Prof Cameron P Simmons

Research Area: Global Health
Scientific Themes: Tropical Medicine & Global Health
Keywords: Dengue virus, Immune response, Anti-virals, Pathogenesis and Genetic susceptibility
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Dengue is the most important arboviral disease of humans and an enormous and growing public health problem. There are no licensed vaccines or anti-virals, no good animal models of disease and no validated methods of identifying patients who might develop complications from their illness. Our research programme is designed to better understand the pathogenesis of dengue and develop better methods of identifying patients at risk of severe disease.
Our studies in dengue patients are conducted in collaboration with clinical partners in hospitals throughout VietNam and particularly the Mekong Delta region. Through genomic studies of the virus and host, combined with investigations of the host immune response, we are gaining a better understanding of the factors associated with severe dengue. From this base we also evaluate new diagnostic tests and anti-viral drugs in randomised, controlled trials. Our research incorporates skills in epidemiology, genomics, bioinformatics, clinical trials, immunology and virology.

Name Department Institution Country
Matt Henn Broad Institute United States
Martin Hibberd Genome Institute of Singapore Singapore
Paul Young University of Queensland Australia
Steve Whitehead Laboratory of Infectious Diseases, NIH United States
Alex Matter Novartis Institute for Tropical Diseases Singapore

Lam PK, Tam DT, Diet TV, Tam CT, Tien NT, Kieu NT, Simmons C, Farrar J et al. 2013. Clinical characteristics of Dengue shock syndrome in Vietnamese children: a 10-year prospective study in a single hospital. Clin Infect Dis, 57 (11), pp. 1577-1586. Read abstract | Read more

Dengue shock syndrome (DSS) is a severe manifestation of dengue virus infection that particularly affects children and young adults. Despite its increasing global importance, there are no prospective studies describing the clinical characteristics, management, or outcomes of DSS. Hide abstract

Lam PK, Tam DTH, Diet TV, Tam CT, Tien NTH, Kieu NTT, Simmons C, Farrar J et al. 2013. Dengue shock syndrome in children: clinical features and a prognostic model for profound shock TROPICAL MEDICINE & INTERNATIONAL HEALTH, 18 pp. 53-54.

Wiszniewski W, Hunter JV, Hanchard NA, Willer JR, Shaw C, Tian Q, Illner A, Wang X et al. 2013. TM4SF20 ancestral deletion and susceptibility to a pediatric disorder of early language delay and cerebral white matter hyperintensities American Journal of Human Genetics, 93 (2), pp. 197-210. Read abstract | Read more

White matter hyperintensities (WMHs) of the brain are important markers of aging and small-vessel disease. WMHs are rare in healthy children and, when observed, often occur with comorbid neuroinflammatory or vasculitic processes. Here, we describe a complex 4 kb deletion in 2q36.3 that segregates with early childhood communication disorders and WMH in 15 unrelated families predominantly from Southeast Asia. The premature brain aging phenotype with punctate and multifocal WMHs was observed in ∼70% of young carrier parents who underwent brain MRI. The complex deletion removes the penultimate exon 3 of TM4SF20, a gene encoding a transmembrane protein of unknown function. Minigene analysis showed that the resultant net loss of an exon introduces a premature stop codon, which, in turn, leads to the generation of a stable protein that fails to target to the plasma membrane and accumulates in the cytoplasm. Finally, we report this deletion to be enriched in individuals of Vietnamese Kinh descent, with an allele frequency of about 1%, embedded in an ancestral haplotype. Our data point to a constellation of early language delay and WMH phenotypes, driven by a likely toxic mechanism of TM4SF20 truncation, and highlight the importance of understanding and managing population-specific low-frequency pathogenic alleles. © 2013 The American Society of Human Genetics. Hide abstract

Farrar JJ, Hien TT, Horstick O, Hung NT, Jaenisch T, Junghanns T, Kroeger A, Laksono IS et al. 2013. Dogma in classifying dengue disease. Am J Trop Med Hyg, 89 (2), pp. 198-201. | Read more

Cuong HQ, Vu NT, Cazelles B, Boni MF, Thai KT, Rabaa MA, Quang LC, Simmons CP, Huu TN, Anders KL. 2013. Spatiotemporal dynamics of dengue epidemics, southern Vietnam. Emerg Infect Dis, 19 (6), pp. 945-953. Read abstract | Read more

An improved understanding of heterogeneities in dengue virus transmission might provide insights into biological and ecologic drivers and facilitate predictions of the magnitude, timing, and location of future dengue epidemics. To investigate dengue dynamics in urban Ho Chi Minh City and neighboring rural provinces in Vietnam, we analyzed a 10-year monthly time series of dengue surveillance data from southern Vietnam. The per capita incidence of dengue was lower in Ho Chi Minh City than in most rural provinces; annual epidemics occurred 1-3 months later in Ho Chi Minh City than elsewhere. The timing and the magnitude of annual epidemics were significantly more correlated in nearby districts than in remote districts, suggesting that local biological and ecologic drivers operate at a scale of 50-100 km. Dengue incidence during the dry season accounted for 63% of variability in epidemic magnitude. These findings can aid the targeting of vector-control interventions and the planning for dengue vaccine implementation. Hide abstract

Nguyet MN, Duong TH, Trung VT, Nguyen TH, Tran CN, Long VT, Dui LET, Nguyen HL et al. 2013. Host and viral features of human dengue cases shape the population of infected and infectious Aedes aegypti mosquitoes. Proc Natl Acad Sci U S A, 110 (22), pp. 9072-9077. Read abstract | Read more

Dengue is the most prevalent arboviral disease of humans. The host and virus variables associated with dengue virus (DENV) transmission from symptomatic dengue cases (n = 208) to Aedes aegypti mosquitoes during 407 independent exposure events was defined. The 50% mosquito infectious dose for each of DENV-1-4 ranged from 6.29 to 7.52 log10 RNA copies/mL of plasma. Increasing day of illness, declining viremia, and rising antibody titers were independently associated with reduced risk of DENV transmission. High early DENV plasma viremia levels in patients were a marker of the duration of human infectiousness, and blood meals containing high concentrations of DENV were positively associated with the prevalence of infectious mosquitoes 14 d after blood feeding. Ambulatory dengue cases had lower viremia levels compared with hospitalized dengue cases but nonetheless at levels predicted to be infectious to mosquitoes. These data define serotype-specific viremia levels that vaccines or drugs must inhibit to prevent DENV transmission. Hide abstract

Boni MF, Chau NV, Dong N, Todd S, Nhat NT, de Bruin E, van Beek J, Hien NT, Simmons CP, Farrar J, Koopmans M. 2013. Population-level antibody estimates to novel influenza A/H7N9. J Infect Dis, 208 (4), pp. 554-558. Read abstract | Read more

There are no contemporary data available describing human immunity to novel influenza A/H7N9. Using 1723 prospectively collected serum samples in southern Vietnam, we tested for antibodies to 5 avian influenza virus antigens, using a protein microarray. General-population antibody titers against subtype H7 virus are higher than antibody titers against subtype H5 and lower than titers against H9. The highest titers were observed for human influenza virus subtypes. Titers to avian influenza virus antigens increased with age and with geometric mean antibody titer to human influenza virus antigens. There were no titer differences between the urban and the rural location in our study. Hide abstract

Bhatt S, Gething PW, Brady OJ, Messina JP, Farlow AW, Moyes CL, Drake JM, Brownstein JS et al. 2013. The global distribution and burden of dengue Nature, 496 (7446), pp. 504-507. Read abstract | Read more

Dengue is a systemic viral infection transmitted between humans by Aedes mosquitoes. For some patients, dengue is a life-threatening illness. There are currently no licensed vaccines or specific therapeutics, and substantial vector control efforts have not stopped its rapid emergence and global spread. The contemporary worldwide distribution of the risk of dengue virus infection and its public health burden are poorly known. Here we undertake an exhaustive assembly of known records of dengue occurrence worldwide, and use a formal modelling framework to map the global distribution of dengue risk. We then pair the resulting risk map with detailed longitudinal information from dengue cohort studies and population surfaces to infer the public health burden of dengue in 2010. We predict dengue to be ubiquitous throughout the tropics, with local spatial variations in risk influenced strongly by rainfall, temperature and the degree of urbanization. Using cartographic approaches, we estimate there to be 390 million (95% credible interval 284-528) dengue infections per year, of which 96 million (67-136) manifest apparently (any level of disease severity). This infection total is more than three times the dengue burden estimate of the World Health Organization. Stratification of our estimates by country allows comparison with national dengue reporting, after taking into account the probability of an apparent infection being formally reported. The most notable differences are discussed. These new risk maps and infection estimates provide novel insights into the global, regional and national public health burden imposed by dengue. We anticipate that they will provide a starting point for a wider discussion about the global impact of this disease and will help to guide improvements in disease control strategies using vaccine, drug and vector control methods, and in their economic evaluation. © 2013 Macmillan Publishers Limited. All rights reserved. Hide abstract

Bhatt S, Gething PW, Brady OJ, Messina JP, Farlow AW, Moyes CL, Drake JM, Brownstein JS et al. 2013. The global distribution and burden of dengue. Nature, 496 (7446), pp. 504-507. Read abstract | Read more

Dengue is a systemic viral infection transmitted between humans by Aedes mosquitoes. For some patients, dengue is a life-threatening illness. There are currently no licensed vaccines or specific therapeutics, and substantial vector control efforts have not stopped its rapid emergence and global spread. The contemporary worldwide distribution of the risk of dengue virus infection and its public health burden are poorly known. Here we undertake an exhaustive assembly of known records of dengue occurrence worldwide, and use a formal modelling framework to map the global distribution of dengue risk. We then pair the resulting risk map with detailed longitudinal information from dengue cohort studies and population surfaces to infer the public health burden of dengue in 2010. We predict dengue to be ubiquitous throughout the tropics, with local spatial variations in risk influenced strongly by rainfall, temperature and the degree of urbanization. Using cartographic approaches, we estimate there to be 390 million (95% credible interval 284-528) dengue infections per year, of which 96 million (67-136) manifest apparently (any level of disease severity). This infection total is more than three times the dengue burden estimate of the World Health Organization. Stratification of our estimates by country allows comparison with national dengue reporting, after taking into account the probability of an apparent infection being formally reported. The most notable differences are discussed. These new risk maps and infection estimates provide novel insights into the global, regional and national public health burden imposed by dengue. We anticipate that they will provide a starting point for a wider discussion about the global impact of this disease and will help to guide improvements in disease control strategies using vaccine, drug and vector control methods, and in their economic evaluation. Hide abstract

Duong V, Henn MR, Simmons C, Ngan C, Y B, Gavotte L, Viari A, Ong S et al. 2013. Complex dynamic of dengue virus serotypes 2 and 3 in Cambodia following series of climate disasters. Infect Genet Evol, 15 pp. 77-86. Read abstract | Read more

The Dengue National Control Program was established in Cambodia in 2000 and has reported between 10,000 and 40,000 dengue cases per year with a case fatality rate ranging from 0.7 to 1.7. In this study 39 DENV-2 and 57 DENV-3 viruses isolated from patients between 2000 and 2008 were fully sequenced. Five DENV2 and four DENV3 distinct lineages with different dynamics were identified. Each lineage was characterized by the presence of specific mutations with no evidence of recombination. In both DENV-2 and DENV-3 the lineages present prior to 2003 were replaced after that date by unrelated lineages. After 2003, DENV-2 lineages D2-3 and D2-4 cocirculated until 2007 when they were almost completely replaced by a lineage D2-5 which emerged from D2-3 Conversely, all DENV-3 lineages remained, diversified and cocirculated with novel lineages emerging. Years 2006 and 2007 were marked by a high prevalence of DENV-3 and 2007 with a large dengue outbreak and a high proportion of patients with severe disease. Selective sweeps in DENV-1 and DENV-2 were linked to immunological escape to a predominately DENV-3-driven immunological response. The complex dynamic of dengue in Cambodia in the last ten years has been associated with a combination of stochastic climatic events, cocirculation, coevolution, adaptation to different vector populations, and with the human population immunological landscape. Hide abstract

Duong V, Simmons C, Gavotte L, Viari A, Ong S, Chantha N, Lennon NJ, Birren BW et al. 2013. Genetic diversity and lineage dynamic of dengue virus serotype 1 (DENV-1) in Cambodia Infection, Genetics and Evolution, 15 pp. 59-68. Read abstract | Read more

