-gamma spot forming units/million PBMCs). This level of effector T-cell responses appears higher than that seen in previously reported studies of CS-based candidate malaria vaccines.

Bejon P, Mwacharo J, Kai O, Mwangi T, Milligan P, Todryk S, Keating S, Lang T et al. 2006. A phase 2b randomised trial of the candidate malaria vaccines FP9 ME-TRAP and MVA ME-TRAP among children in Kenya. PLoS Clin Trials, 1 (6), pp. e29. | Show Abstract | Read more

OBJECTIVE: The objective was to measure the efficacy of the vaccination regimen FFM ME-TRAP in preventing episodes of clinical malaria among children in a malaria endemic area. FFM ME-TRAP is sequential immunisation with two attenuated poxvirus vectors (FP9 and modified vaccinia virus Ankara), which both deliver the pre-erythrocytic malaria antigen construct multiple epitope-thrombospondin-related adhesion protein (ME-TRAP). DESIGN: The trial was randomised and double-blinded. SETTING: The setting was a rural, malaria-endemic area of coastal Kenya. PARTICIPANTS: We vaccinated 405 healthy 1- to 6-year-old children. INTERVENTIONS: Participants were randomised to vaccination with either FFM ME-TRAP or control (rabies vaccine). OUTCOME MEASURES: Following antimalarial drug treatment children were seen weekly and whenever they were unwell during nine months of monitoring. The axillary temperature was measured, and blood films taken when febrile. The primary analysis was time to a parasitaemia of over 2,500 parasites/mul. RESULTS: The regime was moderately immunogenic, but the magnitude of T cell responses was lower than in previous studies. In intention to treat (ITT) analysis, time to first episode was shorter in the FFM ME-TRAP group. The cumulative incidence of febrile malaria was 52/190 (27%) for FFM ME-TRAP and 40/197 (20%) among controls (hazard ratio = 1.52). This was not statistically significant (95% confidence interval [CI] 1.0-2.3; p = 0.14 by log-rank). A group of 346 children were vaccinated according to protocol (ATP). Among these children, the hazard ratio was 1.3 (95% CI 0.8-2.1; p = 0.55 by log-rank). When multiple malaria episodes were included in the analyses, the incidence rate ratios were 1.6 (95% CI 1.1-2.3); p = 0.017 for ITT, and 1.4 (95% CI 0.9-2.1); p = 0.16 for ATP. Haemoglobin and parasitaemia in cross-sectional surveys at 3 and 9 mo did not differ by treatment group. Among children vaccinated with FFM ME-TRAP, there was no correlation between immunogenicity and malaria incidence. CONCLUSIONS: No protection was induced against febrile malaria by this vaccine regimen. Future field studies will require vaccinations with stronger immunogenicity in children living in malarious areas.

Walther M, Woodruff J, Edele F, Jeffries D, Tongren JE, King E, Andrews L, Bejon P et al. 2006. Innate immune responses to human malaria: heterogeneous cytokine responses to blood-stage Plasmodium falciparum correlate with parasitological and clinical outcomes. J Immunol, 177 (8), pp. 5736-5745. | Show Abstract

Taking advantage of a sporozoite challenge model established to evaluate the efficacy of new malaria vaccine candidates, we have explored the kinetics of systemic cytokine responses during the prepatent period of Plasmodium falciparum infection in 18 unvaccinated, previously malaria-naive subjects, using a highly sensitive, bead-based multiplex assay, and relate these data to peripheral parasite densities as measured by quantitative real-time PCR. These data are complemented with the analysis of cytokine production measured in vitro from whole blood or PBMC, stimulated with P. falciparum-infected RBC. We found considerable qualitative and quantitative interindividual variability in the innate responses, with subjects falling into three groups according to the strength of their inflammatory response. One group secreted moderate levels of IFN-gamma and IL-10, but no detectable IL-12p70. A second group produced detectable levels of circulating IL-12p70 and developed very high levels of IFN-gamma and IL-10. The third group failed to up-regulate any significant proinflammatory responses, but showed the highest levels of TGF-beta. Proinflammatory responses were associated with more rapid control of parasite growth but only at the cost of developing clinical symptoms, suggesting that the initial innate response may have far-reaching consequences on disease outcome. Furthermore, the in vitro observations on cytokine kinetics presented here, suggest that intact schizont-stage infected RBC can trigger innate responses before rupture of the infected RBC.

Dunachie SJ, Walther M, Epstein JE, Keating S, Berthoud T, Andrews L, Andersen RF, Bejon P et al. 2006. A DNA prime-modified vaccinia virus ankara boost vaccine encoding thrombospondin-related adhesion protein but not circumsporozoite protein partially protects healthy malaria-naive adults against Plasmodium falciparum sporozoite challenge. Infect Immun, 74 (10), pp. 5933-5942. | Show Abstract | Read more

The safety, immunogenicity, and efficacy of DNA and modified vaccinia virus Ankara (MVA) prime-boost regimes were assessed by using either thrombospondin-related adhesion protein (TRAP) with a multiple-epitope string ME (ME-TRAP) or the circumsporozoite protein (CS) of Plasmodium falciparum. Sixteen healthy subjects who never had malaria (malaria-naive subjects) received two priming vaccinations with DNA, followed by one boosting immunization with MVA, with either ME-TRAP or CS as the antigen. Immunogenicity was assessed by ex vivo gamma interferon (IFN-gamma) enzyme-linked immunospot assay (ELISPOT) and antibody assay. Two weeks after the final vaccination, the subjects underwent P. falciparum sporozoite challenge, with six unvaccinated controls. The vaccines were well tolerated and immunogenic, with the DDM-ME TRAP regimen producing stronger ex vivo IFN-gamma ELISPOT responses than DDM-CS. One of eight subjects receiving the DDM-ME TRAP regimen was completely protected against malaria challenge, with this group as a whole showing significant delay to parasitemia compared to controls (P = 0.045). The peak ex vivo IFN-gamma ELISPOT response in this group correlated strongly with the number of days to parasitemia (P = 0.033). No protection was observed in the DDM-CS group. Prime-boost vaccination with DNA and MVA encoding ME-TRAP but not CS resulted in partial protection against P. falciparum sporozoite challenge in the present study.

Bejon P, Kai OK, Mwacharo J, Keating S, Lang T, Gilbert SC, Peshu N, Marsh K, Hill AV. 2006. Alternating vector immunizations encoding pre-erythrocytic malaria antigens enhance memory responses in a malaria endemic area. Eur J Immunol, 36 (8), pp. 2264-2272. | Show Abstract | Read more

A heterologous prime-boost strategy has been developed to potently induce T cell responses to pre-erythrocytic malaria antigens. Efficacy in the field is likely to depend on both peak immunogenicity and the durability of responses. To improve both immunogenicity and durability of responses, 54 adult males from a malaria endemic area were immunized with different vaccination regimens, systematically varying antigenic insert and the number and sequence of component vaccinations. The component vaccinations were recombinant attenuated viruses, either fowlpox (FP) 9 or modified vaccinia virus Ankara (MVA). These were recombinant for either of two pre-erythrocytic malaria antigens (multiple epitope-thrombospondin-related adhesion protein, ME-TRAP, or circumsporozoite antigen (CS). ELISPOT assays were used to measure the effector and resting memory T cell responses. Sequence, antigen insert and number of vaccinations influenced immunogenicity, but the novel alternating vector immunizations generated the largest resting memory T cell populations. Effector responses were maintained at 84% of the peak response after 270 days. This durability of response is unprecedented. Classical prime-boost vaccination responses were at 5% of the peak after 270 days. Vaccines administered by heterologous prime-boost regimes are being developed for diverse pathogens and cancer. These data suggest these vaccines should also be administered by alternating vector regimens in clinical development.

Bejon P, Mwacharo J, Kai OK, Todryk S, Keating S, Lang T, Gilbert SC, Peshu N, Marsh K, Hill AV. 2006. Immunogenicity of the candidate malaria vaccines FP9 and modified vaccinia virus Ankara encoding the pre-erythrocytic antigen ME-TRAP in 1-6 year old children in a malaria endemic area. Vaccine, 24 (22), pp. 4709-4715. | Show Abstract | Read more

In a phase 1 trial, 22 children in a malaria endemic area were immunised with candidate malaria vaccination regimes. The regimes used two recombinant viral vectors, attenuated fowlpox strain FP9 and modified vaccinia virus Ankara (MVA). Both encoded the pre-erythrocytic malaria antigen construct ME-TRAP. Strong T cell responses were detected by both ex vivo and cultured ELISpot assays. Data from phase 1 trials in adults on anti-vector responses raised by FP9 is presented. These responses partially cross-reacted with MVA, and detectably reduced the immunogenicity of vaccination with MVA. This prompted the comparison of half dose and full dose FP9 priming vaccinations in children. Regimes using half dose FP9 priming tended to be more immunogenic than full dose. The potential for enhanced immunogenicity with half doses of priming vectors warrants further investigation, and larger studies to determine protection against malaria in children are required.

Gilbert SC, Moorthy VS, Andrews L, Pathan AA, McConkey SJ, Vuola JM, Keating SM, Berthoud T, Webster D, McShane H, Hill AV. 2006. Synergistic DNA-MVA prime-boost vaccination regimes for malaria and tuberculosis. Vaccine, 24 (21), pp. 4554-4561. | Show Abstract | Read more

T-cell-mediated responses against the liver-stage of Plasmodium falciparum are critical for protection in the human irradiated sporozoite model and several animal models. Heterologous prime-boost approaches, employing plasmid DNA and viral vector delivery of malarial DNA sequences, have proved particularly promising for maximising T-cell-mediated protection in animal models. The T-cell responses induced by this prime-boost regime, in animals and humans, are substantially greater than the sum of the responses induced by DNA or MVA vaccines used alone, leading to the term introduced here of "synergistic" prime-boost immunisation. The insert in our first generation clinical constructs is known as multiple epitope-thrombospondin-related adhesion protein (ME-TRAP). We have performed an extensive series of phase I/II trials evaluating various prime-boost combination regimens for delivery of ME-TRAP in over 500 malaria-naïve and malaria-exposed individuals. The three delivery vectors are DNA, modified vaccinia virus Ankara (MVA) and, more recently, fowlpox strain 9 (FP9). Administration was intra-epidermal and intramuscular for DNA and intradermal for MVA and FP9. Doses of DNA ranged from 4 microg to 2mg. Doses of MVA were up to 1.5 x 10(8) plaque forming units (pfu) and of FP9, up to 1.0 x 10(8)pfu. Further trials employing bacille Calmette-Guérin (BCG) as the priming agent and MVA expressing antigen 85A of Mycobacterium tuberculosis as the boosting agent has extended the scope of synergistic prime-boost vaccination. In this review we summarise the safety, immunogenicity and efficacy results from these malaria and tuberculosis vaccine clinical trials.

Walther M, Thompson FM, Dunachie S, Keating S, Todryk S, Berthoud T, Andrews L, Andersen RF et al. 2006. Safety, immunogenicity, and efficacy of prime-boost immunization with recombinant poxvirus FP9 and modified vaccinia virus Ankara encoding the full-length Plasmodium falciparum circumsporozoite protein. Infect Immun, 74 (5), pp. 2706-2716. | Show Abstract | Read more

Heterologous prime-boost immunization with DNA and various recombinant poxviruses encoding malaria antigens is capable of inducing strong cell-mediated immune responses and partial protection in human sporozoite challenges. Here we report a series of trials assessing recombinant fowlpox virus and modified vaccinia virus Ankara encoding the Plasmodium falciparum circumsporozoite protein in various prime-boost combinations, doses, and application routes. For the first time, these vaccines were administered intramuscularly and at doses of up to 5 x 10(8) PFU. Vaccines containing this antigen proved safe and induced modest immune responses but showed no evidence of efficacy in a sporozoite challenge.

Bejon P, Peshu N, Gilbert SC, Lowe BS, Molyneux CS, Forsdyke J, Lang T, Hill AV, Marsh K. 2006. Safety profile of the viral vectors of attenuated fowlpox strain FP9 and modified vaccinia virus Ankara recombinant for either of 2 preerythrocytic malaria antigens, ME-TRAP or the circumsporozoite protein, in children and adults in Kenya. Clin Infect Dis, 42 (8), pp. 1102-1110. | Show Abstract | Read more

BACKGROUND: We are developing a heterologous prime-boost vaccine strategy against malaria. This approach uses sequential immunization with different vectors to deliver a common preerythrocytic malaria antigen. Preliminary evidence of efficacy and safety has been previously documented in studies from an area where malaria is nonendemic. Additional safety data from an area where malaria is endemic are now required before larger-scale studies are undertaken to determine the efficacy of this vaccine strategy in the field. Other modified vaccinia virus Ankara (MVA) recombinants and prime-boost immunizations are being developed as vaccines against human immunodeficiency virus (HIV) infection, tuberculosis, and cancer, and MVA is a candidate attenuated smallpox vaccine. METHODS: Candidate vaccines against malaria were intradermally administered to 73 adults (7 of whom were HIV positive) and 22 children in Kenya. These vaccines used the attenuated fowlpox strain FP9 and the MVA recombinant for either of 2 preerythrocytic malaria antigens, multiple preerythrocytic-stage epitopes joined with the preerythrocytic-stage antigen TRAP (ME-TRAP) and the circumsporozoite protein (CS). Adverse events were recorded. RESULTS: Reactogenicity was mild. MVA caused less frequent and less severe cutaneous reaction if given after FP9 priming. Half doses reduced the frequency and the severity of systemic reactogenicity, and particular vaccine lots were associated with different reactogenicities. Unexpectedly, prior immunity to the ME-TRAP antigen appeared to be protective against local reactions after immunization. CONCLUSIONS: Where the final intention is to use MVA after FP9 priming, previous testing of MVA alone overestimates reactogenicity. These recombinant vectors appear to be safe and suitable for use in larger-scale studies of children in Africa and of HIV-positive individuals.

