Rett syndrome (RTT) is a severe form of mental retardation, which is caused by spontaneous mutations in the X-linked gene MECP2. How the loss of MeCP2 function leads to RTT is currently unknown. Mice lacking the Mecp2 gene initially show normal postnatal development but later acquire neurological phenotypes, including heightened anxiety, that resemble RTT. The MECP2 gene encodes a methyl-CpG-binding protein that can act as a transcriptional repressor. Using cDNA microarrays, we found that Mecp2-null animals differentially express several genes that are induced during the stress response by glucocorticoids. Increased levels of mRNAs for serum glucocorticoid-inducible kinase 1 (Sgk) and FK506-binding protein 51 (Fkbp5) were observed before and after onset of neurological symptoms, but plasma glucocorticoid was not significantly elevated in Mecp2-null mice. MeCP2 is bound to the Fkbp5 and Sgk genes in brain and may function as a modulator of glucocorticoid-inducible gene expression. Given the known deleterious effect of glucocorticoid exposure on brain development, our data raise the possibility that disruption of MeCP2-dependent regulation of stress-responsive genes contributes to the symptoms of RTT.
\n \n\n \n \nBACKGROUND: The introduction of the endosymbiotic bacterium, Wolbachia into Aedes aegypti populations is a novel approach to reduce disease transmission. The presence of Wolbachia limits the ability of the mosquito to transmit dengue virus (DENV) and the strength of this effect appears to correlate with Wolbachia densities in the mosquito. There is also some evidence that Wolbachia densities may increase following the consumption of a bloodmeal. Here we have examined whether multiple blood feeds lead to increases in density or associated changes in Wolbachia-mediated blocking of DENV. METHODS: The Wolbachia infected Aedes aegypti mosquito line was used for the study. There were three treatment groups; a non-blood fed control, a second group fed once and a third group fed twice on human blood. All groups were orally infected with DENV-2 and then their midguts and salivary glands were dissected 10-11 days post infection. RNA/DNA was simultaneously extracted from each tissue and subsequently used for DENV RNA copies and Wolbachia density quantification, respectively. RESULTS: We found variation between replicate vector competence experiments and no clear evidence that Wolbachia numbers increased in either the salivary glands or remainder of the body with feeding and hence saw no corresponding improvements in DENV blocking. CONCLUSIONS: Aedes aegypti are \"sip\" feeders returning often to obtain bloodmeals and hence it is important to assess whether repeat blood feeding improved the efficacy of Wolbachia-based DENV blocking. Our work suggests in the laboratory context when Wolbachia densities are high that repeat feeding does not improve blocking and hence this ability should likely be stable with respect to feeding cycle in the field.
\n \n\n \n \nThis study examined the ability of different plasmid vectors encoding H(C) fragment, the non-toxic binding portion of tetanus toxin, to be stably retained by Salmonella enterica var. Typhimurium (Salmonella typhimurium) vaccine strain BRD509 and, upon immunisation, to induce an antibody response against the carried antigen. The H(C) fragment expression cassette containing the transcription/translation signals, H(C) fragment open reading frame and the downstream TrpA terminator, was excised from pTETtac4 and incorporated into the plasmids pIC20H, pBR322, pACYC184 and pRSF1010. The resulting constructs were transferred into attenuated S. typhimurium, BRD509, and the level of H(C) fragment expression was examined by Western blot analysis. The relative stability of each plasmid in S. typhimurium was determined in vitro in the absence of antibiotic selection, and in vivo following immunisation. The ability of each H(C) fragment-expressing strain to induce lipopolysaccharide- and tetanus toxoid-specific antibody responses was assayed by an enzyme-linked immunosorbent assay. These studies showed that all the vaccine vector constructs, except the S. typhimurium carrying the expression vector based on pIC20H, were able to elicit a high titre immune response. The level of tetanus toxoid-specific antibody induced by S. typhimurium directly correlated with the level of in vitro and in vivo stability of the H(C) fragment expression plasmid carried by the bacterium, and not with an increased copy number of the parent plasmid vector.