In Cambodia, dengue virus (DENV) was first isolated in 1963 and has become endemic with peak epidemic during raining season. Since 2000, the Dengue National Control Program has reported from 10,000 to 40,000 cases per year with fatality rates ranging from 0.7 to 1.7. All four dengue serotypes are found circulating in Cambodia with alternative predominance of serotypes DENV-2 and DENV-3. The DENV-1 represents from 5% to 20% of all circulating viruses, depending upon the year. In this work, 79 clinical strains of DENV-1 were isolated between 2000 and 2009 and their genome fully sequenced. Four distinct lineages with different dynamics were identified. The main evolutionary drive was negative selective pressure but each lineage was characterized by the presence of specific mutations acquired through evolution. Coexistence, extinction and replacement of lineages occurred over the 10-year period. Lineages 1, 2 and 3 were all detected since 2000-2002 and disappeared in 2003, 2004-2005 and 2007, respectively. Lineages 1 and 2 displayed different dynamics. Lineage 1 was very diverse whereas lineage 2 was very homogeneous. Lineage 4 which derived from lineage 3 in 2003 remained the only one at the end of the sampling period in 2008-2009 owing to a selective sweep. The lineages dynamic of DENV-1 viruses and consequences for molecular epidemiology are discussed. © 2011 Elsevier B.V. Hide abstract

Tam DT, Simmons CP, Wills BA. 2013. Reply to Halstead and Sayce et al. Clin Infect Dis, 56 (6), pp. 903-904. | Read more

Wiszniewski W, Hunter JV, Hanchard NA, Willer JR, Shaw C, Tian Q, Illner A, Wang X et al. 2013. TM4SF20 ancestral deletion and susceptibility to a pediatric disorder of early language delay and cerebral white matter hyperintensities. Am J Hum Genet, 93 (2), pp. 197-210. Read abstract | Read more

White matter hyperintensities (WMHs) of the brain are important markers of aging and small-vessel disease. WMHs are rare in healthy children and, when observed, often occur with comorbid neuroinflammatory or vasculitic processes. Here, we describe a complex 4 kb deletion in 2q36.3 that segregates with early childhood communication disorders and WMH in 15 unrelated families predominantly from Southeast Asia. The premature brain aging phenotype with punctate and multifocal WMHs was observed in ~70% of young carrier parents who underwent brain MRI. The complex deletion removes the penultimate exon 3 of TM4SF20, a gene encoding a transmembrane protein of unknown function. Minigene analysis showed that the resultant net loss of an exon introduces a premature stop codon, which, in turn, leads to the generation of a stable protein that fails to target to the plasma membrane and accumulates in the cytoplasm. Finally, we report this deletion to be enriched in individuals of Vietnamese Kinh descent, with an allele frequency of about 1%, embedded in an ancestral haplotype. Our data point to a constellation of early language delay and WMH phenotypes, driven by a likely toxic mechanism of TM4SF20 truncation, and highlight the importance of understanding and managing population-specific low-frequency pathogenic alleles. Hide abstract

Whitehorn J, Thi LV, Dui LET, Simmons CP. 2013. Aedes aegypti (L.) survival after exposure to ivermectin. Southeast Asian J Trop Med Public Health, 44 (2), pp. 179-181. Read abstract

Ivermectin has been shown in in vitro studies to have insecticidal properties against Aedes aegypti adults. This study aimed to assess these properties in vivo. Aedes aegypti survival was not affected by acquiring a blood meal from humans both 5 hours and 24 hours after ingestion of a typical dose of ivermectin. Hide abstract

Tam DTH, Simmons CP, Wills BA. 2013. Reply to Halstead and Sayce et al Clinical Infectious Diseases, 56 (6), pp. 903-904. | Read more

Whitehorn J, Chau TN, Nguyet NM, Kien DT, Quyen NT, Trung DT, Pang J, Wills B et al. 2013. Genetic variants of MICB and PLCE1 and associations with non-severe dengue. PLoS One, 8 (3), pp. e59067. Read abstract | Read more

A recent genome-wide association study (GWAS) identified susceptibility loci for dengue shock syndrome (DSS) at MICB rs3132468 and PLCE1 rs3740360. The aim of this study was to define the extent to which MICB (rs3132468) and PLCE1 (rs3740360) were associated with less severe clinical phenotypes of pediatric and adult dengue. Hide abstract

Dung TTN, Phat VV, Nga TVT, My PVT, Duy PT, Campbell JI, Thuy CT, Hoang NVM et al. 2013. The validation and utility of a quantitative one-step multiplex RT real-time PCR targeting Rotavirus A and Norovirus Journal of Virological Methods, 187 (1), pp. 138-143. Read abstract | Read more

Rotavirus (RoV) and Norovirus (NoV) are the main causes of viral gastroenteritis. Currently, there is no validated multiplex real-time PCR that can detect and quantify RoV and NoV simultaneously. The aim of the study was to develop, validate, and internally control a multiplex one-step RT real-time PCR to detect and quantify RoV and NoV in stool samples. PCR sensitivity was assessed by comparing amplification against the current gold standard, enzyme immunoassay (EIA), on stool samples from 94 individuals with diarrhea and 94 individuals without diarrhea. PCR detected 10% more RoV positive samples than EIA in stools samples from patients with diarrhea. PCR detected 23% more NoV genogroup II positive samples from individuals with diarrhea and 9% more from individuals without diarrhea than EIA, respectively. Genotyping of the PCR positive/EIA negative samples suggested the higher rate of PCR positivity, in comparison to EIA, was due to increased sensitivity, rather than nonspecific hybridization. Quantitation demonstrated that the viral loads of RoV and NoV in the stools of diarrheal patients were an order of magnitude greater than in individuals without diarrhea. This internally controlled real-time PCR method is robust, exhibits a high degree of reproducibility, and may have a greater utility and sensitivity than commercial EIA kits. © 2012 Elsevier B.V. Hide abstract

Hanh Tien NT, Lam PK, Duyen HT, Ngoc TV, Ha PT, Kieu NT, Simmons C, Wolbers M, Wills B. 2013. Assessment of microalbuminuria for early diagnosis and risk prediction in dengue infections. PLoS One, 8 (1), pp. e54538. Read abstract | Read more

Dengue is the most important arboviral infection of humans. Following an initial febrile period, a small proportion of infected patients develop a vasculopathy, with children at particular risk for severe vascular leakage and shock. Differentiation between dengue and other common childhood illnesses is difficult during the early febrile phase, and risk prediction for development of shock is poor. The presence of microalbuminuria is recognized as a useful early predictor for subsequent complications in a number of other disorders with vascular involvement. Significant proteinuria occurs in association with dengue shock syndrome and it is possible that early-phase microalbuminuria may be helpful both for diagnosis of dengue and for identification of patients likely to develop severe disease. Hide abstract

Wolbers M, Kleinschmidt I, Simmons CP, Donnelly CA. 2012. Considerations in the design of clinical trials to test novel entomological approaches to dengue control. PLoS Negl Trop Dis, 6 (11), pp. e1937. | Read more

Dung TT, Phat VV, Nga TV, My PV, Duy PT, Campbell JI, Thuy CT, Hoang NV et al. 2013. The validation and utility of a quantitative one-step multiplex RT real-time PCR targeting rotavirus A and norovirus. J Virol Methods, 187 (1), pp. 138-143. Read abstract | Read more

Rotavirus (RoV) and Norovirus (NoV) are the main causes of viral gastroenteritis. Currently, there is no validated multiplex real-time PCR that can detect and quantify RoV and NoV simultaneously. The aim of the study was to develop, validate, and internally control a multiplex one-step RT real-time PCR to detect and quantify RoV and NoV in stool samples. PCR sensitivity was assessed by comparing amplification against the current gold standard, enzyme immunoassay (EIA), on stool samples from 94 individuals with diarrhea and 94 individuals without diarrhea. PCR detected 10% more RoV positive samples than EIA in stools samples from patients with diarrhea. PCR detected 23% more NoV genogroup II positive samples from individuals with diarrhea and 9% more from individuals without diarrhea than EIA, respectively. Genotyping of the PCR positive/EIA negative samples suggested the higher rate of PCR positivity, in comparison to EIA, was due to increased sensitivity, rather than nonspecific hybridization. Quantitation demonstrated that the viral loads of RoV and NoV in the stools of diarrheal patients were an order of magnitude greater than in individuals without diarrhea. This internally controlled real-time PCR method is robust, exhibits a high degree of reproducibility, and may have a greater utility and sensitivity than commercial EIA kits. Hide abstract

Simmons CP, Wolbers M, Nguyen MN, Whitehorn J, Shi PY, Young P, Petric R, Nguyen VV, Farrar J, Wills B. 2012. Therapeutics for dengue: recommendations for design and conduct of early-phase clinical trials. PLoS Negl Trop Dis, 6 (9), pp. e1752. | Read more

Vithana EN, Khor CC, Qiao C, Nongpiur ME, George R, Chen LJ, Do T, Abu-Amero K et al. 2012. Genome-wide association analyses identify three new susceptibility loci for primary angle closure glaucoma. Nat Genet, 44 (10), pp. 1142-1146. Read abstract | Read more

Primary angle closure glaucoma (PACG) is a major cause of blindness worldwide. We conducted a genome-wide association study including 1,854 PACG cases and 9,608 controls across 5 sample collections in Asia. Replication experiments were conducted in 1,917 PACG cases and 8,943 controls collected from a further 6 sample collections. We report significant associations at three new loci: rs11024102 in PLEKHA7 (per-allele odds ratio (OR)=1.22; P=5.33×10(-12)), rs3753841 in COL11A1 (per-allele OR=1.20; P=9.22×10(-10)) and rs1015213 located between PCMTD1 and ST18 on chromosome 8q (per-allele OR=1.50; P=3.29×10(-9)). Our findings, accumulated across these independent worldwide collections, suggest possible mechanisms explaining the pathogenesis of PACG. Hide abstract

Tam DT, Ngoc TV, Tien NT, Kieu NT, Thuy TT, Thanh LT, Tam CT, Truong NT et al. 2012. Effects of short-course oral corticosteroid therapy in early dengue infection in Vietnamese patients: a randomized, placebo-controlled trial. Clin Infect Dis, 55 (9), pp. 1216-1224. Read abstract | Read more

Patients with dengue can experience a variety of serious complications including hypovolemic shock, thrombocytopenia, and bleeding. These problems occur as plasma viremia is resolving and are thought to be immunologically mediated. Early corticosteroid therapy may prevent the development of such complications but could also prolong viral clearance. Hide abstract

Nguyen NM, Tran CN, Phung LK, Duong KT, Huynh HLEA, Farrar J, Nguyen QT, Tran HT et al. 2013. A randomized, double-blind placebo controlled trial of balapiravir, a polymerase inhibitor, in adult dengue patients. J Infect Dis, 207 (9), pp. 1442-1450. Read abstract | Read more

Dengue is the most common arboviral infection of humans. There are currently no specific treatments for dengue. Balapiravir is a prodrug of a nucleoside analogue (called R1479) and an inhibitor of hepatitis C virus replication in vivo. Hide abstract

Chau NVV, Simmons CP, Wills B. 2012. Dengue REPLY NEW ENGLAND JOURNAL OF MEDICINE, 367 (2), pp. 181-181.

Anders KL, Nguyet NM, Quyen NT, Ngoc TV, Tram TV, Gan TT, Tung NT, Dung NT, Chau NV, Wills B, Simmons CP. 2012. An evaluation of dried blood spots and oral swabs as alternative specimens for the diagnosis of dengue and screening for past dengue virus exposure. Am J Trop Med Hyg, 87 (1), pp. 165-170. Read abstract | Read more

Non-invasive specimens for dengue diagnosis may be preferable where venous blood is difficult to collect and/or process, such as community-based or remote settings or when sampling from young children. We evaluated the performance of oral swabs and dried blood spots (DBS), compared with plasma, in diagnosing acute dengue and screening for past dengue virus (DENV) exposure. DENV-specific immunoglobulin (Ig) M, IgG, and NS1 antigen were detected both in oral swabs and DBS from acute patients. Oral swabs were less sensitive (IgM: 68.7%, IgG: 91.9%, NS1: 64.7%), but retained good specificity (100%, 92.3%, 95.8%, respectively) compared with plasma. DBS displayed high sensitivity (IgM: 100%, IgG: 96%, NS1: 100%) and specificity (IgM: 75%, IgG: 93%). DENV RNA was amplified from DBS (sensitivity 95.6%) but not from oral swabs. DENV-IgG (indicative of past flavivirus exposure) were detected with moderate sensitivity (61.1%) but poor specificity (50%) in oral swabs from healthy volunteers. Dried blood spots allow sensitive and specific diagnosis of acute dengue by serological, molecular, and antigen detection methods. Oral swabs may be an adequate alternative where blood cannot be collected. Hide abstract

Dunstan SJ, Rockett KA, Quyen NTN, Teo YY, Thai CQ, Hang NT, Jeffreys A, Clark TG et al. 2012. Variation in human genes encoding adhesion and proinflammatory molecules are associated with severe malaria in the Vietnamese Genes and Immunity, 13 (6), pp. 503-508. Read abstract | Read more