Dunachie SJ, Walther M, Vuola JM, Webster DP, Keating SM, Berthoud T, Andrews L, Bejon P et al. 2006. A clinical trial of prime-boost immunisation with the candidate malaria vaccines RTS,S/AS02A and MVA-CS. Vaccine, 24 (15), pp. 2850-2859. | Show Abstract | Read more

Heterologous prime-boost immunisation with RTS,S/AS02A and the poxvirus MVA-CS was evaluated in 18 healthy malaria-naïve subjects in Oxford. Both priming with RTS,S and boosting MVA-CS, and the reverse, were found to be safe and well tolerated. T cell responses as measured by IFN-gamma ex vivo ELISPOT were induced, but the responses were low to moderate in both groups, with heterologous boosting yielding only small increments in T cell immunogenicity and no increased antibody response. Protection against 3D7 Plasmodium falciparum sporozoite challenge 4 weeks after the final vaccination was equal for both regimens at 33% (95% C.I. 4.3-77.7%), with one subject remaining fully protected on rechallenge at 5 months.

Webster DP, Dunachie S, McConkey S, Poulton I, Moore AC, Walther M, Laidlaw SM, Peto T, Skinner MA, Gilbert SC, Hill AV. 2006. Safety of recombinant fowlpox strain FP9 and modified vaccinia virus Ankara vaccines against liver-stage P. falciparum malaria in non-immune volunteers. Vaccine, 24 (15), pp. 3026-3034. | Show Abstract | Read more

The ability to generate potent antigen-specific T cell responses by vaccination has been a major hurdle in vaccinology. Vaccinia virus and avipox viruses have been shown to be capable of expressing antigens in mammalian cells and can induce a protective immune response against several mammalian pathogens. We report on two such vaccine constructs, modified vaccinia virus Ankara and FP9 (an attenuated fowlpox virus) both expressing the pre-erythrocytic malaria antigen thrombospondin-related adhesion protein and a string of CD8+ epitopes (ME-TRAP). In prime-boost combinations in a mouse model MVA and FP9 are highly immunogenic and induce substantial protective efficacy. A series of human clinical trials using the recombinant MVA and FP9 malaria vaccines encoding ME-TRAP, both independently and in prime-boost combinations with or without the DNA vaccine DNA ME-TRAP, has shown them to be both immunogenic for CD8+ T cells and capable of inducing protective efficacy. We report here a detailed analysis of the safety profiles of these viral vectors and show that anti-vector antibody responses induced by the vectors are generally low to moderate. We conclude that these vectors are safe and show acceptable side effect profiles for prophylactic vaccination.

Graham SP, Pellé R, Honda Y, Mwangi DM, Tonukari NJ, Yamage M, Glew EJ, de Villiers EP et al. 2006. Theileria parva candidate vaccine antigens recognized by immune bovine cytotoxic T lymphocytes. Proc Natl Acad Sci U S A, 103 (9), pp. 3286-3291. | Show Abstract | Read more

East Coast fever, caused by the tick-borne intracellular apicomplexan parasite Theileria parva, is a highly fatal lymphoproliferative disease of cattle. The pathogenic schizont-induced lymphocyte transformation is a unique cancer-like condition that is reversible with parasite removal. Schizont-infected cell-directed CD8(+) cytotoxic T lymphocytes (CTL) constitute the dominant protective bovine immune response after a single exposure to infection. However, the schizont antigens targeted by T. parva-specific CTL are undefined. Here we show the identification of five candidate vaccine antigens that are the targets of MHC class I-restricted CD8(+) CTL from immune cattle. CD8(+) T cell responses to these antigens were boosted in T. parva-immune cattle resolving a challenge infection and, when used to immunize naïve cattle, induced CTL responses that significantly correlated with survival from a lethal parasite challenge. These data provide a basis for developing a CTL-targeted anti-East Coast fever subunit vaccine. In addition, orthologs of these antigens may be vaccine targets for other apicomplexan parasites.

Bejon P, Andrews L, Hunt-Cooke A, Sanderson F, Gilbert SC, Hill AV. 2006. Thick blood film examination for Plasmodium falciparum malaria has reduced sensitivity and underestimates parasite density. Malar J, 5 pp. 104. | Show Abstract | Read more

BACKGROUND: Thick blood films are routinely used to diagnose Plasmodium falciparum malaria. Here, they were used to diagnose volunteers exposed to experimental malaria challenge. METHODS: The frequency with which blood films were positive at given parasite densities measured by PCR were analysed. The poisson distribution was used to calculate the theoretical likelihood of diagnosis. Further in vitro studies used serial dilutions to prepare thick films from malaria cultures at known parasitaemia. RESULTS: Even in expert hands, thick blood films were considerably less sensitive than might have been expected from the parasite numbers measured by quantitative PCR. In vitro work showed that thick films prepared from malaria cultures at known parasitaemia consistently underestimated parasite densities. CONCLUSION: It appears large numbers of parasites are lost during staining. This limits their sensitivity, and leads to erroneous estimates of parasite density.

Moore AC, Gallimore A, Draper SJ, Watkins KR, Gilbert SC, Hill AV. 2005. Anti-CD25 antibody enhancement of vaccine-induced immunogenicity: increased durable cellular immunity with reduced immunodominance. J Immunol, 175 (11), pp. 7264-7273. | Show Abstract

An efficacious vaccine strategy must be capable of inducing strong responses of an appropriate phenotype that are long lasting and sufficiently broad to prevent pathogen escape mechanisms. In the present study, we use anti-CD25 mAb to augment vaccine-induced immunity in mice. We demonstrate that coformulation of Ab and poxviral- or adenoviral-vectored vaccines induces significantly increased T cell responses to a malaria Ag; prior anti-CD25 Ab administration was not required for this effect. Furthermore, this vaccination approach subverts immunodominant epitope hierarchies by enhancing responses to subdominant epitopes induced by recombinant modified vaccinia virus Ankara immunization. Administration of anti-CD25 with a vaccine also induces more durable immunity compared with vaccine alone; significantly higher T cell responses were observed 100 days after the primary immunization. Enhanced immunogenicity is observed for multiple vaccine types with enhanced CD4+ and CD8+ T cell responses induced by bacillus Calmette-Guérin and a recombinant subunit protein vaccine to hepatitis B virus and with multiple Ags of tumor, viral, bacterial, and parasitic origin. Vaccine strategies incorporating anti-CD25 lead to improved protection against pre-erythrocytic malaria challenge. These data underpin new strategies for the design and development of more efficacious vaccines in clinical settings.

Sridhar S, Sandoval AR, Moore A, Gilbert S, Hill AVS. 2005. Effect of adenovirus dose and prime-boost interval on the immune kinetics of the effector immune response IMMUNOLOGY, 116 pp. 109-109.

Reyes-Sandoval A, Sridhar S, Moore AC, Gilbert SC, Gao G, Wilson JM, Hill AV. 2005. Immunogenicity of a pre-erythrocytic-stage malaria insert ME-trap encoded by novel simian adenoviral vectors IMMUNOLOGY, 116 pp. 46-46.

Keating SM, Bejon P, Berthoud T, Vuola JM, Todryk S, Webster DP, Dunachie SJ, Moorthy VS, McConkey SJ, Gilbert SC, Hill AV. 2005. Durable human memory T cells quantifiable by cultured enzyme-linked immunospot assays are induced by heterologous prime boost immunization and correlate with protection against malaria. J Immunol, 175 (9), pp. 5675-5680. | Show Abstract

Immunological memory is a required component of protective antimalarial responses raised by T cell-inducing vaccines. The magnitude of ex vivo IFN-gamma T cell responses is widely used to identify immunogenic vaccines although this response usually wanes and may disappear within weeks. However, protection in the field is likely to depend on durable central memory T cells that are not detected by this assay. To identify longer-lived memory T cells, PBMC from malaria-naive vaccinated volunteers who had received prime boost vaccinations with a combination of DNA and/or viral vectors encoding the multiepitope string-thrombospondin-related adhesion protein Ag were cultured in vitro with Ag for 10 days before the ELISPOT assay. Ex vivo T cell responses peaked at 7 days after the final immunization and declined substantially over 6 mo, but responses identified after T cell culture increased over the 6-mo period after the final immunization. Moreover, individual cultured ELISPOT responses at the day of challenge time point correlated significantly with degree of protection against malaria sporozoite challenge, whereas ex vivo responses did not, despite a correlation between the peak ex vivo response and magnitude of memory responses 6 mo later. This cultured assay identifies long-lasting protective T cell responses and therefore offers an attractive option for assessments of vaccine immunogenicity.

Walther M, Tongren JE, Andrews L, Korbel D, King E, Fletcher H, Andersen RF, Bejon P et al. 2005. Upregulation of TGF-beta, FOXP3, and CD4+CD25+ regulatory T cells correlates with more rapid parasite growth in human malaria infection. Immunity, 23 (3), pp. 287-296. | Show Abstract | Read more

Understanding the regulation of immune responses is central for control of autoimmune and infectious disease. In murine models of autoimmunity and chronic inflammatory disease, potent regulatory T lymphocytes have recently been characterized. Despite an explosion of interest in these cells, their relevance to human disease has been uncertain. In a longitudinal study of malaria sporozoite infection via the natural route, we provide evidence that regulatory T cells have modifying effects on blood-stage infection in vivo in humans. Cells with the characteristics of regulatory T cells are rapidly induced following blood-stage infection and are associated with a burst of TGF-beta production, decreased proinflammatory cytokine production, and decreased antigen-specific immune responses. Both the production of TGF-beta and the presence of CD4+CD25+FOXP3+ regulatory T cells are associated with higher rates of parasite growth in vivo. P. falciparum-mediated induction of regulatory T cells may represent a parasite-specific virulence factor.

Hutchings CL, Gilbert SC, Hill AV, Moore AC. 2005. Novel protein and poxvirus-based vaccine combinations for simultaneous induction of humoral and cell-mediated immunity. J Immunol, 175 (1), pp. 599-606. | Show Abstract

The presence of both cell-mediated and humoral immunity is important in protection from and clearance of a number of infectious pathogens. We describe novel vaccine regimens using combinations of plasmid DNA, poxvirus and protein to induce strong Ag-specific T cell and Ab responses simultaneously in a murine model. Intramuscular (i.m.) immunization with plasmid DNA encoding the middle Ag of hepatitis B (DNA) concurrently with a commercial hepatitis B virus (HBV) vaccine (Engerix-B) followed by boosting immunizations with both modified vaccinia virus Ankara (MVA) encoding the middle Ag of HBV and Engerix-B induced high levels of CD4(+) and CD8(+) T cells and high titer Ab responses to hepatitis B surface Ag (HbsAg). Substitution of Engerix-B with adjuvant-free rHBsAg induced similar T cell responses and greatly enhanced Ab levels. Repeated immunizations with recombinant or nonrecombinant MVA mixed with Ag induced higher titers of Abs compared with immunization with either Ag or Engerix-B further demonstrating this novel adjuvant effect of MVA. The poxviruses NYVAC, fowlpox (FP9) and ALVAC, and to a lesser extent, adenovirus, also displayed similar adjuvant properties when used in combination with rHBsAg. The use of poxviruses as an adjuvant for protein to concurrently induce Ag-specific T cells and Abs could be applied to the development of vaccines for many diseases, including HIV and malaria, where both cell mediated and humoral immunity may be important for protection.

Andrews L, Andersen RF, Webster D, Dunachie S, Walther RM, Bejon P, Hunt-Cooke A, Bergson G, Sanderson F, Hill AV, Gilbert SC. 2005. Quantitative real-time polymerase chain reaction for malaria diagnosis and its use in malaria vaccine clinical trials. Am J Trop Med Hyg, 73 (1), pp. 191-198. | Show Abstract

The demand for an effective malaria vaccine is high, with millions of people being affected by the disease every year. A large variety of potential vaccines are under investigation worldwide, and when tested in clinical trials, researchers need to extract as much data as possible from every vaccinated and control volunteer. The use of quantitative real-time polymerase chain reaction (PCR), carried out in real-time during the clinical trials of vaccines designed to act against the liver stage of the parasite's life cycle, provides more information than the gold standard method of microscopy alone and increases both safety and accuracy. PCR can detect malaria parasites in the blood up to 5 days before experienced microscopists see parasites on blood films, with a sensitivity of 20 parasites/mL blood. This PCR method has so far been used to follow 137 vaccinee and control volunteers in Phase IIa trials in Oxford and on 220 volunteer samples during a Phase IIb field trial in The Gambia.