\n \n\n \n \nBACKGROUND: Various Zika virus (ZIKV) vaccine candidates are currently in development. Nevertheless, unique challenges in clinical development and regulatory pathways may hinder the licensure of high-quality, safe, and effective ZIKV vaccines. DISCUSSION: Implementing phase 3 efficacy trials will be difficult given the challenges of the spatio-temporal heterogeneity of ZIKV transmission, the unpredictability of ZIKV epidemics, the broad spectrum of clinical manifestations making a single definite endpoint difficult, a lack of sensitive and specific diagnostic assays, and the need for inclusion of vulnerable target populations. In addition to a vaccine, drugs for primary prophylaxis, post-exposure prophylaxis, or treatment should also be developed to prevent or mitigate the severity of congenital Zika syndrome. CONCLUSION: Establishing the feasibility of immune correlates and/or surrogates are a priority. Given the challenges in conducting phase 3 trials at a time of waning incidence, human challenge trials should be considered to evaluate efficacy. Continued financial support and engagement of industry partners will be essential to the successful development, licensure, and accessibility of Zika vaccines or therapeutics.
\n \n\n \n \nTwo phase 3 placebo-controlled trials of the CYD-TDV vaccine, evaluated in children aged 2-14 y (CYD14) and 9-16 y (CYD15), demonstrated vaccine efficacy (VE) of 56.5% and 60.8%, respectively, against symptomatic virologically confirmed dengue (VCD). Sieve analyses were conducted to evaluate whether and how VE varied with amino acid sequence features of dengue viruses (DENVs). DENV premembrane/envelope amino acid sequences from VCD endpoint cases were aligned with the vaccine insert sequences, and extensions of the proportional hazards model were applied to assess variation in VE with amino acid mismatch proportion distances from vaccine strains, individual amino acid residues, and phylogenetic genotypes. In CYD14, VE against VCD of any serotype (DENV-Any) decreased significantly with increasing amino acid distance from the vaccine, whereas in CYD15, VE against DENV-Any was distance-invariant. Restricting to the common age range and amino acid distance range between the trials and accounting for differential VE by serotype, however, showed no evidence of VE variation with distance in either trial. In serotype-specific analyses, VE against DENV4 decreased significantly with increasing amino acid distance from the DENV4 vaccine insert and was significantly greater against residue-matched DENV4 at eight signature positions. These effects were restricted to 2- to 8-y-olds, potentially because greater seropositivity of older children at baseline might facilitate a broader protective immune response. The relevance of an antigenic match between vaccine strains and circulating DENVs was also supported by greater estimated VE against serotypes and genotypes for which the circulating DENVs had shorter amino acid sequence distances from the vaccine.
\n \n\n \n \nSalmonella spp. are regarded as facultative intracellular bacterial pathogens which are found inside macrophages (M\u03a6) after i.v. infection. It is generally assumed that M\u03a6 restrict the replication of the bacteria during infection. In this study we examined the in vivo activities of M\u03a6 during experimental S. typhimurium infections, using a selective liposome-based M\u03a6, elimination technique. Unexpectedly, elimination of M\u03a6 prior to infection with virulent S. typhimurium decreased morbidity and mortality, suggesting that M\u03a6 mediate the pathology caused by S. typhimurium. Removal of M\u03a6 during vaccination with attenuated S. typhimurium did not affect protection against challenge with virulent S. typhimurium, suggesting that M\u03a6 are not required for the induction of protective immunity and that other cells must function as antigen-presenting cell to elicit T cell-mediated protection. However, M\u03a6 appeared to be important effecters of protection against challenge infection since elimination of M\u03a6 from vaccinated mice prior to challenge infection with virulent S. typhimurium significantly decreased protection. These results enhance our understanding of the control of S. typhimurium growth in vivo, and moreover suggest that M\u03a6 play a major role in the pathology of virulent S. typhimurium infections. As such, these cells may present a novel target for therapeutic intervention.