The genetic basis for susceptibility to malaria has been studied widely in African populations but less is known of the contribution of specific genetic variants in Asian populations. We genotyped 67 single-nucleotide polymorphisms (SNPs) in 1030 severe malaria cases and 2840 controls from Vietnam. After data quality control, genotyping data of 956 cases and 2350 controls were analysed for 65 SNPs (3 gender confirmation, 62 positioned in/near 42 malarial candidate genes). A total of 14 SNPs were monomorphic and 2 (rs8078340 and rs33950507) were not in Hardy-Weinberg equilibrium in controls (P<0.01). In all, 7/46 SNPs in 6 genes (ICAM1, IL1A, IL17RC, IL13, LTA and TNF) were associated with severe malaria, with 3/7 SNPs in the TNF/LTA region. Genotype-phenotype correlations between SNPs and clinical parameters revealed that genotypes of rs708567 (IL17RC) correlate with parasitemia (P=0.028, r2=0.0086), with GG homozygotes having the lowest parasite burden. Additionally, rs708567 GG homozygotes had a decreased risk of severe malaria (P=0.007, OR=0.78 (95% CI; 0.65-0.93)) and death (P=0.028, OR=0.58 (95% CI; 0.37-0.93)) than those with AA and AG genotypes. In summary, variants in six genes encoding adhesion and proinflammatory molecules are associated with severe malaria in the Vietnamese. Further replicative studies in independent populations will be necessary to confirm these findings. © 2012 Macmillan Publishers Limited. All rights reserved. Hide abstract

Dunstan SJ, Rockett KA, Quyen NT, Teo YY, Thai CQ, Hang NT, Jeffreys A, Clark TG et al. 2012. Variation in human genes encoding adhesion and proinflammatory molecules are associated with severe malaria in the Vietnamese. Genes Immun, 13 (6), pp. 503-508. Read abstract | Read more

The genetic basis for susceptibility to malaria has been studied widely in African populations but less is known of the contribution of specific genetic variants in Asian populations. We genotyped 67 single-nucleotide polymorphisms (SNPs) in 1030 severe malaria cases and 2840 controls from Vietnam. After data quality control, genotyping data of 956 cases and 2350 controls were analysed for 65 SNPs (3 gender confirmation, 62 positioned in/near 42 malarial candidate genes). A total of 14 SNPs were monomorphic and 2 (rs8078340 and rs33950507) were not in Hardy-Weinberg equilibrium in controls (P<0.01). In all, 7/46 SNPs in 6 genes (ICAM1, IL1A, IL17RC, IL13, LTA and TNF) were associated with severe malaria, with 3/7 SNPs in the TNF/LTA region. Genotype-phenotype correlations between SNPs and clinical parameters revealed that genotypes of rs708567 (IL17RC) correlate with parasitemia (P=0.028, r(2)=0.0086), with GG homozygotes having the lowest parasite burden. Additionally, rs708567 GG homozygotes had a decreased risk of severe malaria (P=0.007, OR=0.78 (95% CI; 0.65-0.93)) and death (P=0.028, OR=0.58 (95% CI; 0.37-0.93)) than those with AA and AG genotypes. In summary, variants in six genes encoding adhesion and proinflammatory molecules are associated with severe malaria in the Vietnamese. Further replicative studies in independent populations will be necessary to confirm these findings. Hide abstract

Yacoub S, Grifiths A, Chau TTH, Simmons C, Wills B, Hien TT, Henein M, Farrar J. 2012. Cardiac function and haemodynamics in Vietnemese patients with different dengue severity grades INTERNATIONAL JOURNAL OF INFECTIOUS DISEASES, 16 pp. E119-E119. | Read more

Trung DT, Thao LETT, Dung NM, Ngoc TV, Hien TT, Chau NV, Wolbers M, Tam DT, Farrar J, Simmons C, Wills B. 2012. Clinical features of dengue in a large Vietnamese cohort: intrinsically lower platelet counts and greater risk for bleeding in adults than children. PLoS Negl Trop Dis, 6 (6), pp. e1679. Read abstract | Read more

As dengue spreads to new geographical regions and the force of infection changes in existing endemic areas, a greater breadth of clinical presentations is being recognised. Clinical experience suggests that adults manifest a pattern of complications different from those observed in children, but few reports have described the age-related spectrum of disease in contemporaneous groups of patients recruited at the same geographical location. Hide abstract

Whitehorn J, Rodriguez Roche R, Guzman MG, Martinez E, Gomez WV, Nainggolan L, Laksono IS, Mishra A et al. 2012. Prophylactic platelets in dengue: survey responses highlight lack of an evidence base. PLoS Negl Trop Dis, 6 (6), pp. e1716. Read abstract | Read more

Dengue is the most important arboviral infection of humans. Thrombocytopenia is frequently observed in the course of infection and haemorrhage may occur in severe disease. The degree of thrombocytopenia correlates with the severity of infection, and may contribute to the risk of haemorrhage. As a result of this prophylactic platelet transfusions are sometimes advocated for the prevention of haemorrhage. There is currently no evidence to support this practice, and platelet transfusions are costly and sometimes harmful. We conducted a global survey to assess the different approaches to the use of platelets in dengue. Respondents were all physicians involved with the treatment of patients with dengue. Respondents were asked that their answers reflected what they would do if they were the treating physician. We received responses from 306 physicians from 20 different countries. The heterogeneity of the responses highlights the variation in clinical practice and lack of an evidence base in this area and underscores the importance of prospective clinical trials to address this key question in the clinical management of patients with dengue. Hide abstract

Akbar NA, Allende I, Balmaseda A, Coelho IC, da Cunha RV, Datta B, Devi SS, Farrar J et al. 2012. Regarding "Dengue--how best to classify it". Clin Infect Dis, 54 (12), pp. 1820-1821. | Read more

Tisoncik JR, Korth MJ, Simmons CP, Farrar J, Martin TR, Katze MG. 2012. Into the eye of the cytokine storm. Microbiol Mol Biol Rev, 76 (1), pp. 16-32. Read abstract | Read more

The cytokine storm has captured the attention of the public and the scientific community alike, and while the general notion of an excessive or uncontrolled release of proinflammatory cytokines is well known, the concept of a cytokine storm and the biological consequences of cytokine overproduction are not clearly defined. Cytokine storms are associated with a wide variety of infectious and noninfectious diseases. The term was popularized largely in the context of avian H5N1 influenza virus infection, bringing the term into popular media. In this review, we focus on the cytokine storm in the context of virus infection, and we highlight how high-throughput genomic methods are revealing the importance of the kinetics of cytokine gene expression and the remarkable degree of redundancy and overlap in cytokine signaling. We also address evidence for and against the role of the cytokine storm in the pathology of clinical and infectious disease and discuss why it has been so difficult to use knowledge of the cytokine storm and immunomodulatory therapies to improve the clinical outcomes for patients with severe acute infections. Hide abstract

Whitehorn J, Van Vinh Chau N, Truong NT, Tai LT, Van Hao N, Hien TT, Wolbers M, Merson L et al. 2012. Lovastatin for adult patients with dengue: protocol for a randomised controlled trial. Trials, 13 (1), pp. 203. Read abstract | Read more

Dengue is the most important vector-borne viral infection of man, with approximately 2 billion people living in areas at risk. Infection results in a range of manifestations from asymptomatic infection through to life-threatening shock and haemorrhage. One of the hallmarks of severe dengue is vascular endothelial disruption. There is currently no specific therapy and clinical management is limited to supportive care. Statins are a class of drug initially developed for lipid lowering. There has been considerable recent interest in their effects beyond lipid lowering. These include anti-inflammatory effects at the endothelium. In addition, it is possible that lovastatin may have an anti-viral effect against dengue. Observational data suggest that the use of statins may improve outcomes for such conditions as sepsis and pneumonia. This paper describes the protocol for a randomised controlled trial investigating a short course of lovastatin therapy in adult patients with dengue. Hide abstract

Vithana EN, Khor C-C, Nongpiur ME, Perera SA, Cheng C-Y, Ho C-L, Wu R-Y, Su DH et al. 2012. Genome-wide association analyses identify three new susceptibility loci for primary angle closure glaucoma Nature Genetics, 44 (10), pp. 1142-1146. Read abstract | Read more

Primary angle closure glaucoma (PACG) is a major cause of blindness worldwide. We conducted a genome-wide association study including 1,854 PACG cases and 9,608 controls across 5 sample collections in Asia. Replication experiments were conducted in 1,917 PACG cases and 8,943 controls collected from a further 6 sample collections. We report significant associations at three new loci: rs11024102 in PLEKHA7 (per-allele odds ratio (OR) = 1.22; P = 5.33 × 10), rs3753841 in COL11A1 (per-allele OR = 1.20; P = 9.22 × 10) and rs1015213 located between PCMTD1 and ST18 on chromosome 8q (per-allele OR = 1.50; P = 3.29 × 10). Our findings, accumulated across these independent worldwide collections, suggest possible mechanisms explaining the pathogenesis of PACG. © 2012 Nature America, Inc. All rights reserved. Hide abstract

Van Vinh Chau N, Simmons CP, Wills B. 2012. The authors reply New England Journal of Medicine, 367 (2), pp. 181-181. | Read more

Simmons CP, Farrar JJ, Chau NVV, Wills B. 2012. CURRENT CONCEPTS Dengue NEW ENGLAND JOURNAL OF MEDICINE, 366 (15), pp. 1423-1432. | Read more

Yacoub S, Griffiths A, Hong Chau TT, Simmons CP, Wills B, Hien TT, Henein M, Farrar J. 2012. Cardiac function in Vietnamese patients with different dengue severity grades Critical Care Medicine, 40 (2), pp. 477-483. Read abstract | Read more

Objective: Dengue continues to cause significant global morbidity and mortality. Severe disease is characterized by cardiovascular compromise from capillary leakage. Cardiac involvement in dengue has also been reported but has not been adequately studied. Setting: Hospital for Tropical Diseases, Ho Chi Minh City, Vietnam. Subjects and Design: Seventy-nine patients aged 8-6 yrs with different dengue severity grades were studied using echocardiography including tissue Doppler imaging. The patients were split into severity grades: dengue, dengue with warning signs, and severe dengue. Changes in cardiac functional parameters and hemodynamic indices were monitored over the hospital stay. Intervention: None. Measurements and Main Results; Patients with severe dengue had worse cardiac function compared with dengue in the form of left ventricular systolic dysfunction with increased left myocardial performance index (0.58 [0.26-0.80] vs. 0.38 [0.22-0.70], p = .006). Septal myocardial systolic velocities were reduced (6.4 [4.8-10] vs. 8.1 [6-13] cm/s, p = .01) as well as right ventricular systolic (11.4 [7.5-17] vs. 13.5 [10-17] cm/s, p = .016) and diastolic velocities (13 [8-23] vs. 17 [12-25] cm/s, p = .0026). In the severe group, these parameters improved from hospital admission to discharge; septal myocardial systolic velocities to 8.8 (7-11) cm/s (p = .002), right ventricular myocardial systolic velocities to 15.0 (11.8-23) cm/s, (p = .003), and diastolic velocity to 21 (11-25) cm/s (p = .002). Patients with cardiac impairment were more likely to have significant pleural effusions. Conclusions: Patients with severe dengue have evidence of systolic and diastolic cardiac impairment with septal and right ventricular wall being predominantly affected. Copyright © 2012 by the Society of Critical Care Medicine and Lippincott Williams & Wilkins. Hide abstract

Thai KT, Henn MR, Zody MC, Tricou V, Nguyet NM, Charlebois P, Lennon NJ, Green L et al. 2012. High-resolution analysis of intrahost genetic diversity in dengue virus serotype 1 infection identifies mixed infections. J Virol, 86 (2), pp. 835-843. Read abstract | Read more

Little is known about the rate at which genetic variation is generated within intrahost populations of dengue virus (DENV) and what implications this diversity has for dengue pathogenesis, disease severity, and host immunity. Previous studies of intrahost DENV variation have used a low frequency of sampling and/or experimental methods that do not fully account for errors generated through amplification and sequencing of viral RNAs. We investigated the extent and pattern of genetic diversity in sequence data in domain III (DIII) of the envelope (E) gene in serial plasma samples (n = 49) taken from 17 patients infected with DENV type 1 (DENV-1), totaling some 8,458 clones. Statistically rigorous approaches were employed to account for artifactual variants resulting from amplification and sequencing, which we suggest have played a major role in previous studies of intrahost genetic variation. Accordingly, nucleotide sequence diversities of viral populations were very low, with conservative estimates of the average levels of genetic diversity ranging from 0 to 0.0013. Despite such sequence conservation, we observed clear evidence for mixed infection, with the presence of multiple phylogenetically distinct lineages present within the same host, while the presence of stop codon mutations in some samples suggests the action of complementation. In contrast to some previous studies we observed no relationship between the extent and pattern of DENV-1 genetic diversity and disease severity, immune status, or level of viremia. Hide abstract

James S, Simmons CP, James AA. 2011. Ecology. Mosquito trials. Science, 334 (6057), pp. 771-772. Read abstract | Read more

Field trials of modified mosquitoes present complex but manageable challenges. Hide abstract

Powell TJ, Fox A, Peng Y, Quynh Mai LET, Lien VT, Hang NL, Wang L, Lee LY et al. 2012. Identification of H5N1-specific T-cell responses in a high-risk cohort in vietnam indicates the existence of potential asymptomatic infections. J Infect Dis, 205 (1), pp. 20-27. Read abstract | Read more