Williams A, Goonetilleke NP, McShane H, Clark SO, Hatch G, Gilbert SC, Hill AV. 2005. Boosting with poxviruses enhances Mycobacterium bovis BCG efficacy against tuberculosis in guinea pigs. Infect Immun, 73 (6), pp. 3814-3816. | Show Abstract | Read more

Tuberculosis is rising in the developing world due to poor health care, human immunodeficiency virus type 1 infection, and the low protective efficacy of the Mycobacterium bovis BCG vaccine. A new vaccination strategy that could protect adults in the developing world from tuberculosis could have a huge impact on public health. We show that BCG boosted by poxviruses expressing antigen 85A induced unprecedented 100% protection of guinea pigs from high-dose aerosol challenge with Mycobacterium tuberculosis, suggesting a strategy for enhancing and prolonging the efficacy of BCG.

Webster DP, Dunachie S, Vuola JM, Berthoud T, Keating S, Laidlaw SM, McConkey SJ, Poulton I et al. 2005. Enhanced T cell-mediated protection against malaria in human challenges by using the recombinant poxviruses FP9 and modified vaccinia virus Ankara. Proc Natl Acad Sci U S A, 102 (13), pp. 4836-4841. | Show Abstract | Read more

Malaria is a major global health problem for which an effective vaccine is required urgently. Prime-boost vaccination regimes involving plasmid DNA and recombinant modified vaccinia virus Ankara-encoding liver-stage malaria antigens have been shown to be powerfully immunogenic for T cells and capable of inducing partial protection against experimental malaria challenge in humans, manifested as a delay in time to patent parasitemia. Here, we report that substitution of plasmid DNA as the priming vector with a specific attenuated recombinant fowlpox virus, FP9, vaccine in such prime-boost regimes can elicit complete sterile protection that can last for 20 months. Protection at 20 months was associated with persisting memory but not effector T cell responses. The protective efficacy of various immunization regimes correlated with the magnitude of induced immune responses, supporting the strategy of maximizing durable T cell immunogenicity to develop more effective liver-stage vaccines against Plasmodium falciparum malaria.

Bejon P, Andrews L, Andersen RF, Dunachie S, Webster D, Walther M, Gilbert SC, Peto T, Hill AV. 2005. Calculation of liver-to-blood inocula, parasite growth rates, and preerythrocytic vaccine efficacy, from serial quantitative polymerase chain reaction studies of volunteers challenged with malaria sporozoites. J Infect Dis, 191 (4), pp. 619-626. | Show Abstract | Read more

We calculated the number and growth rate of Plasmodium falciparum parasites emerging in recipients of candidate preerythrocytic malaria vaccines and unvaccinated control subjects undergoing mosquito-bite challenge. This was done to measure vaccine efficacy and to distinguish the effects on blood-stage multiplication from those on liver-stage parasites. Real-time polymerase chain reaction measurements of parasite densities were analyzed by nonlinear regression and mixed-effects models. Substantial reductions in numbers of liver parasites resulted from the use of 2 immunization regimens: FP9 boosted by modified virus Ankara (MVA) encoding the malaria epitope-thrombospondin-related adhesion protein insert (92% reduction) and RTS,S/AS02 used in heterologous prime-boost immunization regimens, with MVA encoding the circumsporozoite protein (97% reduction). Forty-eight-hour growth rates in blood from control subjects were not different from those in blood from any vaccination group (mean, 14.4-fold [95% confidence interval, 11-19-fold]).

Young K, Frodsham A, Doumbo OK, Gupta S, Dolo A, Hu JT, Robson KJ, Crisanti A, Hill AV, Gilbert SC. 2005. Inverse associations of human leukocyte antigen and malaria parasite types in two West African populations. Infect Immun, 73 (2), pp. 953-955. | Show Abstract | Read more

Differences in allelic associations between populations continue to cause difficulties in the mapping and identification of susceptibility genes for complex polygenic diseases. Although well recognized, the basis of such interpopulation differences is poorly understood. We present an example of an inverse allelic association of an immune response genotype to an infectious disease in two neighboring West African populations. In this case, both the key environmental contributor, i.e., the malaria parasite, and a major biological mechanism are well defined. We show that this surprising result fits well with the predictions of a mathematical model describing the population genetics and dynamics of this interaction.

Smith CL, Dunbar PR, Mirza F, Palmowski MJ, Shepherd D, Gilbert SC, Coulie P, Schneider J et al. 2005. Recombinant modified vaccinia Ankara primes functionally activated CTL specific for a melanoma tumor antigen epitope in melanoma patients with a high risk of disease recurrence. Int J Cancer, 113 (2), pp. 259-266. | Show Abstract | Read more

Recombinant plasmid DNA and attenuated poxviruses are under development as cancer and infectious disease vaccines. We present the results of a phase I clinical trial of recombinant plasmid DNA and modified vaccinia Ankara (MVA), both encoding 7 melanoma tumor antigen cytotoxic T lymphocyte (CTL) epitopes. HLA-A*0201-positive patients with surgically treated melanoma received either a "prime-boost" DNA/MVA or a homologous MVA-only regimen. Ex vivo tetramer analysis, performed at multiple time points, provided detailed kinetics of vaccine-driven CTL responses specific for the high-affinity melan-A(26-35) analogue epitope. Melan-A26-35-specific CTL were generated in 2/6 patients who received DNA/MVA (detectable only after the first MVA injection) and 4/7 patients who received MVA only. Ex vivo ELISPOT analysis and in vitro proliferation assays confirmed the effector function of these CTL. Responses were seen in smallpox-vaccinated as well as vaccinia-naive patients, as defined by anti-vaccinia antibody responses demonstrated by ELISA assay. The observations that 1) CTL responses were generated to only 1 of the recombinant epitopes and 2) that the magnitude of these responses (0.029-0.19% CD8(+) T cells) was below the levels usually seen in acute viral infections suggest that to ensure high numbers of CTL specific for multiple recombinant epitopes, a deeper understanding of the interplay between CTL responses specific for the viral vector and recombinant epitopes is required.

Walther M, Dunachie S, Keating S, Vuola JM, Berthoud T, Schmidt A, Maier C, Andrews L et al. 2005. Safety, immunogenicity and efficacy of a pre-erythrocytic malaria candidate vaccine, ICC-1132 formulated in Seppic ISA 720. Vaccine, 23 (7), pp. 857-864. | Show Abstract | Read more

ICC-1132, a recombinant virus-like particle comprising of a modified hepatitis B core protein with a B cell (NANP) and two T cell epitopes of Plasmodium falciparum circumsporozoite protein (CSP), was administered i.m. as a single 50 microg dose in Seppic ISA 720 to 11 volunteers. Local reactogenicity and systemic side effects were acceptable with the predominant finding being mild pain at the injection site. This regimen induced anti-NANP antibodies in 10/11 and modest T cell responses. There was no evidence of protection from experimental challenge with P. falciparum sporozoites. Other formulations and/or multi-dose regimens will be required to enhance the immunogenicity and efficacy of ICC-1132.

Vuola JM, Keating S, Webster DP, Berthoud T, Dunachie S, Gilbert SC, Hill AV. 2005. Differential immunogenicity of various heterologous prime-boost vaccine regimens using DNA and viral vectors in healthy volunteers. J Immunol, 174 (1), pp. 449-455. | Show Abstract

Heterologous prime-boost vaccination has been shown to be an efficient way of inducing T cell responses in animals and in humans. We have used three vaccine vectors, naked DNA, modified vaccinia virus Ankara (MVA), and attenuated fowlpox strain, FP9, for prime-boost vaccination approaches against Plasmodium falciparum malaria in humans. In this study, we characterize, using two types of ELISPOT assays and FACS analysis, cell-mediated immune responses induced by different prime-boost combinations where all vectors encode a multiepitope string fused to the pre-erythrocytic Ag thrombospondin-related adhesion protein. We show that these different vectors need to be used in a specific order for an optimal ex vivo IFN-gamma response. From the different combinations, DNA priming followed by MVA boosting and FP9 priming followed by MVA boosting were most immunogenic and in both cases the IFN-gamma response was of broad specificity and cross-reactive against two P. falciparum strains (3D7 and T9/96). Immunization with all three vectors showed no improvement over optimal two vector regimes. Strong ex vivo IFN-gamma responses peaked 1 wk after the booster dose, but cultured ELISPOT assays revealed longer-lasting T cell memory responses for at least 6 mo. In the DNA-primed vaccinees the IFN-gamma response was mainly due to CD4(+) T cells, whereas in the FP9-primed vaccinees it was mainly due to CD4-dependent CD8(+) T cells. This difference may be of importance for the protective efficacy of these vaccination approaches against various diseases.

Dunachie S, Berthoud T, Keating S, Todryk S, Thompson F, Gilbert S, Fletcher H, Hill A. 2005. Regulatory T cells limit the cellular response to CS and TRAP encoding candidate malaria vaccines in humans [MIM-SD-8756] ACTA TROPICA, 95 pp. S200-S200.

Bejon P, Mwacharo J, Kai O, Lowe B, Todryk S, Peshu N, Gilbert S, Lang T, Marsh K, Hill A. 2005. Safety, immunogenicity and efficacy studies of candidate malaria vaccines FP9 and MVA encoding ME-TRAP in Kenyan children [MIM-PB-224908] ACTA TROPICA, 95 pp. S84-S84.

Dunachie S, Walther M, Keating S, Berthoud T, Thompson F, Todryk S, Carucci D, Sinden R, Gilbert S, Hill A. 2005. TRAP-based vectored vaccines are more immunogenic and protective than CSP-based vectors: Correlation of efficacy with long-lived memory T cells [MIM-AH-359251] ACTA TROPICA, 95 pp. S419-S419.

Moorthy VS, Imoukhuede EB, Milligan P, Bojang K, Keating S, Kaye P, Pinder M, Gilbert SC, Walraven G, Greenwood BM, Hill AS. 2004. A randomised, double-blind, controlled vaccine efficacy trial of DNA/MVA ME-TRAP against malaria infection in Gambian adults. PLoS Med, 1 (2), pp. e33. | Show Abstract | Read more

BACKGROUND: Many malaria vaccines are currently in development, although very few have been evaluated for efficacy in the field. Plasmodium falciparum multiple epitope (ME)- thrombospondin-related adhesion protein (TRAP) candidate vaccines are designed to potently induce effector T cells and so are a departure from earlier malaria vaccines evaluated in the field in terms of their mechanism of action. ME-TRAP vaccines encode a polyepitope string and the TRAP sporozoite antigen. Two vaccine vectors encoding ME-TRAP, plasmid DNA and modified vaccinia virus Ankara (MVA), when used sequentially in a prime-boost immunisation regime, induce high frequencies of effector T cells and partial protection, manifest as delay in time to parasitaemia, in a clinical challenge model. METHODS AND FINDINGS: A total of 372 Gambian men aged 15-45 y were randomised to receive either DNA ME-TRAP followed by MVA ME-TRAP or rabies vaccine (control). Of these men, 296 received three doses of vaccine timed to coincide with the beginning of the transmission season (141 in the DNA/MVA group and 155 in the rabies group) and were followed up. Volunteers were given sulphadoxine/pyrimethamine 2 wk before the final vaccination. Blood smears were collected weekly for 11 wk and whenever a volunteer developed symptoms compatible with malaria during the transmission season. The primary endpoint was time to first infection with asexual P. falciparum. Analysis was per protocol. DNA ME-TRAP and MVA ME-TRAP were safe and well-tolerated. Effector T cell responses to a non-vaccine strain of TRAP were 50-fold higher postvaccination in the malaria vaccine group than in the rabies vaccine group. Vaccine efficacy, adjusted for confounding factors, was 10.3% (95% confidence interval, -22% to +34%; p = 0.49). Incidence of malaria infection decreased with increasing age and was associated with ethnicity. CONCLUSIONS: DNA/MVA heterologous prime-boost vaccination is safe and highly immunogenic for effector T cell induction in a malaria-endemic area. But despite having produced a substantial reduction in liver-stage parasites in challenge studies of non-immune volunteers, this first generation T cell-inducing vaccine was ineffective at reducing the natural infection rate in semi-immune African adults.