\n \n\n \n \nCorynebacterium pseudotuberculosis is an intracellular bacterial pathogen causing a chronic abscessing disease in sheep and goats called caseous lymphadenitis. We are developing this bacterial species as a live vector system to deliver vaccine antigens to the animal immune system. Foreign genes expressed in bacterial hosts can be unstable so we undertook to delete the C. pseudotuberculosis chromosomal recA gene to determine whether a recA- background would reduce the frequency of recombination in cloned DNA. Homologous DNA recombination within an isogenic recA- C. pseudotuberculosis was 10-12-fold lower than that in the recA+ parental strain. Importantly, the recA mutation had no detectable affect upon the virulence of C. pseudotuberculosis in a mouse model. Taken together these results suggest that a recA- background may be useful in the further development of C. pseudotuberculosis as a vaccine vector.
\n \n\n \n \n\u00a9 1998, Faculty of Medicine, Universitas Indonesia. All rights reserved. The successfull testing and development of a new live, attenuated mutant (s) of S. typhi as a typhoid vaccine creates an opportunity for developing novel bivalent or multivalent human vaccines. Development of such vaccines will require careful analysis of optimal expression system (gene dosage, promoter strength,\u2026) to ensure that the recombinant S. typhi elicit a maximal response against the co-expressed heterologous antigen. We have modelled the construction of bivalent vaccines in S. typhimurium using the murine model of typhoid fever. The model antigen used in these studies, the C fragment of tetanus toxin (TT), is highly immunogenic in mice when expressed as a recombinant protein from live, attenuated S. typhimurium. Parameters including background mutation, plasmid copy numbers and stability, promoter strength/regulation and translation effects were examined in this study. Isogenic pur A, aroA, aroA/aroD, htrA and ompR mutants of S. typhimurium SL1344 were compared with the cya/crp mutant of SR-11 for in vivo growth and penetration into central lymphoid organs, and immunisation against C fragment. Protective levels of TT-specific antibody were elicited by all recombinant Salmonellae except the purA mutant. Immunogenicity did not correlate with penetration into the spleen or specific splenic T cell responses, but did correlate with Peyer\u2019s patch colonisation. Maximal immunogenicity of plasmid constructs of C fragment correlated with plasmid stability, and low copy, but stable replicons (eg.pRSF 10 JO) were effective in vaccine delivery. C fragment expressed from Col EI plasmids pBR322 and its derivative pAT153, induced very high levels of TT-specific antibody whereas the high copy derivative pIC20H did not elicit TT- specific immune response and was highly unstable in vivo. In vivo-regulated promoters (ppagC, pnirB andpkatG) were compared with the constitutive promoter ptrc for in vivo expression of the reporter antigen firefly luciferase, and in vivo immunogenicity of C fragment constructs. The strongest in vivo promoter (ptrc) was less effective at immunisation than the in vivo regulated promoter ppagC. Information from these studies will be used to optimally express other heterologous antigens from recombinant S. typhimurium mutants, with the ultimate aim of developing novel bivalent S. typhi vaccines.
\n \n\n \n \n\u00a9 1998, Faculty of Medicine, Universitas Indonesia. All rights reserved. Salmonella enterica var. Typhi and Typhimurium cause similar systemic disease in humans and mice respectively. In mice, resistance to S. Typhimurium is largely controlled by a genetic locus which encodes NRAMP-1 (a protein found within the phagosomal compartment of macrophages. The two phenotypic alleles of NRAMP-I). Itys and ltyR, cause mice to vary in sentivity to S. Typhimurium by 10,000-fold. It is not clear whether there are similar structural alleles in human NRAMP-1, but variability in human NRAMP-I transcription has been reported. The immunology of murine typhoid was, until recently, thought to closely parallel that of human typhoid fever hut recent discrepancies in the behaviour of defined S. Typhimurium and S. Typhi mutants in mice and humans respectively, suggest that there may be some, important differences in the host-pathogen relationships. Our interests have been in defining the key properties of S. Typhimurium as a vaccine vector such that similar properties could be engineered into attenuated S. Typhi. We have studied the expression of C fragment, the protective but non-toxic portion of tetanus toxin, and examined the capacity of different S. Typhimurium mutations, replicons and promoters to deliver protective responses. Mutants were analysed for penetration into central organs eg. liver and spleen, after oral administration, and for their capacity to elicit Salmonellae and tetanus toxoid-specific T and B cell responses. There was a good correlation behveen entry into, and persistence within, the Peyer\u2019s patches and antibody levels, hut no correlation between antibody responses and splenic invasion. There was a positive correlation between splenic T cell responses and splenic invasion. Studies into the role of macrophages and the induction of protection against Salmonellae challenge were also conducted in mice, as part of a