Most reported human H5N1 viral infections have been severe and were detected after hospital admission. A case ascertainment bias may therefore exist, with mild cases or asymptomatic infections going undetected. We sought evidence of mild or asymptomatic H5N1 infection by examining H5N1-specific T-cell and antibody responses in a high-risk cohort in Vietnam. Hide abstract

Midgley CM, Bajwa-Joseph M, Vasanawathana S, Limpitikul W, Wills B, Flanagan A, Waiyaiya E, Tran HB et al. 2011. An In-Depth Analysis of Original Antigenic Sin in Dengue Virus Infection (vol 85, pg 410, 2011) JOURNAL OF VIROLOGY, 85 (22), pp. 12100-12100. | Read more

Khor CC, Chau TN, Pang J, Davila S, Long HT, Ong RT, Dunstan SJ, Wills B et al. 2011. Genome-wide association study identifies susceptibility loci for dengue shock syndrome at MICB and PLCE1. Nat Genet, 43 (11), pp. 1139-1141. Read abstract | Read more

Hypovolemic shock (dengue shock syndrome (DSS)) is the most common life-threatening complication of dengue. We conducted a genome-wide association study of 2,008 pediatric cases treated for DSS and 2,018 controls from Vietnam. Replication of the most significantly associated markers was carried out in an independent Vietnamese sample of 1,737 cases and 2,934 controls. SNPs at two loci showed genome-wide significant association with DSS. We identified a susceptibility locus at MICB (major histocompatibility complex (MHC) class I polypeptide-related sequence B), which was within the broad MHC region on chromosome 6 but outside the class I and class II HLA loci (rs3132468, P(meta) = 4.41 × 10(-11), per-allele odds ratio (OR) = 1.34 (95% confidence interval: 1.23-1.46)). We identified associated variants within PLCE1 (phospholipase C, epsilon 1) on chromosome 10 (rs3765524, P(meta) = 3.08 × 10(-10), per-allele OR = 0.80 (95% confidence interval: 0.75-0.86)). We identify two loci associated with susceptibility to DSS in people with dengue, suggesting possible mechanisms for this severe complication of dengue. Hide abstract

Yacoub S, Griffiths A, Chau TT, Simmons CP, Wills B, Hien TT, Henein M, Farrar J. 2012. Cardiac function in Vietnamese patients with different dengue severity grades. Crit Care Med, 40 (2), pp. 477-483. Read abstract | Read more

Dengue continues to cause significant global morbidity and mortality. Severe disease is characterized by cardiovascular compromise from capillary leakage. Cardiac involvement in dengue has also been reported but has not been adequately studied. Hide abstract

Tricou V, Minh NN, Farrar J, Tran HT, Simmons CP. 2011. Kinetics of viremia and NS1 antigenemia are shaped by immune status and virus serotype in adults with dengue. PLoS Negl Trop Dis, 5 (9), pp. e1309. Read abstract | Read more

Dengue is a major public health problem in tropical and subtropical countries. Exploring the relationships between virological features of infection with patient immune status and outcome may help to identify predictors of disease severity and enable rational therapeutic strategies. Hide abstract

Hue KD, Tuan TV, Thi HT, Bich CT, Anh HH, Wills BA, Simmons CP. 2011. Validation of an internally controlled one-step real-time multiplex RT-PCR assay for the detection and quantitation of dengue virus RNA in plasma. J Virol Methods, 177 (2), pp. 168-173. Read abstract | Read more

Dengue is mosquito-borne virus infection that annually causes ~50 million clinically apparent cases worldwide. An internally controlled one-step real-time multiplex RT-PCR assay was developed for detection and quantitation of DENV RNA in plasma sample by using specific primers and fluorogenic TaqMan probes. All primers and probes targeted sequences near the 3' end of the NS5 gene. The method comprised two multiplex assays and was validated for sensitivity, specificity, linearity, reproducibility and precision. An internal control template was spiked into each clinical specimen to provide quality assurance for each experimental step. The assay allowed for detection of between 0.5 and 3 infectious particles per mL, is rapid and has been operationally characterized in 287 Vietnamese dengue patients from two therapeutic intervention trials at the Hospital for Tropical Diseases, Ho Chi Minh City, Viet Nam. Hide abstract

Whitehorn J, Simmons CP. 2011. The pathogenesis of dengue. Vaccine, 29 (42), pp. 7221-7228. Read abstract | Read more

Dengue is an important cause of childhood and adult morbidity in Asian and Latin American countries and its geographic footprint is growing. The clinical manifestations of dengue are the expression of a constellation of host and viral factors, some acquired, others intrinsic to the individual. The virulence of the virus plus the flavivirus infection history, age, gender and genotype of the host all appear to help shape the severity of infection. Similarly, the characteristics of the innate and acquired host immune response subsequent to infection are also likely determinants of outcome. This review summarises recent developments in the understanding of dengue pathogenesis and their relevance to dengue vaccine development. Hide abstract

Duong V, Simmons C, Gavotte L, Viari A, Ong S, Chantha N, Lennon NJ, Birren BW et al. 2013. Genetic diversity and lineage dynamic of dengue virus serotype 1 (DENV-1) in Cambodia. Infect Genet Evol, 15 pp. 59-68. Read abstract | Read more

In Cambodia, dengue virus (DENV) was first isolated in 1963 and has become endemic with peak epidemic during raining season. Since 2000, the Dengue National Control Program has reported from 10,000 to 40,000 cases per year with fatality rates ranging from 0.7 to 1.7. All four dengue serotypes are found circulating in Cambodia with alternative predominance of serotypes DENV-2 and DENV-3. The DENV-1 represents from 5% to 20% of all circulating viruses, depending upon the year. In this work, 79 clinical strains of DENV-1 were isolated between 2000 and 2009 and their genome fully sequenced. Four distinct lineages with different dynamics were identified. The main evolutionary drive was negative selective pressure but each lineage was characterized by the presence of specific mutations acquired through evolution. Coexistence, extinction and replacement of lineages occurred over the 10-year period. Lineages 1, 2 and 3 were all detected since 2000-2002 and disappeared in 2003, 2004-2005 and 2007, respectively. Lineages 1 and 2 displayed different dynamics. Lineage 1 was very diverse whereas lineage 2 was very homogeneous. Lineage 4 which derived from lineage 3 in 2003 remained the only one at the end of the sampling period in 2008-2009 owing to a selective sweep. The lineages dynamic of DENV-1 viruses and consequences for molecular epidemiology are discussed. Hide abstract

Raghwani J, Rambaut A, Holmes EC, Hang VT, Hien TT, Farrar J, Wills B, Lennon NJ, Birren BW, Henn MR, Simmons CP. 2011. Endemic dengue associated with the co-circulation of multiple viral lineages and localized density-dependent transmission. PLoS Pathog, 7 (6), pp. e1002064. Read abstract | Read more

Dengue is one of the most important infectious diseases of humans and has spread throughout much of the tropical and subtropical world. Despite this widespread dispersal, the determinants of dengue transmission in endemic populations are not well understood, although essential for virus control. To address this issue we performed a phylogeographic analysis of 751 complete genome sequences of dengue 1 virus (DENV-1) sampled from both rural (Dong Thap) and urban (Ho Chi Minh City) populations in southern Viet Nam during the period 2003-2008. We show that DENV-1 in Viet Nam exhibits strong spatial clustering, with likely importation from Cambodia on multiple occasions. Notably, multiple lineages of DENV-1 co-circulated in Ho Chi Minh City. That these lineages emerged at approximately the same time and dispersed over similar spatial regions suggests that they are of broadly equivalent fitness. We also observed an important relationship between the density of the human host population and the dispersion rate of dengue, such that DENV-1 tends to move from urban to rural populations, and that densely populated regions within Ho Chi Minh City act as major transmission foci. Despite these fluid dynamics, the dispersion rates of DENV-1 are relatively low, particularly in Ho Chi Minh City where the virus moves less than an average of 20 km/year. These low rates suggest a major role for mosquito-mediated dispersal, such that DENV-1 does not need to move great distances to infect a new host when there are abundant susceptibles, and imply that control measures should be directed toward the most densely populated urban environments. Hide abstract

Fry SR, Meyer M, Semple MG, Simmons CP, Sekaran SD, Huang JX, McElnea C, Huang CY, Valks A, Young PR, Cooper MA. 2011. The diagnostic sensitivity of dengue rapid test assays is significantly enhanced by using a combined antigen and antibody testing approach. PLoS Negl Trop Dis, 5 (6), pp. e1199. Read abstract | Read more

Serological tests for IgM and IgG are routinely used in clinical laboratories for the rapid diagnosis of dengue and can differentiate between primary and secondary infections. Dengue virus non-structural protein 1 (NS1) has been identified as an early marker for acute dengue, and is typically present between days 1-9 post-onset of illness but following seroconversion it can be difficult to detect in serum. Hide abstract

Török ME, Yen NT, Chau TT, Mai NT, Phu NH, Mai PP, Dung NT, Chau NV et al. 2011. Timing of initiation of antiretroviral therapy in human immunodeficiency virus (HIV)--associated tuberculous meningitis. Clin Infect Dis, 52 (11), pp. 1374-1383. Read abstract | Read more

The optimal time to initiate antiretroviral therapy (ART) in human immunodeficiency virus (HIV)-associated tuberculous meningitis is unknown. Hide abstract

Duyen HT, Ngoc TV, Ha DOT, Hang VT, Kieu NT, Young PR, Farrar JJ, Simmons CP, Wolbers M, Wills BA. 2011. Kinetics of plasma viremia and soluble nonstructural protein 1 concentrations in dengue: differential effects according to serotype and immune status. J Infect Dis, 203 (9), pp. 1292-1300. Read abstract | Read more

We describe the magnitude and kinetics of plasma viremia and nonstructural protein 1 (sNS1) levels in sequential samples from 167 children with acute dengue, enrolled early in a community study in Vietnam. All children recovered fully, and only 5 required hospitalization. Among those with dengue virus type 1 (DENV-1), plasma viremia was significantly greater in primary (49) than secondary (44) infections and took longer to resolve. In primary DENV-2 and 3 infections, viremia was significantly lower than among primary DENV-1 infections. Concentrations of sNS1 were significantly higher for DENV-1 than for DENV-2 after adjusting for viremia, with marked differences in the kinetic profiles between primary and secondary infections. Secondary infection and higher viremia were independent predictors of more severe thrombocytopenia, and higher viremia was associated with a small increase in hemoconcentration. Our findings identify clear serotype and immune-status related effects on the dynamics of dengue viremia and sNS1 responses, together with associations with important clinical parameters. Hide abstract

Khor CC, Pang J, Long HT, Hibberd ML, Davila S, Ong RTH, Chau TNB, Dunstan SJ et al. 2011. Genome-wide association study identifies susceptibility loci for dengue shock syndrome at MICB and PLCE1 Nature Genetics, 43 (11), pp. 1139-1141. Read abstract | Read more

Hypovolemic shock (dengue shock syndrome (DSS)) is the most common life-threatening complication of dengue. We conducted a genome-wide association study of 2,008 pediatric cases treated for DSS and 2,018 controls from Vietnam. Replication of the most significantly associated markers was carried out in an independent Vietnamese sample of 1,737 cases and 2,934 controls. SNPs at two loci showed genome-wide significant association with DSS. We identified a susceptibility locus at MICB (major histocompatibility complex (MHC) class I polypeptide-related sequence B), which was within the broad MHC region on chromosome 6 but outside the class I and class II HLA loci (rs3132468, P = 4.41 × 10 , per-allele odds ratio (OR) = 1.34 (95% confidence interval: 1.23-1.46)). We identified associated variants within PLCE1 (phospholipase C, epsilon 1) on chromosome 10 (rs3765524, P = 3.08 × 10 , per-allele OR = 0.80 (95% confidence interval: 0.75-0.86)). We identify two loci associated with susceptibility to DSS in people with dengue, suggesting possible mechanisms for this severe complication of dengue. © 2011 Nature America, Inc. All rights reserved. Hide abstract

Hue KDT, Tuan TV, Thi HTN, Bich CTN, Anh HHL, Wills BA, Simmons CP. 2011. Validation of an internally controlled one-step real-time multiplex RT-PCR assay for the detection and quantitation of dengue virus RNA in plasma Journal of Virological Methods, 177 (2), pp. 168-173. Read abstract | Read more

Dengue is mosquito-borne virus infection that annually causes ~50 million clinically apparent cases worldwide. An internally controlled one-step real-time multiplex RT-PCR assay was developed for detection and quantitation of DENV RNA in plasma sample by using specific primers and fluorogenic TaqMan probes. All primers and probes targeted sequences near the 3' end of the NS5 gene. The method comprised two multiplex assays and was validated for sensitivity, specificity, linearity, reproducibility and precision. An internal control template was spiked into each clinical specimen to provide quality assurance for each experimental step. The assay allowed for detection of between 0.5 and 3 infectious particles per mL, is rapid and has been operationally characterized in 287 Vietnamese dengue patients from two therapeutic intervention trials at the Hospital for Tropical Diseases, Ho Chi Minh City, Viet Nam. © 2011 Elsevier B.V. Hide abstract