McShane H, Pathan AA, Sander CR, Keating SM, Gilbert SC, Huygen K, Fletcher HA, Hill AV. 2004. Recombinant modified vaccinia virus Ankara expressing antigen 85A boosts BCG-primed and naturally acquired antimycobacterial immunity in humans. Nat Med, 10 (11), pp. 1240-1244. | Show Abstract | Read more

Protective immunity against Mycobacterium tuberculosis depends on the generation of a T(H)1-type cellular immune response, characterized by the secretion of interferon-gamma (IFN-gamma) from antigen-specific T cells. The induction of potent cellular immune responses by vaccination in humans has proven difficult. Recombinant viral vectors, especially poxviruses and adenoviruses, are particularly effective at boosting previously primed CD4(+) and CD8(+) T-cell responses against a number of intracellular pathogens in animal studies. In the first phase 1 study of any candidate subunit vaccine against tuberculosis, recombinant modified vaccinia virus Ankara (MVA) expressing antigen 85A (MVA85A) was found to induce high levels of antigen-specific IFN-gamma-secreting T cells when used alone in bacille Calmette-Guerin (BCG)-naive healthy volunteers. In volunteers who had been vaccinated 0.5-38 years previously with BCG, substantially higher levels of antigen-specific IFN-gamma-secreting T cells were induced, and at 24 weeks after vaccination these levels were 5-30 times greater than in vaccinees administered a single BCG vaccination. Boosting vaccinations with MVA85A could offer a practical and efficient strategy for enhancing and prolonging antimycobacterial immunity in tuberculosis-endemic areas.

Behboudi S, Moore A, Gilbert SC, Nicoll CL, Hill AV. 2004. Dendritic cells infected by recombinant modified vaccinia virus Ankara retain immunogenicity in vivo despite in vitro dysfunction. Vaccine, 22 (31-32), pp. 4326-4331. | Show Abstract | Read more

The administration of recombinant vaccinia virus Ankara (MVA) encoding a CTL epitope (pb9) from a malaria antigen induced activation and maturation of splenic dendritic cells (DCs) in vivo. In contrast, incubation of immature dendritic cells (iDCs) with the MVA, in vitro, resulted in down-regulation of MHC class I molecules and reduced their T-cell stimulatory ability. However, the ability of the infected DC to induce an antigen-specific CTL response, in vivo, remained intact. Furthermore, the administration of recombinant MVA-infected DC, but not pb9 peptide-pulsed DC, boosted and expanded the anti-pb9 CTL response that was primed by pb9 peptide-pulsed DC. These data indicate that despite the ability of poxviruses to impair DC maturation in vivo, the important ability of MVA to boost CD8 T-cell response in vivo is mediated at the level of the infected dendritic cells.

Vordermeier HM, Rhodes SG, Dean G, Goonetilleke N, Huygen K, Hill AV, Hewinson RG, Gilbert SC. 2004. Cellular immune responses induced in cattle by heterologous prime-boost vaccination using recombinant viruses and bacille Calmette-Guérin. Immunology, 112 (3), pp. 461-470. | Show Abstract | Read more

The development of novel vaccine strategies to replace or supplement bacille Calmette-Guérin (BCG) is urgently required. Here we study, in cattle, the use of heterologous prime-boost strategies based on vaccination with BCG and the mycobacterial mycolyl transferase Ag85A (Rv3804c) expressed either in recombinant modified vaccinia virus Ankara (MVA85A) or attenuated fowlpox strain FP9 (FP85A). Five different vaccination schedules were tested in the first experiment: MVA85A followed by BCG (group 1); BCG followed by MVA85A (group 2); BCG followed by FP85A and then MVA85A (group 3); MVA85A followed by MVA85A and then FP85A (group 4); and FP85A followed by FP85A and then MVA85A (group 5). Vaccine-induced levels of cellular immunity were assessed by determining interferon-gamma (IFN-gamma) responses in vitro. Prime-boost protocols, using recombinant MVA and BCG in combination (groups 1-3), resulted in significantly higher frequencies of Ag85-specific IFN-gamma-secreting cells than the two viral vectors used in combination (P=0.0055), or BCG used alone (groups 2 and 3, P=0.04). The T-cell repertoires of the calves in all five groups were significantly broader following heterologous booster immunizations than after the primary immunization. In a second experiment, the effects of BCG\MVA85A heterologous prime-boost vaccination were compared with BCG\BCG homologous revaccination. The results suggested a higher Ag85A-specific response with a wider T-cell repertoire in the MVA85A-boosted calves than in the BCG\BCG-vaccinated calves. In conclusion therefore, the present report demonstrates the effectiveness of heterologous prime-boost strategies based on recombinant MVA and BCG to induce strong cellular immune responses in cattle and prioritise such vaccination strategies for rapid assessment of protective efficacy in this natural target species of tuberculosis.

Anderson RJ, Hannan CM, Gilbert SC, Laidlaw SM, Sheu EG, Korten S, Sinden R, Butcher GA, Skinner MA, Hill AV. 2004. Enhanced CD8+ T cell immune responses and protection elicited against Plasmodium berghei malaria by prime boost immunization regimens using a novel attenuated fowlpox virus. J Immunol, 172 (5), pp. 3094-3100. | Show Abstract

Sterile immunity can be provided against the pre-erythrocytic stages of malaria by IFN-gamma-secreting CD8(+) T cells that recognize parasite-infected hepatocytes. In this study, we have investigated the use of attenuated fowlpox virus (FPV) strains as recombinant vaccine vectors for eliciting CD8(+) T cells against Plasmodium berghei. The gene encoding the P. berghei circumsporozoite (PbCS) protein was inserted into an FPV vaccine strain licensed for use in chickens, Webster's FPV, and the novel FPV vaccine strain FP9 by homologous recombination. The novel FP9 strain proved more potent as a vaccine for eliciting CD8(+) T cell responses against the PbCS Ag. Sequential immunization with rFP9 and recombinant modified vaccinia virus Anakara (MVA) encoding the PbCS protein, administered by clinically acceptable routes, elicited potent CD8(+) T cell responses against the PbCS protein. This immunization regimen elicited substantial protection against a stringent liver-stage challenge with P. berghei and was more immunogenic and protective than DNA/MVA prime/boost immunization. However, further improvement was not achieved by sequential (triple) immunization with a DNA vaccine, FP9, and MVA.

Prieur E, Gilbert SC, Schneider J, Moore AC, Sheu EG, Goonetilleke N, Robson KJ, Hill AV. 2004. A Plasmodium falciparum candidate vaccine based on a six-antigen polyprotein encoded by recombinant poxviruses. Proc Natl Acad Sci U S A, 101 (1), pp. 290-295. | Show Abstract | Read more

To generate broadly protective T cell responses more similar to those acquired after vaccination with radiation-attenuated Plasmodium falciparum sporozoites, we have constructed candidate subunit malaria vaccines expressing six preerythrocytic antigens linked together to produce a 3240-aa-long polyprotein (L3SEPTL). This polyprotein was expressed by a plasmid DNA vaccine vector (DNA) and by two attenuated poxvirus vectors, modified vaccinia virus Ankara (MVA) and fowlpox virus of the FP9 strain. MVAL3SEPTL boosted anti-thrombospondin-related adhesive protein (anti-TRAP) and anti-liver stage antigen 1 (anti-LSA1) CD8(+) T cell responses when primed by single antigen TRAP- or LSA1-expressing DNAs, respectively, but not by DNA-L3SEPTL. However, prime boost regimes involving two heterologous viral vectors expressing L3SEPTL induced a strong cellular response directed against an LSA1 peptide located in the C-terminal region of the polyprotein. Peptide-specific T cells secreted IFN-gamma and were cytotoxic. IFN-gamma-secreting T cells specific for each of the six antigens were induced after vaccination with L3SEPTL, supporting the use of polyprotein inserts to induce multispecific T cells against P. falciparum. The use of polyprotein constructs in nonreplicating poxviruses should broaden the target antigen range of vaccine-induced immunity and increase the number of potential epitopes available for immunogenetically diverse human populations.

Taracha EL, Bishop R, Musoke AJ, Hill AV, Gilbert SC. 2003. Heterologous priming-boosting immunization of cattle with Mycobacterium tuberculosis 85A induces antigen-specific T-cell responses. Infect Immun, 71 (12), pp. 6906-6914. | Show Abstract | Read more

Heterologous priming-boosting vaccination regimens involving priming with plasmid DNA antigen constructs and inoculating (boosting) with the same recombinant antigen expressed in replication-attenuated poxviruses have recently been demonstrated to induce immunity, based on CD4(+)- and CD8(+)-T-cell responses, against several diseases in both rodents and primates. We show that similar priming-boosting vaccination strategies using the 85A antigen of Mycobacterium tuberculosis are effective in inducing antigen-specific gamma interferon-secreting CD4(+) and CD8(+) T cells, detected by a bovine enzyme-linked immunospot assay, in Bos indicus cattle. T-cell responses induced by priming with either plasmid DNA or fowlpox virus 85A constructs were enhanced by boosting with modified vaccinia virus Ankara expressing the same antigen administered intradermally. On the basis of the data, it appears that intradermal priming was more effective than intramuscular delivery of the priming dose for boosting with the modified vaccinia virus Ankara strain in cattle. Using either fowlpox virus or DNA priming, there was a significant bias toward induction of CD4(+)- rather than CD8(+)-T-cell responses. These data illustrate the general applicability of priming-boosting vaccination strategies for induction of antigen-specific T-cell responses and suggest that the method may be useful for development of veterinary vaccines.

McConkey SJ, Reece WH, Moorthy VS, Webster D, Dunachie S, Butcher G, Vuola JM, Blanchard TJ et al. 2003. Enhanced T-cell immunogenicity of plasmid DNA vaccines boosted by recombinant modified vaccinia virus Ankara in humans. Nat Med, 9 (6), pp. 729-735. | Show Abstract | Read more

In animals, effective immune responses against malignancies and against several infectious pathogens, including malaria, are mediated by T cells. Here we show that a heterologous prime-boost vaccination regime of DNA either intramuscularly or epidermally, followed by intradermal recombinant modified vaccinia virus Ankara (MVA), induces high frequencies of interferon (IFN)-gamma-secreting, antigen-specific T-cell responses in humans to a pre-erythrocytic malaria antigen, thrombospondin-related adhesion protein (TRAP). These responses are five- to tenfold higher than the T-cell responses induced by the DNA vaccine or recombinant MVA vaccine alone, and produce partial protection manifest as delayed parasitemia after sporozoite challenge with a different strain of Plasmodium falciparum. Such heterologous prime-boost immunization approaches may provide a basis for preventative and therapeutic vaccination in humans.

Moorthy VS, McConkey S, Roberts M, Gothard P, Arulanantham N, Degano P, Schneider J, Hannan C et al. 2003. Safety of DNA and modified vaccinia virus Ankara vaccines against liver-stage P. falciparum malaria in non-immune volunteers. Vaccine, 21 (17-18), pp. 1995-2002. | Show Abstract | Read more

A series of phase I clinical studies were conducted to evaluate the safety of plasmid DNA and modified vaccinia virus Ankara malaria vaccines. The vaccines each encoded a polyepitope string fused to whole Plasmodium falciparum TRAP antigen. Forty-three healthy adult volunteers received the vaccines alone or in DNA/MVA prime-boost combinations. The DNA vaccine was administered either intramuscularly by needle or intradermally by a needleless delivery device. The MVA vaccine was administered intradermally by needle. The vaccines were well-tolerated by all three routes and in various DNA/MVA immunisation regimes. There were no severe or serious adverse events.

Palmowski MJ, Choi EM, Hermans IF, Gilbert SC, Chen JL, Gileadi U, Salio M, Van Pel A et al. 2002. Competition between CTL narrows the immune response induced by prime-boost vaccination protocols. J Immunol, 168 (9), pp. 4391-4398. | Show Abstract

Recombinant vaccines encoding strings of virus- or tumor-derived peptides and/or proteins are currently being designed for use against both cancer and infectious diseases. These vaccines aim to induce cytotoxic immune responses against several Ags simultaneously. We developed a novel tetramer-based technique, based on chimeric HLA A2/H-2K(b) H chains, to directly monitor the CTL response to such vaccines in HLA-A2 transgenic mice. We found that priming and boosting with the same polyepitope construct induced immune responses that were dominated by CTL of a single specificity. When a mixture of viruses encoding single proteins was used to boost the polyepitope primed response, CTL of multiple specificities were simultaneously expanded to highly effective levels in vivo. In addition, we show that a preexisting response to one of the epitopes encoded within a polyepitope construct significantly impaired the ability of the vaccine to expand CTL of other specificities. Our findings define a novel vaccination strategy optimized for the induction of an effective polyvalent cytotoxic response.

Gilbert SC, Schneider J, Hannan CM, Hu JT, Plebanski M, Sinden R, Hill AV. 2002. Enhanced CD8 T cell immunogenicity and protective efficacy in a mouse malaria model using a recombinant adenoviral vaccine in heterologous prime-boost immunisation regimes. Vaccine, 20 (7-8), pp. 1039-1045. | Show Abstract | Read more

Recombinant replication-defective adenovirus expressing the CS gene from Plasmodium berghei (Ad-PbCS) was found to induce a strong CD8(+) T cell response after intra-dermal or -muscular immunisation. Boosting of an adenovirus-primed immune response with the replication-impaired poxvirus, modified vaccinia virus Ankara (MVA) led to enhanced immunogenicity and substantial protective efficacy. The recombinant adenoviral vaccine was capable of boosting to protective levels a CD8(+) T cell response primed by either a plasmid DNA vaccine, a recombinant Ty virus-like particle vaccine or recombinant MVA each expressing the same epitope or antigen. Complete protective efficacy after intradermal immunisation was observed with the adenovirus prime-MVA boost regime. This study identifies recombinant replication-defective adenovirus as an alternative to recombinant replication-defective poxviruses as boosting agents for the induction of strong protective CD8(+) T cell responses.