larger program to define the cells responsible for inducing and effecting immunity in murine typhoid. Mice were depleted of macrophages prior to immunisation and/or prior to challenge using the Cl2MDP-macrophage depletion techique. Mice depleted of macrophages prior to and during immunisation with S. Typhimurium aroA/aroD (ie. BRD509) expressing C fragment were highly protected against challenge with S. Typhimurim SL 1344; this protection was reduced when macrophages were removed prior to challenge. Macrophage depletion did not reduce antibody responses against tetanus toxoid but, paradoxically, macrophage depletion increased T cell responses specific for C fragment. This results suggest that macrophages are not the primary antigen presenting cell (APC) involved in immunity elicited by BRD509. However, macrophages appear to be important effectors in protection againts virulent S Typhimurium challenge infection. The results from these studies indicate that Salmonella entry into macrophages is not essential for homologous or heterologous responses elicited by mutants of S. Typhimurium and that much of the inductive processes with respect to immunity against virulent Salmonellae, and the heterologous antigens they are engineered to express, may occur in the Peyer\u2019s patch. Clearly, the relation between Salmonellae and host cells within the Peyer\u2019s patch should be explored further in the murine model and attempts should be made to define the S. Typhi-macrophage relationship in humans.
\n \n\n \n \nTransgenic animals may have advantages over existing biopharmaceutical production methods with respect to the quantity and quality of the source material, which may subsequently reduce production costs and simplify downstream processing. In addition, the attractions of transgenic animals as a means of production include the ability to produce adequate quantities of materials required at a scale of production which may otherwise be prohibitively expensive because of the significant amounts needed for therapy. Indeed, high level expression of complex molecules in an appropriately modified form has already been achieved for a number of clinically relevant proteins. Notwithstanding these successes, the lack of livestock embryonic stem cells means that the generation of transgenic animals is currently relatively expensive and imprecise. Indeed, the isolation and characterization of these cells for livestock species would represent a significant step forward for the technology. Finally, in terms of production of novel or improved human therapeutic products by the biopharmaceutical industry, a choice must now be made between mammalian cell culture, with its proven track record of producing safe and effective products, and transgenic animal technology. Transgenic animal technology may be superior in production yields and costs, but in Australia at least, has yet to clear regulatory hurdles and has disadvantages in terms of time required for product development.
\n \n\n \n \nInitiation of attaching-effacing lesions, which characterize infections with rabbit enteropathogenic Escherichia coli (REPEC), requires bacteria to adhere to the intestinal epithelium. This adherence is reflected in vitro by the affinity of these E. coli strains for various types of eukaryotic cells. TnphoA mutants of REPEC 83/39 (O15:H-) which had lost the ability to adhere to HEp-2 epithelial cells, guinea pig ileal brush borders, and mouse erythrocytes were generated. DNA sequencing of the region surrounding the inactivating transposon insertions within a 95-kb plasmid, designated pRAP for REPEC adherence plasmid, revealed extensive homology between that region and the structural genes of enterotoxigenic E. coli operons encoding the K88 and CS31A fimbrial adhesins and the genes for the afr2 adhesin from REPEC B10 (O103:H2). Seven genes of the ral operon (for REPEC adherence locus), including three putative minor fimbrial subunit genes (ralC, ralF, and ralH), a major fimbrial subunit gene (ralG), a gene of unknown function (ralI), and genes for two fimbrial subunit chaperones (ralD and ralE), were sequenced. When inoculated perorally into weanling rabbits, a mutant with a TnphoA insertion in the ralE gene showed a 10-fold reduction in colonizing ability, with only 1 of 10 rabbits excreting bacteria compared to all 5 of those infected with the wild-type parent strain (P = 0.002). The severity of the diarrheal illness caused by the mutant strain was also reduced. Western blotting of surface protein extracts of strain 83/39 with hyperimmune anti-83/39 antiserum, adsorbed with the ralE mutant, revealed a 32-kDa protein which was absent from protein extracts of two nonadherent mutants. The adsorbed antiserum also bound to the surface of strain 83/39 but not to nonadherent mutants, as detected by immunogold labeling. These results indicate that the ral operon of REPEC 83/39 contains genes necessary for the biosynthesis of fine fimbriae which are responsible for in vitro adherence of the bacteria and play a role in their colonization of, and hence virulence for, rabbits. The putative major fimbrial subunit is a protein with an observed molecular size of approximately 32 kDa which, when assembled, appears to form a capsule of fimbriae surrounding the bacterium similar to that described for CS31A.