Whitehorn J, Simmons CP. 2011. The pathogenesis of dengue Vaccine, 29 (42), pp. 7221-7228. Read abstract | Read more

Dengue is an important cause of childhood and adult morbidity in Asian and Latin American countries and its geographic footprint is growing. The clinical manifestations of dengue are the expression of a constellation of host and viral factors, some acquired, others intrinsic to the individual. The virulence of the virus plus the flavivirus infection history, age, gender and genotype of the host all appear to help shape the severity of infection. Similarly, the characteristics of the innate and acquired host immune response subsequent to infection are also likely determinants of outcome. This review summarises recent developments in the understanding of dengue pathogenesis and their relevance to dengue vaccine development. © 2011 Elsevier Ltd. Hide abstract

Hue KDT, Tuan TV, Thi HTN, Bich CTN, Anh HHL, Wills BA, Simmons CP. 2011. Validation of an internally controlled one-step real-time multiplex RT-PCR assay for the detection and quantitation of dengue virus RNA in plasma Journal of Virological Methods,

Tran CB, Nguyen HT, Phan HT, Tran NV, Wills B, Farrar J, Santangelo JD, Simmons CP. 2011. The seroprevalence and seroincidence of enterovirus71 infection in infants and children in Ho Chi Minh City, Viet Nam. PLoS One, 6 (7), pp. e21116. Read abstract | Read more

Enterovirus 71 (EV71)-associated hand, foot and mouth disease has emerged as a serious public health problem in South East Asia over the last decade. To better understand the prevalence of EV71 infection, we determined EV71 seroprevalence and seroincidence amongst healthy infants and children in Ho Chi Minh City, Viet Nam. In a cohort of 200 newborns, 55% of cord blood samples contained EV71 neutralizing antibodies and these decayed to undetectable levels by 6 months of age in 98% of infants. The EV71 neutralizing antibody seroconversion rate was 5.6% in the first year and 14% in the second year of life. In children 5-15 yrs of age, seroprevalence of EV71 neutralizing antibodies was 84% and in cord blood it was 55%. Taken together, these data suggest EV71 force of infection is high and highlights the need for more research into its epidemiology and pathogenesis in high disease burden countries. Hide abstract

Fox A, Le NM, Simmons CP, Wolbers M, Wertheim HF, Pham TK, Tran TH, Trinh TM et al. 2011. Immunological and viral determinants of dengue severity in hospitalized adults in Ha Noi, Viet Nam. PLoS Negl Trop Dis, 5 (3), pp. e967. Read abstract | Read more

The relationships between the infecting dengue serotype, primary and secondary infection, viremia and dengue severity remain unclear. This cross-sectional study examined these interactions in adult patients hospitalized with dengue in Ha Noi. Hide abstract

Anders KL, Nguyet NM, Chau NV, Hung NT, Thuy TT, Lien LEB, Farrar J, Wills B, Hien TT, Simmons CP. 2011. Epidemiological factors associated with dengue shock syndrome and mortality in hospitalized dengue patients in Ho Chi Minh City, Vietnam. Am J Trop Med Hyg, 84 (1), pp. 127-134. Read abstract | Read more

Understanding trends in dengue disease burden and risk factors for severe disease can inform health service allocation, clinical management, and planning for vaccines and therapeutics. Dengue admissions at three tertiary hospitals in Ho Chi Minh City, Vietnam, increased between 1996 and 2009, peaking at 22,860 in 2008. Children aged 6-10 years had highest risk of dengue shock syndrome (DSS); however, mortality was highest in younger children and decreased with increasing age (odds ratio [OR] = 0.52, 95% confidence interval [CI] = 0.36-0.75 in 6- to 10- year-old children and OR = 0.27, 95% CI = 0.16-0.44 in 11- to 15-year-old children compared with 1- to 5-year-old children). Males were overrepresented among dengue cases; however, girls had higher risk of DSS (OR = 1.19, 95% CI = 1.14-1.24) and death (OR = 1.57, 95% CI = 1.14-2.17). Young children with dengue had greatest risk of death and should be targeted in dengue vaccine and drug trials. The increased risk of severe outcomes in girls warrants further attention in studies of pathogenesis, health-seeking behavior, and clinical care. Hide abstract

Hoang LT, Lynn DJ, Henn M, Birren BW, Lennon NJ, Le PT, Duong KT, Nguyen TT et al. 2010. The early whole-blood transcriptional signature of dengue virus and features associated with progression to dengue shock syndrome in Vietnamese children and young adults. J Virol, 84 (24), pp. 12982-12994. Read abstract | Read more

Dengue is a pantropic public health problem. In children, dengue shock syndrome (DSS) is the most common life-threatening complication. The ability to predict which patients may develop DSS may improve triage and treatment. To this end, we conducted a nested case-control comparison of the early host transcriptional features in 24 DSS patients and 56 sex-, age-, and virus serotype-matched uncomplicated (UC) dengue patients. In the first instance, we defined the "early dengue" profile. The transcriptional signature in acute rather than convalescent samples (≤72 h post-illness onset) was defined by an overabundance of interferon-inducible transcripts (31% of the 551 overabundant transcripts) and canonical gene ontology terms that included the following: response to virus, immune response, innate immune response, and inflammatory response. Pathway and network analyses identified STAT1, STAT2, STAT3, IRF7, IRF9, IRF1, CEBPB, and SP1 as key transcriptional factors mediating the early response. Strikingly, the only difference in the transcriptional signatures of early DSS and UC dengue cases was the greater abundance of several neutrophil-associated transcripts in patients who progressed to DSS, a finding supported by higher plasma concentrations of several canonical proteins associated with neutrophil degranulation (bactericidal/permeability-increasing protein [BPI], elastase 2 [ELA2], and defensin 1 alpha [DEF1A]). Elevated levels of neutrophil-associated transcripts were independent of the neutrophil count and also of the genotype of the infecting virus, as genome-length sequences of dengue virus serotype 1 (DENV-1) (n = 15) and DENV-2 (n = 3) sampled from DSS patients were phylogenetically indistinguishable from those sampled from uncomplicated dengue patients (32 DENV-1 and 9 DENV-2 sequences). Collectively, these data suggest a hitherto unrecognized association between neutrophil activation, pathogenesis, and the development of DSS and point to future strategies for guiding prognosis. Hide abstract

Midgley CM, Bajwa-Joseph M, Vasanawathana S, Limpitikul W, Wills B, Flanagan A, Waiyaiya E, Tran HB et al. 2011. An in-depth analysis of original antigenic sin in dengue virus infection. J Virol, 85 (1), pp. 410-421. Read abstract | Read more

The evolution of dengue viruses has resulted in four antigenically similar yet distinct serotypes. Infection with one serotype likely elicits lifelong immunity to that serotype, but generally not against the other three. Secondary or sequential infections are common, as multiple viral serotypes frequently cocirculate. Dengue infection, although frequently mild, can lead to dengue hemorrhagic fever (DHF) which can be life threatening. DHF is more common in secondary dengue infections, implying a role for the adaptive immune response in the disease. There is currently much effort toward the design and implementation of a dengue vaccine but these efforts are made more difficult by the challenge of inducing durable neutralizing immunity to all four viruses. Domain 3 of the dengue virus envelope protein (ED3) has been suggested as one such candidate because it contains neutralizing epitopes and it was originally thought that relatively few cross-reactive antibodies are directed to this domain. In this study, we performed a detailed analysis of the anti-ED3 response in a cohort of patients suffering either primary or secondary dengue infections. The results show dramatic evidence of original antigenic sin in secondary infections both in terms of binding and enhancement activity. This has important implications for dengue vaccine design because heterologous boosting is likely to maintain the immunological footprint of the first vaccination. On the basis of these findings, we propose a simple in vitro enzyme-linked immunosorbent assay (ELISA) to diagnose the original dengue infection in secondary dengue cases. Hide abstract

Trung DT, Thao LETT, Hien TT, Hung NT, Vinh NN, Hien PT, Chinh NT, Simmons C, Wills B. 2010. Liver involvement associated with dengue infection in adults in Vietnam. Am J Trop Med Hyg, 83 (4), pp. 774-780. Read abstract | Read more

Globally, the number of adults hospitalized with dengue has increased markedly in recent years. It has been suggested that hepatic dysfunction is more significant in this group than among children. We describe the spectrum and evolution of disease manifestations among 644 adults with dengue who were prospectively recruited on admission to a major infectious disease hospital in southern Vietnam and compare them with a group of patients with similar illnesses not caused by dengue. Transaminase levels increased in virtually all dengue patients and correlated with other markers of disease severity. However, peak enzyme values usually occurred later than other complications. Clinically severe liver involvement was infrequent and idiosyncratic, but usually resulted in severe bleeding. Chronic co-infection with hepatitis B was associated with modestly but significantly increased levels of alanine aminotransferase, but did not otherwise impact the clinical picture. Hide abstract

Guzman MG, Jaenisch T, Gaczkowski R, Ty Hang VT, Sekaran SD, Kroeger A, Vazquez S, Ruiz D et al. 2010. Multi-country evaluation of the sensitivity and specificity of two commercially-available NS1 ELISA assays for dengue diagnosis. PLoS Negl Trop Dis, 4 (8), pp. e811-e811. Read abstract | Read more

Early diagnosis of dengue can assist patient triage and management and prevent unnecessary treatments and interventions. Commercially available assays that detect the dengue virus protein NS1 in the plasma/serum of patients offers the possibility of early and rapid diagnosis. Hide abstract

Tran TH, Nguyen TD, Nguyen TT, Ninh TT, Tran NB, Nguyen VM, Tran TT, Cao TT et al. 2010. A randomised trial evaluating the safety and immunogenicity of the novel single oral dose typhoid vaccine M01ZH09 in healthy Vietnamese children. PLoS One, 5 (7), pp. e11778. Read abstract | Read more

The emergence of drug resistant typhoid fever is a major public health problem, especially in Asia. An oral single dose typhoid vaccine would have major advantages. M01ZH09 is a live oral single dose candidate typhoid vaccine containing Salmonella enterica serovar Typhi (Ty2 aroC(-)ssaV(-)) ZH9 with two independently attenuating deletions. Studies in healthy adults demonstrated immunogenicity and an acceptable safety profile. Hide abstract

Thai KT, Cazelles B, Nguyen NV, Vo LT, Boni MF, Farrar J, Simmons CP, van Doorn HR, de Vries PJ. 2010. Dengue dynamics in Binh Thuan province, southern Vietnam: periodicity, synchronicity and climate variability. PLoS Negl Trop Dis, 4 (7), pp. e747. Read abstract | Read more

Dengue is a major global public health problem with increasing incidence and geographic spread. The epidemiology is complex with long inter-epidemic intervals and endemic with seasonal fluctuations. This study was initiated to investigate dengue transmission dynamics in Binh Thuan province, southern Vietnam. Hide abstract

Rabaa MA, Ty Hang VT, Wills B, Farrar J, Simmons CP, Holmes EC. 2010. Phylogeography of recently emerged DENV-2 in southern Viet Nam. PLoS Negl Trop Dis, 4 (7), pp. e766. Read abstract | Read more

Revealing the dispersal of dengue viruses (DENV) in time and space is central to understanding their epidemiology. However, the processes that shape DENV transmission patterns at the scale of local populations are not well understood, particularly the impact of such factors as human population movement and urbanization. Herein, we investigated trends in the spatial dynamics of DENV-2 transmission in the highly endemic setting of southern Viet Nam. Through a phylogeographic analysis of 168 full-length DENV-2 genome sequences obtained from hospitalized dengue cases from 10 provinces in southern Viet Nam, we reveal substantial genetic diversity in both urban and rural areas, with multiple lineages identified in individual provinces within a single season, and indicative of frequent viral migration among communities. Focusing on the recently introduced Asian I genotype, we observed particularly high rates of viral exchange between adjacent geographic areas, and between Ho Chi Minh City, the primary urban center of this region, and populations across southern Viet Nam. Within Ho Chi Minh City, patterns of DENV movement appear consistent with a gravity model of virus dispersal, with viruses traveling across a gradient of population density. Overall, our analysis suggests that Ho Chi Minh City may act as a source population for the dispersal of DENV across southern Viet Nam, and provides further evidence that urban areas of Southeast Asia play a primary role in DENV transmission. However, these data also indicate that more rural areas are also capable of maintaining virus populations and hence fueling DENV evolution over multiple seasons. Hide abstract

Thai KT, Phuong HL, Thanh Nga TT, Giao PT, Hung LEQ, Van Nam N, Binh TQ, Simmons C et al. 2010. Clinical, epidemiological and virological features of Dengue virus infections in Vietnamese patients presenting to primary care facilities with acute undifferentiated fever. J Infect, 60 (3), pp. 229-237. Read abstract | Read more