Gilbert SC. 2001. Virus-like particles as vaccine adjuvants. Mol Biotechnol, 19 (2), pp. 169-177. | Show Abstract | Read more

Virus-like particles (VLPs) consist of one or more viral coat proteins that assemble into particles. They can be taken up by antigen presenting cells (APC), peptides derived from them are presented on MHC class I molecules at the cell surface, and thereby prime a CD8+ T cell response, either against the particle-forming protein itself (such as Hepatitis B surface antigen) or additional peptide sequences that are produced as fusions with the particle-forming protein. This article describes the preparation of Ty-VLPs in Saccharomyces cerevisiae, a system that can easily be handled in the laboratory or scaled up for manufacture, and is safe in use.

Schneider J, Langermans JA, Gilbert SC, Blanchard TJ, Twigg S, Naitza S, Hannan CM, Aidoo M et al. 2001. A prime-boost immunisation regimen using DNA followed by recombinant modified vaccinia virus Ankara induces strong cellular immune responses against the Plasmodium falciparum TRAP antigen in chimpanzees. Vaccine, 19 (32), pp. 4595-4602. | Show Abstract | Read more

Two chimpanzees were vaccinated intramuscularly against malaria using plasmid DNA expressing the pre-erythrocytic antigens thrombospondin related adhesion protein (PfTRAP) and liver stage specific antigen-1 (PfLSA-1) of Plasmodium falciparum together with GM-CSF protein. A recombinant modified vaccinia virus Ankara (MVA) expressing PfTRAP was injected intramuscularly 6 weeks later to boost the immune response. This sequence of antigen delivery induced a specific and long-lasting T cell and antibody response to PfTRAP as detected by ELISPOT assay and ELISA. Antibody responses were detected after four DNA injections, and were boosted by injection of recombinant MVA expressing PfTRAP. Interferon-gamma secreting antigen-specific T cells were detected in both animals, but only after boosting with recombinant MVA. By screening a panel of PfTRAP-derived peptides, an epitope was identified that was recognized by cytotoxic T lymphocytes in one of the chimpanzees studied. T cells specific for this epitope were present in PBMCs and liver-infiltrating lymphocytes at a frequency of between 1 in 200 and 1 in 500. The high immunogenicity of this prime-boost regimen in chimpanzees supports further assessment of this delivery strategy for the induction of protection against P. falciparum malaria in humans.

McShane H, Brookes R, Gilbert SC, Hill AV. 2001. Enhanced immunogenicity of CD4(+) t-cell responses and protective efficacy of a DNA-modified vaccinia virus Ankara prime-boost vaccination regimen for murine tuberculosis. Infect Immun, 69 (2), pp. 681-686. | Show Abstract | Read more

DNA vaccines whose DNA encodes a variety of antigens from Mycobacterium tuberculosis have been evaluated for immunogenicity and protective efficacy. CD8(+) T-cell responses and protection achieved in other infectious disease models have been optimized by using a DNA immunization to prime the immune system and a recombinant virus encoding the same antigen(s) to boost the response. A DNA vaccine (D) and recombinant modified vaccinia virus Ankara (M) in which the DNA encodes early secreted antigenic target 6 and mycobacterial protein tuberculosis 63 synthesized, and each was found to generate specific gamma interferon (IFN-gamma)-secreting CD4(+) T cells. Enhanced CD4(+) IFN-gamma T-cell responses were produced by both D-M and M-D immunization regimens. Significantly higher levels of IFN-gamma were seen with a D-D-D-M immunization regimen. The most immunogenic regimens were assessed in a challenge study and found to produce protection equivalent to that produced by Mycobacterium bovis BCG. Thus, heterologous prime-boost regimens boost CD4(+) as well as CD8(+) T-cell responses, and the use of heterologous constructs encoding the same antigen(s) may improve the immunogenicity and protective efficacy of DNA vaccines against tuberculosis and other diseases.

Ansell J, Hu JT, Gilbert SC, Hamilton KA, Hill AV, Lindsay SW. 2000. Improved method for distinguishing the human source of mosquito blood meals between close family members. Trans R Soc Trop Med Hyg, 94 (5), pp. 572-574. | Show Abstract | Read more

We have developed a simple and relatively cheap method to distinguish the origin of mosquito blood meals between close family members, effective for both laboratory and field samples. Each blood meal was squashed on to filter paper and eluted overnight with 0.5 mL phosphate-buffered saline. Deoxyribonucleic acid (DNA) was extracted using a chemical matrix (Insta-gene) which bound to everything from the blood meal except DNA, which remained in the supernatant. Following extractions, reference DNA samples taken directly from finger-prick blood of human subjects and those from blood meals of unknown origin were amplified with human microsatellite markers using a thermal cycler. Polymerase chain reaction products were then run on an ABI gel (Automated Biosystems) to obtain a genotype for each sample. The DNA from each mosquito blood meal was then matched to an individual host. With laboratory samples, human DNA which had been extracted from mosquito blood meals up to 12 h after feeding could be used. One important application of this method will be to identify which members of a community are most at risk from vector-borne diseases. It also has numerous potential applications in studies of insect biting behaviour in both human and veterinary science.

Aidoo M, Lalvani A, Gilbert SC, Hu JT, Daubersies P, Hurt N, Whittle HC, Druihle P, Hill AV. 2000. Cytotoxic T-lymphocyte epitopes for HLA-B53 and other HLA types in the malaria vaccine candidate liver-stage antigen 3. Infect Immun, 68 (1), pp. 227-232. | Show Abstract | Read more

The development of an effective preerythrocytic vaccine against Plasmodium falciparum malaria is likely to require inclusion of components from several preerythrocytic antigens. The association of HLA-B53 with resistance to severe malaria in West Africa provided evidence that HLA class I-restricted CD8(+) T-cell responses play a role in protective immunity in African children, supporting data from rodent models of malaria. Previously, a single epitope from liver-stage-specific antigen 1 (LSA-1) has been shown to be recognized by HLA-B53-specific cytotoxic T lymphocytes (CTL), but HLA-B53 epitopes were not found in four other antigens. In this study we measured CTL responses to peptides from the recently sequenced antigen liver-stage antigen 3 (LSA-3) and identified in it a new epitope restricted by HLA-B53. Several CTL epitopes restricted by other class I types were also identified within LSA-3 in studies in The Gambia and Tanzania. CTL were also identified to an additional P. falciparum antigen, exported protein 1 (Exp-1), the homologue of which is a protective antigen in a rodent model of malaria. These findings emphasize the diversity of P. falciparum antigens recognized by CD8(+) T cells in humans and support the inclusion of components from several antigens in new CTL-inducing vaccines against malaria.

Hill AV, Reece W, Gothard P, Moorthy V, Roberts M, Flanagan K, Plebanski M, Hannan C et al. 2000. DNA-based vaccines for malaria: a heterologous prime-boost immunisation strategy. Dev Biol (Basel), 104 pp. 171-179. | Show Abstract

A generic approach to inducing high level CD8+ T cell responses would be of value for prophylactic and therapeutic immunisation against several infectious diseases. However, it has been very difficult to achieve such immune responses using available vaccination strategies. Malaria is one of several diseases against which a new generation of better CD8+ T cell-inducing vaccines might be useful and is unusual in that it allows assessment of vaccine efficacy in small numbers of volunteers in carefully controlled challenge studies. Here we review the identification of a heterologous prime-boost regime using DNA priming and recombinant modified vaccinia Ankara (MVA) boosting that induces high level T cell responses in both mice and non-human primates. Clinical trials to determine whether this prime-boost approach is immunogenic in humans are in progress.

Dégano P, Schneider J, Hannan CM, Gilbert SC, Hill AV. 1999. Gene gun intradermal DNA immunization followed by boosting with modified vaccinia virus Ankara: enhanced CD8+ T cell immunogenicity and protective efficacy in the influenza and malaria models. Vaccine, 18 (7-8), pp. 623-632. | Show Abstract | Read more

In influenza and malaria, CD8+ T cells play an important role in protective immunity in mice. An immunization strategy consisting of DNA priming followed by boosting with recombinant modified vaccinia virus Ankara (MVA) induces complete protection, associated with high levels of CD8+ T cells, against Plasmodium berghei sporozoite challenge in mice. Intradermal delivery of DNA with a gene gun requires smaller amounts of DNA than intramuscular injection, in order to induce similar levels of immune responses. The present study compares both routes for the induction of specific CD8+ T cell responses and protection using different prime-boost immunization regimes in the influenza and the malaria models. In the DNA/MVA regime, equally high CD8+ T cell responses and levels of protection are achieved using ten times less DNA when delivered with a gene gun compared to intramuscular injection.

Schneider J, Gilbert SC, Hannan CM, Dégano P, Prieur E, Sheu EG, Plebanski M, Hill AV. 1999. Induction of CD8+ T cells using heterologous prime-boost immunisation strategies. Immunol Rev, 170 (1), pp. 29-38. | Show Abstract | Read more

One of the current challenges in vaccine design is the development of antigen delivery systems or vaccination strategies that induce high protective levels of CD8+ T cells. These cells are crucial for protection against certain tumours and intracellular pathogens such as the liver-stage parasite of malaria. A liver-stage malaria vaccine should therefore include CD8+ T-cell-inducing components. This review provides an overview of prime-boost immunisation strategies that result in protective CD8+ T-cell responses against malaria with an emphasis on work from our laboratory. Possible mechanisms explaining why heterologous prime-boost strategies, in particular boosting with replication-impaired recombinant poxviruses, are so effective are discussed.

Plebanski M, Lee EA, Hannan CM, Flanagan KL, Gilbert SC, Gravenor MB, Hill AV. 1999. Altered peptide ligands narrow the repertoire of cellular immune responses by interfering with T-cell priming. Nat Med, 5 (5), pp. 565-571. | Show Abstract | Read more

Variation in epitopes of infectious pathogens inhibits various effector functions of T lymphocytes through antagonism of the T-cell receptor. However, a more powerful strategy for immune evasion would be to prevent the induction of T-cell responses. We report here mutual 'interference' with the priming of human T-cell responses by a pair of naturally occurring variants of a malaria cytotoxic T-cell epitope. Interference with priming also occurs in vivo for a murine malaria T-cell epitope. Reshaping of the T-cell repertoire by such immune interference during naive T-cell induction may provide a general mechanism for observed patterns of immunodominance and persistence by many polymorphic pathogens.

Gilbert SC, Schneider J, Plebanski M, Hannan CM, Blanchard TJ, Smith GL, Hill AV. 1999. Ty virus-like particles, DNA vaccines and Modified Vaccinia Virus Ankara; comparisons and combinations. Biol Chem, 380 (3), pp. 299-303. | Show Abstract | Read more

Three types of vaccine, all expressing the same antigen from Plasmodium berghei, or a CD8+ T cell epitope from that antigen, were compared for their ability to induce CD8+ T cell responses in mice. Higher levels of lysis and numbers of IFN-gamma secreting T cells were primed with Ty virus-like particles and Modified Vaccinia Virus Ankara (MVA) than with DNA vaccines, but none of the vaccines were able to protect immunised mice from infectious challenge even after repeated doses. However, when the immune response was primed with one type of vaccine (Ty-VLPs or DNA) and boosted with another (MVA) complete protection against infection was achieved. Protection correlated with very high levels of IFN-gamma secreting T cells and lysis. This method of vaccination uses delivery systems and routes that can be used in humans and could provide a generally applicable regime for the induction of high levels of CD8+ T cells.

Plebanski M, Gilbert SC, Schneider J, Hannan CM, Layton G, Blanchard T, Becker M, Smith G, Butcher G, Sinden RE, Hill AV. 1998. Protection from Plasmodium berghei infection by priming and boosting T cells to a single class I-restricted epitope with recombinant carriers suitable for human use. Eur J Immunol, 28 (12), pp. 4345-4355. | Show Abstract | Read more

The desirability of inducing cytotoxic T cell responses to defined epitopes in humans has led to the development of a variety of recombinant delivery systems. Recombinant protein particles derived from a yeast retrotransposon (Ty) and the modified Ankara vaccinia (MVA) virus can deliver large epitope strings or even whole proteins. Both have previously been administered safely in humans. Immunization with recombinant Ty and MVA containing a single Plasmodium berghei class I-binding epitope provided 95% sterile protection against malaria in mice. The sequence of immunization, Ty followed by MVA, was critical to elicit high levels of IFN-gamma-producing cells and protection. The reciprocal sequence (MVA/TY) or homologous boosting was not protective. Both constructs (Ty and MVA) contain the H-2Kd-restricted pb9 CTL epitope from the circumsporozoite protein of P. berghei among a string of 8-15 human P. falciparum-derived CTL epitopes restricted through 7 common HLA alleles as well as widely recognized CD4 T cell epitopes. Thus, the novel recombinant Ty/MVA prime/boost combination with these constructs provides a safe alternative for evaluation for human vaccination against P. falciparum malaria.