\n \n\n \n \nCorynebacterium pseudotuberculosis, a gram-positive intracellular bacterial pathogen, is the etiological agent of the disease caseous lymphadenitis (CLA) in both sheep and goats. Attenuated mutants of C. pseudotuberculosis have the potential to act as novel live veterinary vaccine vectors. We have cloned and sequenced the aroB and aroQ genes from C. pseudotuberculosis C231. By allelic exchange, aroQ mutants of both C231, designated CS100, and a pld mutant strain TB521, designated CS200, were constructed. Infection of BALB/c mice indicated that introduction of the aroQ mutation into C231 and TB521 attenuated both strains. In sublethally infected BALB/c mice, both CS100 and CS200 were cleared from spleens and livers by day 8 postinfection. The in vivo persistence of these strains was increased when the intact aroQ gene was supplied on a plasmid in trans. Mice infected with TB521 harbored bacteria in organs at least till day 8 postinfection without ill effect. When used as a vaccine, only the maximum tolerated dose of CS100 had the capacity to protect mice from homologous challenge. Vaccination with TB521 also elicited protective immunity, and this was associated with gamma interferon (IFN-gamma) production from splenocytes stimulated 7 days postvaccination. The role of IFN-gamma in controlling primary infections with C. pseudotuberculosis was examined in mice deficient for the IFN-gamma receptor (IFN-gammaR(-/-) mice). IFN-gammaR(-/-) mice cleared an infection with CS100 but were significantly more susceptible than control littermates to infection with C231 or TB521. These studies support an important role for IFN-gamma in control of primary C. pseudotuberculosis infections and indicate that aroQ mutants remain attenuated even in immunocompromised animals. This is the first report of an aroQ mutant of a bacterial pathogen, and the results may have implications for the construction of aromatic mutants of Mycobacterium tuberculosis for use as vaccines.
\n \n\n \n \nDNA vaccines are an exciting development in vaccine technology which may have a special role in preventing viral infections and as 'theracines' for cancer. Their use in preventing bacterial infections has, by comparison, been less well documented. While it is unlikely that traditional, highly successful and cheap vaccines for diseases such as diphtheria will be replaced by DNA vaccines, naked DNA may be particularly appropriate for preventing bacterial infections where cytotoxic T cells confer protection, or where a Th1 type T cell response mediates resistance. For example, DNA vaccines containing different mycobacterial antigens have been shown to inhibit overt infections by Mycobacterium tuberculosis in rodent models. The use of DNA vaccines in bacterial infections may be complicated by fundamental differences between prokaryotic and eukaryotic genes and gene products, including mRNA stability, codon bias, secondary structures surrounding native start sequences and glycosylation. These problems can be solved by re-synthesis of bacterial genes to produce 'new' sequences which are more highly expressed by eukaryotic cells.