To explore clinical and virological characteristics and describe the epidemiology of dengue in patients who presented with acute undifferentiated fever (AUF) at primary health centers (PHC) in Binh Thuan Province, Vietnam. Hide abstract

Flohr C, Tuyen LN, Quinnell RJ, Lewis S, Minh TT, Campbell J, Simmons C, Telford G et al. 2010. Reduced helminth burden increases allergen skin sensitization but not clinical allergy: a randomized, double-blind, placebo-controlled trial in Vietnam. Clin Exp Allergy, 40 (1), pp. 131-142. Read abstract | Read more

Observational evidence suggests that infection with helminths protects against allergic disease and allergen skin sensitization. It is postulated that such effects are mediated by helminth-induced cytokine responses, in particular IL-10. Hide abstract

Tricou V, Vu HT, Quynh NV, Nguyen CV, Tran HT, Farrar J, Wills B, Simmons CP. 2010. Comparison of two dengue NS1 rapid tests for sensitivity, specificity and relationship to viraemia and antibody responses. BMC Infect Dis, 10 (1), pp. 142. Read abstract | Read more

Dengue is a major public health problem in tropical and subtropical countries. Rapid and easy diagnosis of dengue can assist patient triage and care-management. The detection of DENV NS1 on rapid lateral flow tests offers a fast route to a presumptive dengue diagnosis but careful evaluations are urgently needed as more and more people use them. Hide abstract

Fox A, Horby P, Ha NH, Hoa LENM, Lam NT, Simmons C, Farrar J, Van Kinh N, Wertheim H. 2010. Influenza A H5N1 and HIV co-infection: case report. BMC Infect Dis, 10 (1), pp. 167. Read abstract | Read more

The role of adaptive immunity in severe influenza is poorly understood. The occurrence of influenza A/H5N1 in a patient with HIV provided a rare opportunity to investigate this. Hide abstract

Vu TT, Holmes EC, Duong V, Nguyen TQ, Tran TH, Quail M, Churcher C, Parkhill J et al. 2010. Emergence of the Asian 1 genotype of dengue virus serotype 2 in viet nam: in vivo fitness advantage and lineage replacement in South-East Asia. PLoS Negl Trop Dis, 4 (7), pp. e757. Read abstract | Read more

A better description of the extent and structure of genetic diversity in dengue virus (DENV) in endemic settings is central to its eventual control. To this end we determined the complete coding region sequence of 187 DENV-2 genomes and 68 E genes from viruses sampled from Vietnamese patients between 1995 and 2009. Strikingly, an episode of genotype replacement was observed, with Asian 1 lineage viruses entirely displacing the previously dominant Asian/American lineage viruses. This genotype replacement event also seems to have occurred within DENV-2 in Thailand and Cambodia, suggestive of a major difference in viral fitness. To determine the cause of this major evolutionary event we compared both the infectivity of the Asian 1 and Asian/American genotypes in mosquitoes and their viraemia levels in humans. Although there was little difference in infectivity in mosquitoes, we observed significantly higher plasma viraemia levels in paediatric patients infected with Asian 1 lineage viruses relative to Asian/American viruses, a phenotype that is predicted to result in a higher probability of human-to-mosquito transmission. These results provide a mechanistic basis to a marked change in the genetic structure of DENV-2 and more broadly underscore that an understanding of DENV evolutionary dynamics can inform the development of vaccines and anti-viral drugs. Hide abstract

Tricou V, Minh NN, Van TP, Lee SJ, Farrar J, Wills B, Tran HT, Simmons CP. 2010. A randomized controlled trial of chloroquine for the treatment of dengue in Vietnamese adults. PLoS Negl Trop Dis, 4 (8), pp. e785. Read abstract | Read more

There is currently no licensed antiviral drug for treatment of dengue. Chloroquine (CQ) inhibits the replication of dengue virus (DENV) in vitro. Hide abstract

Buchy P, Vong S, Chu S, Garcia JM, Hien TT, Hien VM, Channa M, Ha DOQ et al. 2010. Kinetics of neutralizing antibodies in patients naturally infected by H5N1 virus. PLoS One, 5 (5), pp. e10864. Read abstract | Read more

Little is known about the kinetics of anti-H5 neutralizing antibodies in naturally H5N1-infected patients with severe clinical illness or asymptomatic infection. Hide abstract

Chau TN, Anders KL, Lien LEB, Hung NT, Hieu LT, Tuan NM, Thuy TT, Phuong LET et al. 2010. Clinical and virological features of Dengue in Vietnamese infants. PLoS Negl Trop Dis, 4 (4), pp. e657. Read abstract | Read more

Infants account for a small proportion of the overall dengue case burden in endemic countries but can be clinically more difficult to manage. The clinical and laboratory features in infants with dengue have not been extensively characterised. Hide abstract

Hang VTT, Holmes EC, Veasna D, Quy NT, Hien TT, Quail M, Churcher C, Parkhill J et al. 2010. Emergence of the Asian 1 genotype of dengue Virus serotype 2 in viet Nam: in vivo fitness advantage and lineage replacement in South-East Asia PLoS Neglected Tropical Diseases, 4 (7), Read abstract | Read more

A better description of the extent and structure of genetic diversity in dengue virus (DENV) in endemic settings is central to its eventual control. To this end we determined the complete coding region sequence of 187 DENV-2 genomes and 68 E genes from viruses sampled from Vietnamese patients between 1995 and 2009. Strikingly, an episode of genotype replacement was observed, with Asian 1 lineage viruses entirely displacing the previously dominant Asian/American lineage viruses. This genotype replacement event also seems to have occurred within DENV- 2 in Thailand and Cambodia, suggestive of a major difference in viral fitness. To determine the cause of this major evolutionary event we compared both the infectivity of the Asian 1 and Asian/American genotypes in mosquitoes and their viraemia levels in humans. Although there was little difference in infectivity in mosquitoes, we observed significantly higher plasma viraemia levels in paediatric patients infected with Asian 1 lineage viruses relative to Asian/ American viruses, a phenotype that is predicted to result in a higher probability of human-to-mosquito transmission. These results provide a mechanistic basis to a marked change in the genetic structure of DENV-2 and more broadly underscore that an understanding of DENV evolutionary dynamics can inform the development of vaccines and antiviral drugs. © 2010 Ty Hang et al. Hide abstract

Wikramaratna PS, Simmons CP, Gupta S, Recker M. 2010. The effects of tertiary and quaternary infections on the epidemiology of dengue. PLoS One, 5 (8), pp. e12347. Read abstract | Read more

The epidemiology of dengue is characterised by irregular epidemic outbreaks and desynchronised dynamics of its four co-circulating virus serotypes. Whilst infection by one serotype appears to convey life-long protection to homologous infection, it is believed to be a risk factor for severe disease manifestations upon secondary, heterologous infection due to the phenomenon of Antibody-Dependent Enhancement (ADE). Subsequent clinical infections are rarely reported and, since the majority of dengue infections are generally asymptomatic, it is not clear if and to what degree tertiary or quaternary infections contribute to dengue epidemiology. Here we investigate the effect of third and subsequent infections on the transmission dynamics of dengue and show that although the qualitative patterns are largely equivalent, the system more readily exhibits the desynchronised serotype oscillations and multi-annual epidemic outbreaks upon their inclusion. More importantly, permitting third and fourth infections significantly increases the force of infection without resorting to high basic reproductive numbers. Realistic age-prevalent patterns and seroconversion rates are therefore easier reconciled with a low value of dengue's transmission potential if allowing for more than two infections; this should have important consequences for dengue control and intervention measures. Hide abstract

Chau TN, Hieu NT, Anders KL, Wolbers M, Lien LEB, Hieu LT, Hien TT, Hung NT, Farrar J, Whitehead S, Simmons CP. 2009. Dengue virus infections and maternal antibody decay in a prospective birth cohort study of Vietnamese infants. J Infect Dis, 200 (12), pp. 1893-1900. Read abstract | Read more

Dengue hemorrhagic fever can occur in primary dengue virus (DENV) infection of infants. The decay of maternally derived DENV immunoglobulin (Ig) G and the incidence of DENV infection were determined in a prospectively studied cohort of 1244 Vietnamese infants. Higher concentrations of total IgG and DENV-reactive IgG were found in cord plasma relative to maternal plasma. Maternally derived DENV-neutralizing and E protein-reactive IgG titers declined to below measurable levels in >90% of infants by 6 months of age. In contrast, IgG reactive with whole DENV virions persisted until 12 months of age in 20% of infants. Serological surveillance identified 10 infants with asymptomatic DENV infection for an incidence of 1.7 cases per 100 person-years. DENV-neutralizing antibodies remained measurable for > or = 1 year after infection. These results suggest that whereas DENV infection in infants is frequently subclinical, there is a window between 4 and 12 months of age where virion-binding but nonneutralizing IgG could facilitate antibody-dependent enhancement. Hide abstract

Mai NT, Tuan TV, Wolbers M, Hoang DM, Nga TV, Chau TT, Chuong LV, Sinh DX et al. 2009. Immunological and biochemical correlates of adjunctive dexamethasone in Vietnamese adults with bacterial meningitis. Clin Infect Dis, 49 (9), pp. 1387-1392. Read abstract | Read more

Adjunctive treatment to improve outcome from bacterial meningitis has centered on dexamethasone. Among Vietnamese patients with bacterial meningitis, cerebrospinal fluid (CSF) opening pressure and CSF:plasma glucose ratios were significantly improved and levels of CSF cytokines interleukin (IL)-6, IL-8, and IL-10 and were all statistically significantly lower after treatment in patients who were randomized to dexamethasone, compared with levels in patients who received placebo. Hide abstract

Simmons CP, Farrar J. 2009. Changing patterns of dengue epidemiology and implications for clinical management and vaccines. PLoS Med, 6 (9), pp. e1000129. | Read more

Guzman MG, Jaenisch T, Gaczkowski R, Hang VTT, Devi S, Horstick O, Kroeger A, Simmons CP. 2009. Multi-country evaluation of the sensitivity and specificity of two commercially available NS1 ELISA assays for dengue diagnosis TROPICAL MEDICINE & INTERNATIONAL HEALTH, 14 pp. 83-84.

Recker M, Blyuss KB, Simmons CP, Hien TT, Wills B, Farrar J, Gupta S. 2009. Immunological serotype interactions and their effect on the epidemiological pattern of dengue. Proc Biol Sci, 276 (1667), pp. 2541-2548. Read abstract | Read more

Long-term epidemiological data reveal multi-annual fluctuations in the incidence of dengue fever and dengue haemorrhagic fever, as well as complex cyclical behaviour in the dynamics of the four serotypes of the dengue virus. It has previously been proposed that these patterns are due to the phenomenon of the so-called antibody-dependent enhancement (ADE) among dengue serotypes, whereby viral replication is increased during secondary infection with a heterologous serotype; however, recent studies have implied that this positive reinforcement cannot account for the temporal patterns of dengue and that some form of cross-immunity or external forcing is necessary. Here, we show that ADE alone can produce the observed periodicities and desynchronized oscillations of individual serotypes if its effects are decomposed into its two possible manifestations: enhancement of susceptibility to secondary infections and increased transmissibility from individuals suffering from secondary infections. This decomposition not only lowers the level of enhancement necessary for realistic disease patterns but also reduces the risk of stochastic extinction. Furthermore, our analyses reveal a time-lagged correlation between serotype dynamics and disease incidence rates, which could have important implications for understanding the irregular pattern of dengue epidemics. Hide abstract

Khurana S, Suguitan AL, Rivera Y, Simmons CP, Lanzavecchia A, Sallusto F, Manischewitz J, King LR, Subbarao K, Golding H. 2009. Antigenic fingerprinting of H5N1 avian influenza using convalescent sera and monoclonal antibodies reveals potential vaccine and diagnostic targets. PLoS Med, 6 (4), pp. e1000049. Read abstract | Read more

Transmission of highly pathogenic avian H5N1 viruses from poultry to humans have raised fears of an impending influenza pandemic. Concerted efforts are underway to prepare effective vaccines and therapies including polyclonal or monoclonal antibodies against H5N1. Current efforts are hampered by the paucity of information on protective immune responses against avian influenza. Characterizing the B cell responses in convalescent individuals could help in the design of future vaccines and therapeutics. Hide abstract

Long HT, Hibberd ML, Hien TT, Dung NM, Van Ngoc T, Farrar J, Wills B, Simmons CP. 2009. Patterns of gene transcript abundance in the blood of children with severe or uncomplicated dengue highlight differences in disease evolution and host response to dengue virus infection. J Infect Dis, 199 (4), pp. 537-546. Read abstract | Read more

DNA microarrays and specific reverse-transcription polymerase chain reaction assays were used to reveal transcriptional patterns in the blood of children presenting with dengue shock syndrome (DSS) and well-matched patients with uncomplicated dengue. The transcriptome of patients with acute uncomplicated dengue was characterized by a metabolically demanding "host-defense" profile; transcripts related to oxidative metabolism, interferon signaling, protein ubiquination, apoptosis, and cytokines were prominent. In contrast, the transcriptome of patients with DSS was surprisingly benign, particularly with regard to transcripts derived from apoptotic and type I interferon pathways. These data highlight significant heterogeneity in the type or timing of host transcriptional immune responses precipitated by dengue virus infection independent of the duration of illness. In particular, they suggest that, if transcriptional events in the blood compartment contribute to capillary leakage leading to hypovolemic shock, they occur before cardiovascular decompensation, a finding that has implications for rational adjuvant therapy in this syndrome. Hide abstract