Gilbert SC, Hill AV. 1998. The SPf66 Malaria Vaccine: Where Do We Go From Here? Parasitol Today, 14 (6), pp. 251.

Schneider J, Gilbert SC, Blanchard TJ, Hanke T, Robson KJ, Hannan CM, Becker M, Sinden R, Smith GL, Hill AV. 1998. Enhanced immunogenicity for CD8+ T cell induction and complete protective efficacy of malaria DNA vaccination by boosting with modified vaccinia virus Ankara. Nat Med, 4 (4), pp. 397-402. | Show Abstract | Read more

Immunization with irradiated sporozoites can protect against malaria infection and intensive efforts are aimed at reproducing this effect with subunit vaccines. A particular sequence of subunit immunization with pre-erythrocytic antigens of Plasmodium berghei, consisting of single dose priming with plasmid DNA followed by a single boost with a recombinant modified vaccinia virus Ankara (MVA) expressing the same antigen, induced unprecedented complete protection against P. berghei sporozoite challenge in two strains of mice. Protection was associated with very high levels of splenic peptide-specific interferon-gamma-secreting CD8+ T cells and was abrogated when the order of immunization was reversed. DNA priming followed by MVA boosting may provide a general immunization regime for induction of high levels of CD8+ T cells.

Hanke T, Blanchard TJ, Schneider J, Hannan CM, Becker M, Gilbert SC, Hill AV, Smith GL, McMichael A. 1998. Enhancement of MHC class I-restricted peptide-specific T cell induction by a DNA prime/MVA boost vaccination regime. Vaccine, 16 (5), pp. 439-445. | Show Abstract | Read more

Human immunodeficiency virus (HIV) vaccine candidates were previously constructed as a string of cytotoxic T lymphocyte (CTL) epitopes delivered and expressed using DNA and modified virus Ankara (MVA; an attenuated vaccinia virus) vectors. These vaccines were shown to induce interferon (IFN)-gamma-producing and cytolytic CD8+ T cells after a single vaccine administration. In the course of this work, immunization protocols were sought which would improve the levels of induced HIV-specific T cells. It was found that previous immunological exposure to MVA reduced the efficiency of subsequent priming and boosting using the same vaccine vehicle. However, a combined regime whereby the animals were first primed with the DNA vaccine and then boosted with MVA was the most potent protocol for the induction of both interferon-gamma-producing and cytolytic T cells against two CTL epitopes simultaneously. The general applicability of this novel vaccination method for induction of major histocompatibility complex class I-restricted T cells is discussed.

Gilbert SC, Plebanski M, Gupta S, Morris J, Cox M, Aidoo M, Kwiatkowski D, Greenwood BM, Whittle HC, Hill AV. 1998. Association of malaria parasite population structure, HLA, and immunological antagonism. Science, 279 (5354), pp. 1173-1177. | Show Abstract | Read more

Host-parasite coevolution has been likened to a molecular arms race, with particular parasite genes evolving to evade specific host defenses. Study of the variants of an antigenic epitope of Plasmodium falciparum that induces a cytotoxic T cell response supports this view. In African children with malaria, the variants present are influenced by the presence of a human leukocyte antigen (HLA) type that restricts the immune response to this epitope. The distribution of parasite variants may be further influenced by the ability of cohabiting parasite strains to facilitate each other's survival by down-regulating cellular immune responses, using altered peptide ligand antagonism.

Hanke T, Schneider J, Gilbert SC, Hill AV, McMichael A. 1998. DNA multi-CTL epitope vaccines for HIV and Plasmodium falciparum: immunogenicity in mice. Vaccine, 16 (4), pp. 426-435. | Show Abstract | Read more

The potential of building multi-cytotoxic T lymphocyte (CTL) epitope antigens in combination with the nucleic acid immunization technology is explored for development of acquired immunodeficiency syndrome (AIDS) and malaria vaccines. A novel minimal vector pTH for direct gene transfer was constructed for efficient expression of vaccine antigens and used as a vehicle for human immunodeficiency virus (HIV)- and Plasmodium falciparum-derived polyepitope genes. Two murine epitopes were included into these constructs to allow for testing of vaccine immunogenicity in small animals. The results showed that a single DNA injection generated CTL responses in all 15 vaccinated mice. The elicited CTL precursor frequencies were estimated in an interferon-gamma (IFN-gamma)-based ELISPOT assay and found to be an average of 300 (range 4-1346) peptide-responding cells per 10(6) splenocytes.

Kerry-Williams SM, Gilbert SC, Evans LR, Ballance DJ. 1998. Disruption of the Saccharomyces cerevisiae YAP3 gene reduces the proteolytic degradation of secreted recombinant human albumin. Yeast, 14 (2), pp. 161-169. | Show Abstract | Read more

Expression of recombinant human albumin (rHA) in Saccharomyces cerevisiae resulted in secretion of both mature albumin and a 45 kDa degradation product, comprising an N-terminal fragment of rHA with heterogeneous C-termini between residues 403 and 409 (Geisow et al., 1991). Site-directed mutagenesis of the human albumin gene (HA) to change Arg410 to Ala (R410A) caused a significant reduction in the amount of fragment produced. Mutation of the adjacent dibasic site Lys413 Lys414 had little effect in isolation, but in combination with the R410A mutation resulted in a further reduction in the amount of rHA fragment produced. This reduction could be duplicated with nature-identical rHA by disruption of the gene for an aspartyl protease (YAP3), alone or in conjunction with disruption of the KEX2 gene. Disruption of KEX2 alone did not result in any improvement in the degree of degradation of the rHA. Reduced degradation was also observed when an rHA-human growth hormone fusion protein was secreted from a yap3 strain, suggesting that such strains may have a general utility for heterologous protein secretion.

Hanke T, Blanchard TJ, Schneider J, Ogg GS, Tan R, Becker M, Gilbert SC, Hill AV, Smith GL, McMichael A. 1998. Immunogenicities of intravenous and intramuscular administrations of modified vaccinia virus Ankara-based multi-CTL epitope vaccine for human immunodeficiency virus type 1 in mice. J Gen Virol, 79 ( Pt 1) (1), pp. 83-90. | Show Abstract | Read more

A vaccine against human immunodeficiency virus (HIV) is still awaited. Although the correlates of protection remain elusive, it is likely that CD8+ T cells play an important role in the control of this infection. To firmly establish the importance of these cells in protective immunity, a means of efficient elicitation of CD8+ T cell responses in the absence of antibody is needed and, when available, might represent a crucial step towards a protective vaccine. Here, a novel vaccine candidate was constructed as a multi-cytotoxic T lymphocyte (CTL) epitope gene delivered and expressed using modified vaccinia virus Ankara (MVA). The immunogen consists of 20 human, one murine and three rhesus macaque epitopes. The non-human epitopes were included so that the vaccine can be tested for immunogenicity and optimal vaccination doses, routes and regimes in experimental animals. Mice were immunized intravenously (i.v.) or intramuscularly (i.m.) using a single dose of 10(6) p.f.u. of the recombinant MVA and the induction of CTL was assessed. It was demonstrated that both administration routes induced specific CTL responses and that the i.v. route was moderately more immunogenic than the i.m. route. The frequencies of ex vivo splenocytes producing interferon-y upon MHC class I-restricted peptide stimulation were determined using an ELISPOT assay. Also, the correct processing and presentation of some HLA-restricted epitopes in human cells was confirmed.

Bellamy R, Ruwende C, McAdam KP, Thursz M, Sumiya M, Summerfield J, Gilbert SC, Corrah T, Kwiatkowski D, Whittle HC, Hill AV. 1998. Mannose binding protein deficiency is not associated with malaria, hepatitis B carriage nor tuberculosis in Africans. QJM, 91 (1), pp. 13-18. | Show Abstract | Read more

We retrospectively studied MBP genotypes in patients with malaria, tuberculosis (TB), and persistent hepatitis B virus (HBV) carriage, in clinics and hospitals in The Gambia. Children under 10 years with cerebral malaria and/or severe malarial anaemia, were compared with children with symptomatic, mild malaria, and controls of the same age and ethnicity. Adult TB cases with smear-positive pulmonary TB were compared with healthy blood donors from the same ethnic groups. Malaria cases and controls were tested for hepatitis B core antibody (anti-HBc) and surface antigen (HBsAg). TB patients were tested for HIV antibodies. Genotyping used sequence-specific oligonucleotide analysis to identify MBP variant alleles. Overall, 46% (944/2041) of patients and controls were homozygous for the wild-type MBP allele, 45% (922/2041) were carriers of a single variant allele and 8.6% (175/2041) had two variant alleles. Neither homozygotes nor heterozygotes for MBP variants were at increased risk of clinical malaria, persistent HBV carriage or TB. The most common mutation in Africans, the codon 57 variant allele, was weakly associated with resistance to TB (221/794 in TB cases and 276/844 in controls, p = 0.037). MBP deficiency is not a significant risk factor for persistent HBV, severe malaria nor pulmonary TB in West Africa.

Gilbert SC, Plebanski M, Harris SJ, Allsopp CE, Thomas R, Layton GT, Hill AV. 1997. A protein particle vaccine containing multiple malaria epitopes. Nat Biotechnol, 15 (12), pp. 1280-1284. | Show Abstract | Read more

Ty virus-like particles consist of a single protein species that can be produced in yeast. Recombinant Ty-VLPs carrying a string of up to 15 defined cytotoxic T lymphocyte (CTL) epitopes from Plasmodium species prime protective CTL responses in mice following a single administration without adjuvant. Effective processing of epitopes from the string was demonstrated in vitro and in vivo and was not affected by flanking sequences.

Hill AV, Jepson A, Plebanski M, Gilbert SC. 1997. Genetic analysis of host-parasite coevolution in human malaria. Philos Trans R Soc Lond B Biol Sci, 352 (1359), pp. 1317-1325. | Show Abstract | Read more

Recent twin studies of clinical malaria and immune responses to malaria antigens have underscored the importance of both major histocompatability complex (MHC) and non-MHC genes in determining variable susceptibility and immune responsiveness. By using a combination of whole genome genetic linkage studies of families and candidate genes analysis, non-MHC genes are being mapped and identified. Human leucocyte antigen (HLA) genotype was found to affect susceptibility to severe malaria in a large study of West African children. T lymphocytes that may mediate such resistance have been identified and their target antigens and epitopes characterized. Some of these epitopes show substantial polymorphism, which appears to result from immune selection pressure. Natural variant epitopes have been found to escape T-cell recognition in cytolytic and other T-cell assays. More recently a novel immune escape mechanism has been described in viral infections, altered peptide ligand antagonism, whereby variants of a T-cell epitope can downregulate or ablate a T cell response to the index peptide. The likely implications of such immune escape mechanisms for the population structure of malaria parasites, for HLA associations with malaria infection and disease, and for the design of new malaria vaccines, are discussed. The evolutionary consequences of such molecular interactions can be assessed by using mathematical models that capture the dynamic of variable host and parasite molecules. Combined genetic, immunological and mathematical analysis of host and parasite variants in natural populations can identify some mechanisms driving host-parasite coevolution.

Gilbert SC, Hill AV. 1997. Protein particle vaccines against malaria. Parasitol Today, 13 (8), pp. 302-306. | Show Abstract | Read more

Many viral coat proteins retain the ability to assemble into virus-like particles when produced as recombinant proteins. These small particles are highly immunogenic, and in many cases can be used to carry epitopes or antigens from other pathogens. Most particle-forming proteins can tolerate only small additions or alterations to their sequence, but Hepatitis B virus surface antigen (HBsAg) and the yeast-derived Ty particle are exceptionel in their ability to form particles with long N- or C-terminal extensions. Both have been used to produce hybrid particles carrying Plasmodium sequences. These have been shown to be highly immunogenic in animal studies and also in human phase I trials, in the case of HBsAg. Recently, six out of seven human volunteers were protected against sporozoite challenge by a recombinant HBsAg particle vaccine, the most encouraging result to date for any pre-erythrocytic malaria vaccine. Here, Sarah Gilbert and Adrian Hill review the prospects for the future development of protein particle vaccines against malaria.

Allsopp CE, Plebanski M, Gilbert S, Sinden RE, Harris S, Frankel G, Dougan G, Hioe C et al. 1996. Comparison of numerous delivery systems for the induction of cytotoxic T lymphocytes by immunization. Eur J Immunol, 26 (8), pp. 1951-1959. | Show Abstract | Read more

A variety of vaccine delivery systems including peptides with various adjuvants, recombinant particles, live recombinant viruses and bacteria and plasmid DNA were tested for their ability to induce CD8+ cytotoxic T lymphocytes (CTL) against a well-defined epitope (amino acids 252-260) from the circumsporozoite (CS) protein of Plasmodium berghei. We compared routes of immunization that would be applicable for the administration of a malaria vaccine in humans. The majority of these vaccines did not induce high CTL responses in the spleens of immunized mice. However, both a yeast-derived Ty virus-like particle expressing the optimal nine-amino acid epitope SYIPSAEKI from the CS protein (CSP-VLP) and a lipid-tailed peptide of this same sequence induced high levels of the major histocompatibility complex (MHC) class I-restricted CTL with one and three subcutaneous immunizations, respectively. Moreover, these CTL were able to recognize naturally processed antigen expressed by a recombinant vaccinia virus. The levels of CTL induced by CSP-VLP could be augmented by co-immunization with certain cytokines. Target cells pulsed with CSP-VLP were recognized and lysed, showing that the particles were effectively processed and presented through MHC class I presentation pathway. The levels of CTL induced using CSP-VLP and lipopeptides are comparable to those observed after immunization with multiple doses of irradiated sporozoites.