\n \n\n \n \nSalmonella spp. are regarded as facultative intracellular bacterial pathogens which are found inside macrophages (Mphi) after i. v. infection. It is generally assumed that Mphi restrict the replication of the bacteria during infection. In this study we examined the in vivo activities of Mphi during experimental S. typhimurium infections, using a selective liposome-based Mphi elimination technique. Unexpectedly, elimination of Mphi prior to infection with virulent S. typhimurium decreased morbidity and mortality, suggesting that Mphi mediate the pathology caused by S. typhimurium. Removal of Mphi) during vaccination with attenuated S. typhimurium did not affect protection against challenge with virulent S. typhimurium, suggesting that Mphi are not required for the induction of protective immunity and that other cells must function as antigen-presenting cell to elicit T cell-mediated protection. However, Mphi appeared to be important effectors of protection against challenge infection since elimination of Mphi from vaccinated mice prior to challenge infection with virulent S. typhimurium significantly decreased protection. These results enhance our understanding of the control of S. typhimurium growth in vivo, and moreover suggest that Mphi play a major role in the pathology of virulent S. typhimurium infections. As such, these cells may present a novel target for therapeutic intervention.
\n \n\n \n \nWe compared the abilities of different Salmonella enterica var. Typhimurium (S. typhimurium) strains harboring mutations in the genes aroA, aroAD, purA, ompR, htrA, and cya crp to present the heterologous antigen, C fragment of tetanus toxin, to the mouse immune system. Plasmid pTETtac4, encoding C fragment, was transferred into the various S. typhimurium mutants, and the levels of antigen expression were found to be equivalent. After primary oral immunization of BALB/c mice, all attenuated strains were capable of penetrating the gut epithelium and colonizing the Peyer's patches and spleens of mice. Of all strains compared, the delta purA mutant colonized and persisted in the Peyer's patches at the lowest level, whereas the delta htrA mutant colonized and persisted in the spleen at the lowest level. The level of specific antibody elicited by the different strains against either S. typhimurium lipopolysaccharide or tetanus toxoid was strain dependent and did not directly correlate to the mutants' ability to colonize the spleen. The level of immunoglobulin G1 (IgG1) and IgG2a antibody specific for tetanus toxoid was determined in mice immunized with four S. typhimurium mutants. The level of antigen-specific IgG1 and IgG2a was significantly lower in animals immunized with S. typhimurium delta purA. Antigen-specific T-cell proliferation assays indicated a degree of variability in the capacity of some strains to elicit T cells to the heterologous antigen. Cytokine profiles (gamma interferon and interleukin-5) revealed that the four S. typhimurium mutants tested induced a Th1-type immune response. Mice were challenged with a lethal dose of tetanus toxin 96 days after oral immunization. With the exception of the S. typhimurium delta purA mutant, all strains elicited a protective immune response. These data indicate that the level of total Ig specific for the carried antigen, C fragment, does not correlate with the relative invasiveness of the vector, but it is determined by the carrier mutation and the background of the S. typhimurium strain.
\n \n\n \n \nCorynebacterium pseudotuberculosis, a gram-positive facultative intracellular bacterial pathogen, is the etiological agent of the economically important disease caseous lymphadenitis (CLA) in both sheep and goats. Attenuated mutants of C. pseudotuberculosis have the potential to act as novel vaccines against CLA and as veterinary vaccine vectors. In this report, we have assessed the virulence of both aroQ and pld mutants of C. pseudotuberculosis in sheep and concurrently their capacity to act as vaccines against homologous challenge. The results suggest that aroQ mutants of C. pseudotuberculosis are attenuated with regard to both lymph node persistence and vaccination site reactogenicity. Immunologically, aroQ mutants failed to elicit detectable specific gamma interferon (IFN-gamma)-secreting lymphocytes and induced low levels of antibodies to C. pseudotuberculosis culture supernatant antigens. Following subcutaneous vaccination, the immune responses induced by aroQ mutants did not protect sheep from infection with the wild-type strain but did appear to reduce the clinical severity of disease resulting from challenge. Conversely, an attenuated C. pseudotuberculosis strain expressing an enzymatically inactive phospholipase D exotoxin, when used as a vaccine, elicited a protective immune response. Protection appeared to correlate with in vivo persistence of the vaccine strain, the induction of IFN-gamma-secreting lymphocytes, and relatively high levels of antibodies to culture supernatant antigens. The results suggest that aroQ mutants of C. pseudotuberculosis may be overly attenuated for use as a CLA vaccines or as vaccine vectors.
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