Hang VT, Nguyet NM, Trung DT, Tricou V, Yoksan S, Dung NM, Van Ngoc T, Hien TT, Farrar J, Wills B, Simmons CP. 2009. Diagnostic accuracy of NS1 ELISA and lateral flow rapid tests for dengue sensitivity, specificity and relationship to viraemia and antibody responses. PLoS Negl Trop Dis, 3 (1), pp. e360. Read abstract | Read more

Dengue is a public health problem in many countries. Rapid diagnosis of dengue can assist patient triage and management. Detection of the dengue viral protein, NS1, represents a new approach to dengue diagnosis. Hide abstract

Thuong NT, Dunstan SJ, Chau TT, Thorsson V, Simmons CP, Quyen NT, Thwaites GE, Thi Ngoc Lan N et al. 2008. Identification of tuberculosis susceptibility genes with human macrophage gene expression profiles. PLoS Pathog, 4 (12), pp. e1000229. Read abstract | Read more

Although host genetics influences susceptibility to tuberculosis (TB), few genes determining disease outcome have been identified. We hypothesized that macrophages from individuals with different clinical manifestations of Mycobacterium tuberculosis (Mtb) infection would have distinct gene expression profiles and that polymorphisms in these genes may also be associated with susceptibility to TB. We measured gene expression levels of >38,500 genes from ex vivo Mtb-stimulated macrophages in 12 subjects with 3 clinical phenotypes: latent, pulmonary, and meningeal TB (n = 4 per group). After identifying differentially expressed genes, we confirmed these results in 34 additional subjects by real-time PCR. We also used a case-control study design to examine whether polymorphisms in differentially regulated genes were associated with susceptibility to these different clinical forms of TB. We compared gene expression profiles in Mtb-stimulated and unstimulated macrophages and identified 1,608 and 199 genes that were differentially expressed by >2- and >5-fold, respectively. In an independent sample set of 34 individuals and a subset of highly regulated genes, 90% of the microarray results were confirmed by RT-PCR, including expression levels of CCL1, which distinguished the 3 clinical groups. Furthermore, 6 single nucleotide polymorphisms (SNPs) in CCL1 were found to be associated with TB in a case-control genetic association study with 273 TB cases and 188 controls. To our knowledge, this is the first identification of CCL1 as a gene involved in host susceptibility to TB and the first study to combine microarray and DNA polymorphism studies to identify genes associated with TB susceptibility. These results suggest that genome-wide studies can provide an unbiased method to identify critical macrophage response genes that are associated with different clinical outcomes and that variation in innate immune response genes regulate susceptibility to TB. Hide abstract

Simmons C, Farrar J. 2008. Insights into inflammation and influenza. N Engl J Med, 359 (15), pp. 1621-1623. | Read more

Lee LY, Ha DOLA, Simmons C, de Jong MD, Chau NV, Schumacher R, Peng YC, McMichael AJ et al. 2008. Memory T cells established by seasonal human influenza A infection cross-react with avian influenza A (H5N1) in healthy individuals. J Clin Invest, 118 (10), pp. 3478-3490. Read abstract | Read more

The threat of avian influenza A (H5N1) infection in humans remains a global health concern. Current influenza vaccines stimulate antibody responses against the surface glycoproteins but are ineffective against strains that have undergone significant antigenic variation. An alternative approach is to stimulate pre-existing memory T cells established by seasonal human influenza A infection that could cross-react with H5N1 by targeting highly conserved internal proteins. To determine how common cross-reactive T cells are, we performed a comprehensive ex vivo analysis of cross-reactive CD4+ and CD8+ memory T cell responses to overlapping peptides spanning the full proteome of influenza A/Viet Nam/CL26/2005 (H5N1) and influenza A/New York/232/2004 (H3N2) in healthy individuals from the United Kingdom and Viet Nam. Memory CD4+ and CD8+ T cells isolated from the majority of participants exhibited human influenza-specific responses and showed cross-recognition of at least one H5N1 internal protein. Participant CD4+ and CD8+ T cells recognized multiple synthesized influenza peptides, including peptides from the H5N1 strain. Matrix protein 1 (M1) and nucleoprotein (NP) were the immunodominant targets of cross-recognition. In addition, cross-reactive CD4+ and CD8+ T cells recognized target cells infected with recombinant vaccinia viruses expressing either H5N1 M1 or NP. Thus, vaccine formulas inducing heterosubtypic T cell-mediated immunity may confer broad protection against avian and human influenza A viruses. Hide abstract

Chau TN, Quyen NT, Thuy TT, Tuan NM, Hoang DM, Dung NT, Lien LEB, Quy NT et al. 2008. Dengue in Vietnamese infants--results of infection-enhancement assays correlate with age-related disease epidemiology, and cellular immune responses correlate with disease severity. J Infect Dis, 198 (4), pp. 516-524. Read abstract | Read more

The pathogenesis of severe dengue is not well understood. Maternally derived subneutralizing levels of dengue virus-reactive IgG are postulated to be a critical risk factor for severe dengue during infancy. In this study, we found that, in healthy Vietnamese infants, there was a strong temporal association between the Fc-dependent, dengue virus infection-enhancing activity of neat plasma and the age-related epidemiology of severe dengue. We then postulated that disease severity in infants with primary infections would be associated with a robust immune response, possibly as a consequence of higher viral burdens in vivo. Accordingly, in infants hospitalized with acute dengue, the activation phenotype of peripheral-blood NK cells and CD8+ and CD4+ T cells correlated with overall disease severity, but HLA-A*1101-restricted NS3(133-142)-specific CD8+ T cells were not measurable until early convalescence. Plasma levels of cytokines/chemokines were generally higher in infants with dengue shock syndrome. Collectively, these data support a model of dengue pathogenesis in infants whereby antibody-dependent enhancement of infection explains the age-related case epidemiology and could account for antigen-driven immune activation and its association with disease severity. These results also highlight potential risks in the use of live attenuated dengue vaccines in infants in countries where dengue is endemic. Hide abstract

Moran E, Simmons C, Vinh Chau N, Luhn K, Wills B, Dung NP, Thao LETT, Hien TT, Farrar J, Rowland-Jones S, Dong T. 2008. Preservation of a critical epitope core region is associated with the high degree of flaviviral cross-reactivity exhibited by a dengue-specific CD4+ T cell clone. Eur J Immunol, 38 (4), pp. 1050-1057. Read abstract | Read more

Dengue is a member of the Flaviviridae, a large group of related viruses some of which co-circulate in certain regions (e.g. dengue and Yellow fever in South America). Immune responses cross-reactive between different dengue serotypes are important in the pathogenesis of dengue disease but it is not known whether previous infection with one flavivirus might affect the clinical course of subsequent infections with other members of the family. CD4+ T cells have been shown to be important in the production of cytokines in response to dengue infection and can demonstrate significant epitope cross-reactivity. Here, we describe the generation and characterisation of CD4+ T cell clones from a patient experiencing acute dengue infection. These clones were DRB1*15+ and recognised epitope variants not only within other dengue viruses but certain other flaviviruses. This cross-reactivity was dependent upon the presence of a five-amino acid core region, consistent with structural observations of class II MHC binding to TCR demonstrating that only a subset of residues within an epitope bound to a class II molecule are "read out" by the TCR. This capacity of CD4+ T cell clones to recognise a given epitope despite considerable variation between viruses may be of pathological significance, particularly in regions where related viruses co-circulate. Hide abstract

Thwaites G, Caws M, Chau TT, D'Sa A, Lan NT, Huyen MN, Gagneux S, Anh PT et al. 2008. Relationship between Mycobacterium tuberculosis genotype and the clinical phenotype of pulmonary and meningeal tuberculosis. J Clin Microbiol, 46 (4), pp. 1363-1368. Read abstract | Read more

We used large sequence polymorphisms to determine the genotypes of 397 isolates of Mycobacterium tuberculosis from human immunodeficiency virus-uninfected Vietnamese adults with pulmonary (n = 235) or meningeal (n = 162) tuberculosis. We compared the pretreatment radiographic appearances of pulmonary tuberculosis and the presentation, response to treatment, and outcome of tuberculous meningitis between the genotypes. Multivariate analysis identified variables independently associated with genotype and outcome. A higher proportion of adults with pulmonary tuberculosis caused by the Euro-American genotype had consolidation on chest X-ray than was the case with disease caused by other genotypes (P = 0.006). Multivariate analysis revealed that meningitis caused by the East Asian/Beijing genotype was independently associated with a shorter duration of illness before presentation and fewer cerebrospinal fluid (CSF) leukocytes. Older age, fewer CSF leukocytes, and the presence of hemiplegia (but not strain lineage) were independently associated with death or severe disability, although the East Asian/Beijing genotype was strongly associated with drug-resistant tuberculosis. The genotype of M. tuberculosis influenced the presenting features of pulmonary and meningeal tuberculosis. The association between the East Asian/Beijing lineage and disease progression and CSF leukocyte count suggests the lineage may alter the presentation of meningitis by influencing the intracerebral inflammatory response. In addition, increased drug resistance among bacteria of the East Asian/Beijing lineage might influence the response to treatment. This study suggests the genetic diversity of M. tuberculosis has important clinical consequences. Hide abstract

Tanner L, Schreiber M, Low JG, Ong A, Tolfvenstam T, Lai YL, Ng LC, Leo YS et al. 2008. Decision tree algorithms predict the diagnosis and outcome of dengue fever in the early phase of illness. PLoS Negl Trop Dis, 2 (3), pp. e196. Read abstract | Read more

Dengue is re-emerging throughout the tropical world, causing frequent recurrent epidemics. The initial clinical manifestation of dengue often is confused with other febrile states confounding both clinical management and disease surveillance. Evidence-based triage strategies that identify individuals likely to be in the early stages of dengue illness can direct patient stratification for clinical investigations, management, and virological surveillance. Here we report the identification of algorithms that differentiate dengue from other febrile illnesses in the primary care setting and predict severe disease in adults. Hide abstract

Thuong NTT, Dunstan SJ, Simmons CP, Quyen NTH, Farrar JJ, Chau TTH, Thorsson V, Aderem A et al. 2008. Identification of tuberculosis susceptibility genes with human macrophage gene expression profiles PLoS Pathogens, 4 (12), Read abstract | Read more

Although host genetics influences susceptibility to tuberculosis (TB), few genes determining disease outcome have been identified. We hypothesized that macrophages from individuals with different clinical manifestations of Mycobacterium tuberculosis (Mtb) infection would have distinct gene expression profiles and that polymorphisms in these genes may also be associated with susceptibility to TB. We measured gene expression levels of >38,500 genes from ex vivo Mtb-stimulated macrophages in 12 subjects with 3 clinical phenotypes: latent, pulmonary, and meningeal TB (n = 4 per group). After identifying differentially expressed genes, we confirmed these results in 34 additional subjects by real-time PCR. We also used a case-control study design to examine whether polymorphisms in differentially regulated genes were associated with susceptibility to these different clinical forms of TB. We compared gene expression profiles in Mtb-stimulated and unstimulated macrophages and identified 1,608 and 199 genes that were differentially expressed by >2- and >5-fold, respectively. In an independent sample set of 34 individuals and a subset of highly regulated genes, 90% of the microarray results were confirmed by RT-PCR, including expression levels of CCL1, which distinguished the 3 clinical groups. Furthermore, 6 single nucleotide polymorphisms (SNPs) in CCL1 were found to be associated with TB in a case-control genetic association study with 273 TB cases and 188 controls. To our knowledge, this is the first identification of CCL1 as a gene involved in host susceptibility to TB and the first study to combine microarray and DNA polymorphism studies to identify genes associated with TB susceptibility. These results suggest that genome-wide studies can provide an unbiased method to identify critical macrophage response genes that are associated with different clinical outcomes and that variation in innate immune response genes regulate susceptibility to TB. © 2008 Thuong et al. Hide abstract

Simmons CP, Chau TN, Thuy TT, Tuan NM, Hoang DM, Thien NT, Lien LEB, Quy NT et al. 2007. Maternal antibody and viral factors in the pathogenesis of dengue virus in infants. J Infect Dis, 196 (3), pp. 416-424. Read abstract | Read more

The pathogenesis of dengue in infants is poorly understood. We postulated that dengue severity in infants would be positively associated with markers of viral burden and that maternally derived, neutralizing anti-dengue antibody would have decayed before the age at which infants with dengue presented to the hospital. In 75 Vietnamese infants with primary dengue, we found significant heterogeneity in viremia and NS1 antigenemia at hospital presentation, and these factors were independent of disease grade or continuous measures of disease severity. Neutralizing antibody titers, predicted in each infant at the time of their illness, suggested that the majority of infants (65%) experienced dengue hemorrhagic fever when the maternally derived neutralizing antibody titer had declined to <1 : 20. Collectively, these data have important implications for dengue vaccine research because they suggest that viral burden may not solely explain severe dengue in infants and that neutralizing antibody is a reasonable but not absolute marker of protective immunity in infants. Hide abstract