Ruwende C, Khoo SC, Snow RW, Yates SN, Kwiatkowski D, Gupta S, Warn P, Allsopp CE, Gilbert SC, Peschu N. 1995. Natural selection of hemi- and heterozygotes for G6PD deficiency in Africa by resistance to severe malaria. Nature, 376 (6537), pp. 246-249. | Show Abstract | Read more

Glucose-6-phosphate dehydrogenase (G6PD) deficiency, the most common enzymopathy of humans, affects over 400 million people. The geographical correlation of its distribution with the historical endemicity of malaria suggests that this disorder has risen in frequency through natural selection by malaria. However, attempts to confirm that G6PD deficiency is protective in case-control studies of malaria have yielded conflicting results. Hence, for this X-linked disorder, it is unclear whether both male hemizygotes and female heterozygotes are protected or, as frequently suggested, only females. Furthermore, how much protection may be afforded is unknown. Here we report that, in two large case-control studies of over 2,000 African children, the common African form of G6PD deficiency (G6PD A-) is associated with a 46-58% reduction in risk of severe malaria for both female heterozygotes and male hemizygotes. A mathematical model incorporating the measured selective advantage against malaria suggests that a counterbalancing selective disadvantage, associated with this enzyme deficiency, has retarded its rise in frequency in malaria-endemic regions. Although G6PD deficiency is now regarded as a generally benign disorder, in earlier environmental conditions it could have been significantly disadvantageous.

Gilbert SC, van Urk H, Greenfield AJ, McAvoy MJ, Denton KA, Coghlan D, Jones GD, Mead DJ. 1994. Increase in copy number of an integrated vector during continuous culture of Hansenula polymorpha expressing functional human haemoglobin. Yeast, 10 (12), pp. 1569-1580. | Show Abstract | Read more

Recombinant human haemoglobin A (rHbA) was produced by a leucine-requiring strain of Hansenula polymorpha which had been transformed with an integration vector containing the Saccharomyces cerevisiae LEU2 gene and cDNAs for the expression of alpha and beta globin each driven by the H. polymorpha MOX promoter. After 40 generations in a chemostat it was found that the integrated vector had become amplified in the host strain. In some cases this led to an increase in LEU2 gene dosage, but a loss of globin expression cassettes. In other cases the globin gene dosage also increased. These changes coincided with an increase in rHbA production in the culture, which was reversed when the dilution rate was increased. Isolates from a chemostat culture producing elevated levels of rHbA were grown in fed-batch fermentations, resulting in higher productivities than when inoculated with the parent strain. The rHbA produced was purified and characterized. Oxygen binding studies and electrospray mass spectrometry showed that the rHbA had been processed and assembled correctly, and behaved as a fully functional co-operative tetramer.

Hill AV, Yates SN, Allsopp CE, Gupta S, Gilbert SC, Lalvani A, Aidoo M, Davenport M, Plebanski M. 1994. Human leukocyte antigens and natural selection by malaria. Philos Trans R Soc Lond B Biol Sci, 346 (1317), pp. 379-385. | Show Abstract | Read more

The extraordinary polymorphism of human leukocyte antigens (HLA) poses a question as to how this remarkable diversity arose and is maintained. The explanation that infectious pathogens are largely responsible is theoretically attractive but clear and consistent associations between HLA alleles and major infectious diseases have rarely been identified. Large case-control studies of HLA types in African children with severe malaria indicate that HLA associations with this parasitic infection do exist and it is becoming possible to investigate the underlying mechanisms by identification of peptide epitopes in parasite antigens. Such analysis reveals how the magnitude and detectability of HLA associations may be influenced by numerous genetic and environmental factors. These complex interactions will give rise to variation over time and space in the selective pressures exerted by infectious diseases and this fluctuation may, in itself, contribute to the maintenance of HLA polymorphism.

EVANS C, RATLEDGE C, GILBERT S. 1985. A RAPID SCREENING METHOD FOR LIPID-ACCUMULATING YEAST USING A REPLICA-PRINTING TECHNIQUE JOURNAL OF MICROBIOLOGICAL METHODS, 4 (3-4), pp. 203-210. | Read more

RATLEDGE C, GILBERT S. 1985. CARNITINE ACETYLTRANSFERASE ACTIVITY IN OLEAGINOUS YEASTS FEMS MICROBIOLOGY LETTERS, 27 (3), pp. 273-275. | Read more

Ewer K, Rampling T, Venkatraman N, Bowyer G, Wright D, Lambe T, Imoukhuede EB, Payne R et al. 2016. A Monovalent Chimpanzee Adenovirus Ebola Vaccine Boosted with MVA. N Engl J Med, 374 (17), pp. 1635-1646. | Show Abstract | Read more

BACKGROUND: The West African outbreak of Ebola virus disease that peaked in 2014 has caused more than 11,000 deaths. The development of an effective Ebola vaccine is a priority for control of a future outbreak. METHODS: In this phase 1 study, we administered a single dose of the chimpanzee adenovirus 3 (ChAd3) vaccine encoding the surface glycoprotein of Zaire ebolavirus (ZEBOV) to 60 healthy adult volunteers in Oxford, United Kingdom. The vaccine was administered in three dose levels--1×10(10) viral particles, 2.5×10(10) viral particles, and 5×10(10) viral particles--with 20 participants in each group. We then assessed the effect of adding a booster dose of a modified vaccinia Ankara (MVA) strain, encoding the same Ebola virus glycoprotein, in 30 of the 60 participants and evaluated a reduced prime-boost interval in another 16 participants. We also compared antibody responses to inactivated whole Ebola virus virions and neutralizing antibody activity with those observed in phase 1 studies of a recombinant vesicular stomatitis virus-based vaccine expressing a ZEBOV glycoprotein (rVSV-ZEBOV) to determine relative potency and assess durability. RESULTS: No safety concerns were identified at any of the dose levels studied. Four weeks after immunization with the ChAd3 vaccine, ZEBOV-specific antibody responses were similar to those induced by rVSV-ZEBOV vaccination, with a geometric mean titer of 752 and 921, respectively. ZEBOV neutralization activity was also similar with the two vaccines (geometric mean titer, 14.9 and 22.2, respectively). Boosting with the MVA vector increased virus-specific antibodies by a factor of 12 (geometric mean titer, 9007) and increased glycoprotein-specific CD8+ T cells by a factor of 5. Significant increases in neutralizing antibodies were seen after boosting in all 30 participants (geometric mean titer, 139; P<0.001). Virus-specific antibody responses in participants primed with ChAd3 remained positive 6 months after vaccination (geometric mean titer, 758) but were significantly higher in those who had received the MVA booster (geometric mean titer, 1750; P<0.001). CONCLUSIONS: The ChAd3 vaccine boosted with MVA elicited B-cell and T-cell immune responses to ZEBOV that were superior to those induced by the ChAd3 vaccine alone. (Funded by the Wellcome Trust and others; ClinicalTrials.gov number, NCT02240875.).

Busquets N, Lorenzo G, Lopez-Gil E, Rivas R, Solanes D, Galindo-Cardiel I, Xavier Abad F, Rodriguez F et al. 2014. Efficacy assessment of an MVA vectored Rift Valley Fever vaccine in lambs ANTIVIRAL RESEARCH, 108 pp. 165-172. | Read more

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Antrobus RD, Berthoud TK, Mullarkey CE, Hoschler K, Coughlan L, Zambon M, Hill AVS, Gilbert SC. 2014. Coadministration of seasonal influenza vaccine and MVA-NP+M1 simultaneously achieves potent humoral and cell-mediated responses Molecular Therapy, 22 (1), pp. 233-238. | Show Abstract | Read more

Current seasonal influenza vaccines have reduced immunogenicity and are of suboptimal efficacy in older adults. We have previously shown that the novel candidate vaccine MVA-NP+M1 is able to boost memory T cell responses in adults aged 50-85 years. Preclinical studies have demonstrated that viral vectored vaccines can act as adjuvants when coadministered with protein-based vaccines. We have conducted a phase I clinical trial to compare the coadministration of seasonal influenza vaccine and MVA-NP+M1 with seasonal influenza vaccine alone in adults aged 50 years and above. This combination of vaccines was safe and well tolerated. T cell responses to internal influenza proteins were boosted to significantly higher levels in the group receiving MVA-NP+M1 compared with the group receiving seasonal influenza vaccine alone. Rates of seroprotection and seroconversion against the three vaccine strains were similar in both groups; however, there was a significant increase in the geometric mean titer ratio for the H3N2 component of seasonal influenza vaccine in the coadministration group. While some vaccine combinations result in immune interference, the coadministration of MVA-NP+M1 alongside seasonal influenza vaccine is shown here to increase some influenza strain-specific antibody responses and boost memory T cells capable of recognizing a range of influenza A subtypes. © The American Society of Gene & Cell Therapy.

Antrobus RD, Coughlan L, Berthoud TK, Dicks MD, Hill AV, Lambe T, Gilbert SC. 2014. Clinical assessment of a novel recombinant simian adenovirus ChAdOx1 as a vectored vaccine expressing conserved Influenza A antigens. Mol Ther, 22 (3), pp. 668-674. | Show Abstract | Read more

Adenoviruses are potent vectors for inducing and boosting cellular immunity to encoded recombinant antigens. However, the widespread seroprevalence of neutralizing antibodies to common human adenovirus serotypes limits their use. Simian adenoviruses do not suffer from the same drawbacks. We have constructed a replication-deficient chimpanzee adenovirus-vectored vaccine expressing the conserved influenza antigens, nucleoprotein (NP), and matrix protein 1 (M1). Here, we report safety and T-cell immunogenicity following vaccination with this novel recombinant simian adenovirus, ChAdOx1 NP+M1, in a first in human dose-escalation study using a 3+3 study design, followed by boosting with modified vaccinia virus Ankara expressing the same antigens in some volunteers. We demonstrate ChAdOx1 NP+M1 to be safe and immunogenic. ChAdOx1 is a promising vaccine vector that could be used to deliver vaccine antigens where strong cellular immune responses are required for protection.

Ewer KJ, O'Hara GA, Duncan CJ, Collins KA, Sheehy SH, Reyes-Sandoval A, Goodman AL, Edwards NJ et al. 2013. Protective CD8+ T-cell immunity to human malaria induced by chimpanzee adenovirus-MVA immunisation. Nat Commun, 4 pp. 2836. | Show Abstract | Read more

Induction of antigen-specific CD8(+) T cells offers the prospect of immunization against many infectious diseases, but no subunit vaccine has induced CD8(+) T cells that correlate with efficacy in humans. Here we demonstrate that a replication-deficient chimpanzee adenovirus vector followed by a modified vaccinia virus Ankara booster induces exceptionally high frequency T-cell responses (median >2400 SFC/10(6) peripheral blood mononuclear cells) to the liver-stage Plasmodium falciparum malaria antigen ME-TRAP. It induces sterile protective efficacy against heterologous strain sporozoites in three vaccinees (3/14, 21%), and delays time to patency through substantial reduction of liver-stage parasite burden in five more (5/14, 36%), P=0.008 compared with controls. The frequency of monofunctional interferon-γ-producing CD8(+) T cells, but not antibodies, correlates with sterile protection and delay in time to patency (P(corrected)=0.005). Vaccine-induced CD8(+) T cells provide protection against human malaria, suggesting that a major limitation of previous vaccination approaches has been the insufficient magnitude of induced T cells.

Boyd AC, Ruiz-Hernandez R, Peroval MY, Carson C, Balkissoon D, Staines K, Turner AV, Hill AV, Gilbert SC, Butter C. 2013. Towards a universal vaccine for avian influenza: protective efficacy of modified Vaccinia virus Ankara and Adenovirus vaccines expressing conserved influenza antigens in chickens challenged with low pathogenic avian influenza virus. Vaccine, 31 (4), pp. 670-675. | Show Abstract | Read more

Current vaccines targeting surface proteins can drive antigenic variation resulting either in the emergence of more highly pathogenic viruses or of antigenically distinct viruses that escape control by vaccination and thereby persist in the host population. Influenza vaccines typically target the highly mutable surface proteins and do not provide protection against heterologous challenge. Vaccines which induce immune responses against conserved influenza epitopes may confer protection against heterologous challenge. We report here the results of vaccination with recombinant modified Vaccinia virus Ankara (MVA) and Adenovirus (Ad) expressing a fusion construct of nucleoprotein and matrix protein (NP+M1). Prime and boost vaccination regimes were trialled in different ages of chicken and were found to be safe and immunogenic. Interferon-γ (IFN-γ) ELISpot was used to assess the cellular immune response post secondary vaccination. In ovo Ad prime followed by a 4 week post hatch MVA boost was identified as the most immunogenic regime in one outbred and two inbred lines of chicken. Following vaccination, one inbred line (C15I) was challenged with low pathogenic avian influenza (LPAI) H7N7 (A/Turkey/England/1977). Birds receiving a primary vaccination with Ad-NP+M1 and a secondary vaccination with MVA-NP+M1 exhibited reduced cloacal shedding as measured by plaque assay at 7 days post infection compared with birds vaccinated with recombinant viruses containing irrelevant antigen. This preliminary indication of efficacy demonstrates proof of concept in birds; induction of T cell responses in chickens by viral vectors containing internal influenza antigens may be a productive strategy for the development of vaccines to induce heterologous protection against influenza in poultry.

Gilbert SC. 2013. Advances in the development of universal influenza vaccines. Influenza Other Respir Viruses, 7 (5), pp. 750-758. | Show Abstract | Read more

Despite the widespread availability and use of influenza vaccines, influenza still poses a considerable threat to public health. Vaccines against seasonal influenza do not offer protection against pandemic viruses, and vaccine efficacy against seasonal viruses is reduced in seasons when the vaccine composition is not a good match for the predominant circulating viruses. Vaccine efficacy is also reduced in older adults, who are one of the main target groups for vaccination. The continual threat of pandemic influenza, with the known potential for rapid spread around the world and high mortality rates, has prompted researchers to develop a number of novel approaches to providing immunity to this virus, focusing on target antigens which are highly conserved between different influenza A virus subtypes. Several of these have now been taken into clinical development, and this review discusses the progress that has been made, as well as considering the requirements for licensing these new vaccines and how they might be used in the future.

Lambe T, Spencer AJ, Mullarkey CE, Antrobus RD, Yu LM, de Whalley P, Thompson BA, Jones C et al. 2012. T-cell responses in children to internal influenza antigens, 1 year after immunization with pandemic H1N1 influenza vaccine, and response to revaccination with seasonal trivalent-inactivated influenza vaccine. Pediatr Infect Dis J, 31 (6), pp. e86-e91. | Show Abstract | Read more

BACKGROUND: During seasonal influenza epidemics, 5-15% of the population are affected with an illness having a nontrivial mortality, morbidity and economic burden. Inactivated influenza vaccines are routinely used to prevent influenza infection, primarily by inducing humoral immunity. In addition, trivalent-inactivated influenza vaccines have previously been shown to boost influenza-specific T-cell responses in a small percentage of adults. We investigate here the influenza-specific T-cell response, in children, 1 year after pandemic H1N1 vaccination and the ability to boost the T-cell response with trivalent-inactivated influenza immunization. METHODS: Peripheral blood mononuclear cells (PBMCs) were isolated from children previously vaccinated with pandemic H1N1 vaccine, pre- and postseasonal 2010-2011 trivalent influenza vaccine (TIV) vaccination. Samples were analyzed by interferon-gamma enzyme-linked immunosorbent spot for reactogenicity toward internal influenza antigens (nucleoprotein, matrix protein 1 and nonstructural protein 1). RESULTS: Basal ex vivo T-cell responses to nucleoprotein, matrix protein 1 and nonstructural protein 1 measured by interferon-gamma enzyme-linked immunosorbent spot assay were significantly higher in those children who had previously received an AS03B-adjuvanted split virion pandemic vaccine 12 months earlier rather than a nonadjuvanted whole virion vaccine. Boosting of these responses, 21 days after 2010/2011 seasonal TIV vaccination was observed regardless of age or prior pandemic vaccination regime, although boosting was greater in those groups with the lowest initial response. CONCLUSIONS: We show here that children previously vaccinated with the 2009 pandemic H1N1 vaccine have measurable T-cell responses 1 year after vaccination. The magnitudes of these responses are dependent on both age of vaccine and type of pandemic H1N1 vaccine used. After 2010/2011 seasonal TIV vaccination, these T-cell responses undergo a small but significant boost.

Lillie PJ, Berthoud TK, Powell TJ, Lambe T, Mullarkey C, Spencer AJ, Hamill M, Peng Y et al. 2012. Preliminary assessment of the efficacy of a T-cell-based influenza vaccine, MVA-NP+M1, in humans. Clin Infect Dis, 55 (1), pp. 19-25. | Show Abstract | Read more

BACKGROUND: The novel influenza vaccine MVA-NP+M1 is designed to boost cross-reactive T-cell responses to internal antigens of the influenza A virus that are conserved across all subtypes, providing protection against both influenza disease and virus shedding against all influenza A viruses. Following a phase 1 clinical study that demonstrated vaccine safety and immunogenicity, a phase 2a vaccination and influenza challenge study has been conducted in healthy adult volunteers. METHODS: Volunteers with no measurable serum antibodies to influenza A/Wisconsin/67/2005 received either a single vaccination with MVA-NP+M1 or no vaccination. T-cell responses to the vaccine antigens were measured at enrollment and again prior to virus challenge. All volunteers underwent intranasal administration of influenza A/Wisconsin/67/2005 while in a quarantine unit and were monitored for symptoms of influenza disease and virus shedding. RESULTS: Volunteers had a significantly increased T-cell response to the vaccine antigens following a single dose of the vaccine, with an increase in cytolytic effector molecules. Intranasal influenza challenge was undertaken without safety issues. Two of 11 vaccinees and 5 of 11 control subjects developed laboratory-confirmed influenza (symptoms plus virus shedding). Symptoms of influenza were less pronounced in the vaccinees and there was a significant reduction in the number of days of virus shedding in those vaccinees who developed influenza (mean, 1.09 days in controls, 0.45 days in vaccinees, P = .036). CONCLUSIONS: This study provides the first demonstration of clinical efficacy of a T-cell-based influenza vaccine and indicates that further clinical development should be undertaken. CLINICAL TRIALS REGISTRATION: NCT00993083.

Antrobus RD, Lillie PJ, Berthoud TK, Spencer AJ, McLaren JE, Ladell K, Lambe T, Milicic A, Price DA, Hill AV, Gilbert SC. 2012. A T cell-inducing influenza vaccine for the elderly: safety and immunogenicity of MVA-NP+M1 in adults aged over 50 years. PLoS One, 7 (10), pp. e48322. | Show Abstract | Read more

BACKGROUND: Current influenza vaccines have reduced immunogenicity and are of uncertain efficacy in older adults. We assessed the safety and immunogenicity of MVA-NP+M1, a viral-vectored influenza vaccine designed to boost memory T cell responses, in a group of older adults. METHODS: Thirty volunteers (aged 50-85) received a single intramuscular injection of MVA-NP+M1 at a dose of 1·5×10(8) plaque forming units (pfu). Safety and immunogenicity were assessed over a period of one year. The frequency of T cells specific for nucleoprotein (NP) and matrix protein 1 (M1) was determined by interferon-gamma (IFN-γ) ELISpot, and their phenotypic and functional properties were characterized by polychromatic flow cytometry. In a subset of M1-specific CD8(+) T cells, T cell receptor (TCR) gene expression was evaluated using an unbiased molecular approach. RESULTS: Vaccination with MVA-NP+M1 was well tolerated. ELISpot responses were boosted significantly above baseline following vaccination. Increases were detected in both CD4(+) and CD8(+) T cell subsets. Clonality studies indicated that MVA-NP+M1 expanded pre-existing memory CD8(+) T cells, which displayed a predominant CD27(+)CD45RO(+)CD57(-)CCR7(-) phenotype both before and after vaccination. CONCLUSIONS: MVA-NP+M1 is safe and immunogenic in older adults. Unlike seasonal influenza vaccination, the immune responses generated by MVA-NP+M1 are similar between younger and older individuals. A T cell-inducing vaccine such as MVA-NP+M1 may therefore provide a way to circumvent the immunosenescence that impairs routine influenza vaccination. TRIAL REGISTRATION: ClinicalTrials.gov NCT00942071.

de Cassan SC, Forbes EK, Douglas AD, Milicic A, Singh B, Gupta P, Chauhan VS, Chitnis CE, Gilbert SC, Hill AV, Draper SJ. 2011. The requirement for potent adjuvants to enhance the immunogenicity and protective efficacy of protein vaccines can be overcome by prior immunization with a recombinant adenovirus. J Immunol, 187 (5), pp. 2602-2616. | Show Abstract | Read more

A central goal in vaccinology is the induction of high and sustained Ab responses. Protein-in-adjuvant formulations are commonly used to achieve such responses. However, their clinical development can be limited by the reactogenicity of some of the most potent preclinical adjuvants and the cost and complexity of licensing new adjuvants for human use. Also, few adjuvants induce strong cellular immunity, which is important for protection against many diseases, such as malaria. We compared classical adjuvants such as aluminum hydroxide to new preclinical adjuvants and adjuvants in clinical development, such as Abisco 100, CoVaccine HT, Montanide ISA720, and stable emulsion-glucopyranosyl lipid A, for their ability to induce high and sustained Ab responses and T cell responses. These adjuvants induced a broad range of Ab responses when used in a three-shot protein-in-adjuvant regimen using the model Ag OVA and leading blood-stage malaria vaccine candidate Ags. Surprisingly, this range of Ab immunogenicity was greatly reduced when a protein-in-adjuvant vaccine was used to boost Ab responses primed by a human adenovirus serotype 5 vaccine recombinant for the same Ag. This human adenovirus serotype 5-protein regimen also induced a more cytophilic Ab response and demonstrated improved efficacy of merozoite surface protein-1 protein vaccines against a Plasmodium yoelii blood-stage challenge. This indicates that the differential immunogenicity of protein vaccine adjuvants may be largely overcome by prior immunization with recombinant adenovirus, especially for adjuvants that are traditionally considered poorly immunogenic in the context of subunit vaccination and may circumvent the need for more potent chemical adjuvants.

Berthoud TK, Hamill M, Lillie PJ, Hwenda L, Collins KA, Ewer KJ, Milicic A, Poyntz HC et al. 2011. Potent CD8+ T-cell immunogenicity in humans of a novel heterosubtypic influenza A vaccine, MVA-NP+M1. Clin Infect Dis, 52 (1), pp. 1-7. | Show Abstract | Read more

BACKGROUND: Influenza A viruses cause occasional pandemics and frequent epidemics. Licensed influenza vaccines that induce high antibody titers to the highly polymorphic viral surface antigen hemagglutinin must be re-formulated and readministered annually. A vaccine providing protective immunity to the highly conserved internal antigens could provide longer-lasting protection against multiple influenza subtypes. METHODS: We prepared a Modified Vaccinia virus Ankara (MVA) vector encoding nucleoprotein and matrix protein 1 (MVA-NP+M1) and conducted a phase I clinical trial in healthy adults. RESULTS: The vaccine was generally safe and well tolerated, with significantly fewer local side effects after intramuscular rather than intradermal administration. Systemic side effects increased at the higher dose in both frequency and severity, with 5 out of 8 volunteers experiencing severe nausea/vomiting, malaise, or rigors. Ex vivo T-cell responses to NP and M1 measured by IFN-γ ELISPOT assay were significantly increased after vaccination (prevaccination median of 123 spot-forming units/million peripheral blood mononuclear cells, postvaccination peak response median 339, 443, and 1443 in low-dose intradermal, low-dose intramuscular, and high-dose intramuscular groups, respectively), and the majority of the antigen-specific T cells were CD8(+). CONCLUSIONS: We conclude that the vaccine was both safe and remarkably immunogenic, leading to frequencies of responding T cells that appear to be much higher than those induced by any other influenza vaccination approach. Further studies will be required to find the optimum dose and to assess whether the increased T-cell response to conserved influenza proteins results in protection from influenza disease.

Improving viral vectored vaccines

Replication-deficient viral vectored vaccines have proved to be safe and highly immunogenic delivery systems, and have been tested in clinical trials of novel vaccines against malaria, HIV, TB, influenza, and other diseases. Starting in 2015 an adenovirus-vectored Ebola vaccine was fast-tracked through clinical development by the Jenner Institute. However there are still improvements that can be made with the viral vectors we are now using. We can increase expression levels of antigens, ...

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Influenza vaccines – Broad protection through vaccination

Influenza vaccines – Broad protection through vaccinationCompared with the 1918 influenza pandemic, which claimed an estimated 15-50 million lives worldwide, mortality associated with the recent H1N1 pandemic was fortunately low. However, the 2009 pandemic was unusual in that children and young adults were more susceptible to infection. Cross-reactive antibodies induced pre-1950 were found to be protective in the elderly [Skountzou et al., 2010]. However, the predominant humoral immune response ...

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Emerging pathogens- vaccine development

Despite therapeutic advances, the continued emergence and re-emergence of novel infectious pathogens can have devastating healthcare impacts. Increased global interdependence and the ease of human, animal and trade movements facilitate transmission and present multiple opportunities for pathogen spread.There are a number of novel and dangerous pathogens with recognised pandemic potential, including but not limited to, Ebola, Marburg, Rift Valley fever, Lassa Fever, MERS-CoV, Nipah, and Crimean ...

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