Farrar J, Focks D, Gubler D, Barrera R, Guzman MG, Simmons C, Kalayanarooj S, Lum L et al. 2007. Editorial: Towards a global dengue research agenda TROPICAL MEDICINE & INTERNATIONAL HEALTH, 12 (6), pp. 695-699. | Read more

Lühn K, Simmons CP, Moran E, Dung NT, Chau TN, Quyen NT, Thao LETT, Van Ngoc T et al. 2007. Increased frequencies of CD4+ CD25(high) regulatory T cells in acute dengue infection. J Exp Med, 204 (5), pp. 979-985. Read abstract | Read more

Dengue virus infection is an increasingly important tropical disease, causing 100 million cases each year. Symptoms range from mild febrile illness to severe hemorrhagic fever. The pathogenesis is incompletely understood, but immunopathology is thought to play a part, with antibody-dependent enhancement and massive immune activation of T cells and monocytes/macrophages leading to a disproportionate production of proinflammatory cytokines. We sought to investigate whether a defective population of regulatory T cells (T reg cells) could be contributing to immunopathology in severe dengue disease. CD4(+)CD25(high)FoxP3(+) T reg cells of patients with acute dengue infection of different severities showed a conventional phenotype. Unexpectedly, their capacity to suppress T cell proliferation and to secrete interleukin-10 was not altered. Moreover, T reg cells suppressed the production of vasoactive cytokines after dengue-specific stimulation. Furthermore, T reg cell frequencies and also T reg cell/effector T cell ratios were increased in patients with acute infection. A strong indication that a relative rise of T reg cell/effector T cell ratios is beneficial for disease outcome comes from patients with mild disease in which this ratio is significantly increased (P < 0.0001) in contrast to severe cases (P = 0.2145). We conclude that although T reg cells expand and function normally in acute dengue infection, their relative frequencies are insufficient to control the immunopathology of severe disease. Hide abstract

Simmons CP, Popper S, Dolocek C, Chau TN, Griffiths M, Dung NT, Long TH, Hoang DM et al. 2007. Patterns of host genome-wide gene transcript abundance in the peripheral blood of patients with acute dengue hemorrhagic fever. J Infect Dis, 195 (8), pp. 1097-1107. Read abstract | Read more

Responses by peripheral blood leukocytes may contribute to the pathogenesis of dengue hemorrhagic fever (DHF). We used DNA microarrays to reveal transcriptional patterns in the blood of 14 adults with DHF. Acute DHF was defined by an abundance of transcripts from cell cycle- and endoplasmic reticulum (ER)-related genes, suggesting a proliferative response accompanied by ER stress. Transcript-abundance levels for immunoresponse-associated genes, including cell surface markers, immunoglobulin, and innate response elements, were also elevated. Twenty-four genes were identified for which transcript abundance distinguished patients with dengue shock syndrome (DSS) from those without DSS. All the gene transcripts associated with DSS, many of which are induced by type I interferons, were less abundant in patients with DSS than in those without DSS. To our knowledge, these data provide the first snapshot of gene-expression patterns in peripheral blood during acute dengue and suggest that DSS is associated with attenuation of selected aspects of the innate host response. Hide abstract

Thwaites GE, Macmullen-Price J, Tran TH, Pham PM, Nguyen TD, Simmons CP, White NJ, Tran TH, Summers D, Farrar JJ. 2007. Serial MRI to determine the effect of dexamethasone on the cerebral pathology of tuberculous meningitis: an observational study. Lancet Neurol, 6 (3), pp. 230-236. Read abstract | Read more

Adjunctive dexamethasone increases survival from tuberculous meningitis, but the underlying mechanism is unclear. We aimed to determine the effect of dexamethasone on cerebral MRI changes and their association with intracerebral inflammatory responses and clinical outcome in adults treated for tuberculous meningitis. Hide abstract

Simmons CP, Bernasconi NL, Suguitan AL, Mills K, Ward JM, Chau NV, Hien TT, Sallusto F et al. 2007. Prophylactic and therapeutic efficacy of human monoclonal antibodies against H5N1 influenza. PLoS Med, 4 (5), pp. e178. Read abstract | Read more

New prophylactic and therapeutic strategies to combat human infections with highly pathogenic avian influenza (HPAI) H5N1 viruses are needed. We generated neutralizing anti-H5N1 human monoclonal antibodies (mAbs) and tested their efficacy for prophylaxis and therapy in a murine model of infection. Hide abstract

Dong T, Moran E, Vinh Chau N, Simmons C, Luhn K, Peng Y, Wills B, Phuong Dung N et al. 2007. High pro-inflammatory cytokine secretion and loss of high avidity cross-reactive cytotoxic T-cells during the course of secondary dengue virus infection. PLoS One, 2 (12), pp. e1192. Read abstract | Read more

Dengue is one of the most important human diseases transmitted by an arthropod vector and the incidence of dengue virus infection has been increasing - over half the world's population now live in areas at risk of infection. Most infections are asymptomatic, but a subset of patients experience a potentially fatal shock syndrome characterised by plasma leakage. Severe forms of dengue are epidemiologically associated with repeated infection by more than one of the four dengue virus serotypes. Generally attributed to the phenomenon of antibody-dependent enhancement, recent observations indicate that T-cells may also influence disease phenotype. Hide abstract

Dong T, Moran E, Luhn K, Peng Y, McMichael A, Rowland-Jones S, Simmons C, Wills B et al. 2007. High pro-inflammatory cytokine secretion and loss of high avidity cross-reactive cytotoxic T-cells during the course of secondary dengue virus infection PLoS ONE, 2 (12), Read abstract | Read more

Background: Dengue is one of the most important human diseases transmitted by an arthropod vector and the incidence of dengue virus infection has been increasing - over half the world's population now live in areas at risk of infection. Most infections are asymptomatic, but a subset of patients experience a potentially fatal shock syndrome characterised by plasma leakage. Severe forms of dengue are epidemiologically associated with repeated infection by more than one of the four dengue virus serotypes. Generally attributed to the phenomenon of antibody-dependent enhancement, recent observations indicate that T-cells may also influence disease phenotype. Methods and Findings: Virus-specific cytotoxic T lymphocytes (CTL) showing high level cross reactivity between dengue serotypes could be expanded from blood samples taken during the acute phase of secondary dengue infection. These could not be detected in convalescence when only CTL populations demonstrating significant serotype specificity were identified. Dengue cross-reactive CTL clones derived from these patients were of higher avidity than serotype-specific clones and produced much higher levels of both type 1 and certain type 2 cytokines, many previously implicated in dengue pathogenesis. Conclusion: Dengue serotype cross-reactive CTL clones showing high avidity for antigen produce higher levels of inflammatory cytokines than serotype-specific clones. That such cells cannot be expanded from convalescent samples suggests that they maybe depleted, perhaps as a consequence of activation-induced cell death. Such high avidity cross-reactive memory CTL may produce inflammatory cytokines during the course of secondary infection, contributing to the pathogenesis of vascular leak. These cells appear to be subsequently deleted leaving a more serotype-specific memory CTL pool. Further studies are needed to relate these cellular observations to disease phenotype in a large group of patients. If confirmed they have significant implications for understanding the role of virus-specific CTL in pathogenesis of dengue disease. © 2007 Dong et al. Hide abstract

Nguyen D, Dong T, Nguyen VVC, Nguyen MD, Wills B, Rowland-Jones S, Farrar J, Simmons C. 2006. Spectrum and kinetic of T cell responses to dengue virus epitopes and the influence of hla polymorphisms in dengue disease pathogenesis AMERICAN JOURNAL OF TROPICAL MEDICINE AND HYGIENE, 75 (5), pp. 289-290.

Hoang LT, Hibberd ML, Farrar J, Simmons CP. 2006. Global gene expression profiles during acute dengue revealed by microarray analysis AMERICAN JOURNAL OF TROPICAL MEDICINE AND HYGIENE, 75 (5), pp. 290-290.

Popper S, Simmons CP, Dolecek C, Chau TNB, Griffiths M, Dung NTP, Long TH, Hoang DM et al. 2006. Gene expression programs in adults with acute dengue infections AMERICAN JOURNAL OF TROPICAL MEDICINE AND HYGIENE, 75 (5), pp. 290-290.

Hawn TR, Dunstan SJ, Thwaites GE, Simmons CP, Thuong NT, Lan NT, Quy HT, Chau TT et al. 2006. A polymorphism in Toll-interleukin 1 receptor domain containing adaptor protein is associated with susceptibility to meningeal tuberculosis. J Infect Dis, 194 (8), pp. 1127-1134. Read abstract | Read more

Although meningitis is the most severe form of infection caused by Mycobacterium tuberculosis, the immunopathogenesis of this disease is poorly understood. We tested the hypothesis that polymorphisms in Toll-interleukin 1 receptor domain containing adaptor protein (TIRAP), an adaptor protein that mediates signals from Toll-like receptors activated by mycobacteria, are associated with susceptibility to tuberculosis (TB). Hide abstract

de Jong MD, Simmons CP, Thanh TT, Hien VM, Smith GJ, Chau TN, Hoang DM, Chau NV et al. 2006. Fatal outcome of human influenza A (H5N1) is associated with high viral load and hypercytokinemia. Nat Med, 12 (10), pp. 1203-1207. Read abstract | Read more

Avian influenza A (H5N1) viruses cause severe disease in humans, but the basis for their virulence remains unclear. In vitro and animal studies indicate that high and disseminated viral replication is important for disease pathogenesis. Laboratory experiments suggest that virus-induced cytokine dysregulation may contribute to disease severity. To assess the relevance of these findings for human disease, we performed virological and immunological studies in 18 individuals with H5N1 and 8 individuals infected with human influenza virus subtypes. Influenza H5N1 infection in humans is characterized by high pharyngeal virus loads and frequent detection of viral RNA in rectum and blood. Viral RNA in blood was present only in fatal H5N1 cases and was associated with higher pharyngeal viral loads. We observed low peripheral blood T-lymphocyte counts and high chemokine and cytokine levels in H5N1-infected individuals, particularly in those who died, and these correlated with pharyngeal viral loads. Genetic characterization of H5N1 viruses revealed mutations in the viral polymerase complex associated with mammalian adaptation and virulence. Our observations indicate that high viral load, and the resulting intense inflammatory responses, are central to influenza H5N1 pathogenesis. The focus of clinical management should be on preventing this intense cytokine response, by early diagnosis and effective antiviral treatment. Hide abstract

Simmons CP, Thwaites GE, Quyen NT, Torok E, Hoang DM, Chau TT, Mai PP, Lan NT et al. 2006. Pretreatment intracerebral and peripheral blood immune responses in Vietnamese adults with tuberculous meningitis: diagnostic value and relationship to disease severity and outcome. J Immunol, 176 (3), pp. 2007-2014. Read abstract

Tuberculous meningitis (TBM) is the most devastating form of tuberculosis. Both intracerebral and peripheral blood immune responses may be relevant to pathogenesis, diagnosis, and outcome. In this study, the relationship between pretreatment host response, disease phenotype, and outcome in Vietnamese adults with TBM was examined. Before treatment, peripheral blood IFN-gamma ELISPOT responses to the Mycobacterium tuberculosis Ags ESAT-6, CFP-10, and purified protein derivative (PPD) were a poor diagnostic predictor of TBM. Cerebrospinal fluid IL-6 concentrations at presentation were independently associated with severe disease presentation, suggesting an immunological correlate of neurological damage before treatment. Surprisingly however, elevated cerebrospinal fluid inflammatory cytokines were not associated with death or disability in HIV-negative TBM patients at presentation. HIV coinfection attenuated multiple cerebrospinal fluid inflammatory indices. Low cerebrospinal fluid IFN-gamma concentrations were independently associated with death in HIV-positive TBM patients, implying that IFN-gamma contributes to immunity and survival. Collectively, these results reveal the effect of HIV coinfection on the pathogenesis of TBM and suggest that intracerebral immune responses, at least in HIV-negative cases, may not be as intimately associated with disease outcome as previously thought. Hide abstract

Thwaites GE, Duc Bang N, Huy Dung N, Thi Quy H, Thi Tuong Oanh D, Thi Cam Thoa N, Quang Hien N, Tri Thuc N et al. 2005. The influence of HIV infection on clinical presentation, response to treatment, and outcome in adults with Tuberculous meningitis. J Infect Dis, 192 (12), pp. 2134-2141. Read abstract | Read more

Tuberculous meningitis occurs more commonly in human immunodeficiency virus (HIV)-infected individuals than in HIV-uninfected individuals, but whether HIV infection alters the presentation and outcome of tuberculous meningitis is unknown. Hide abstract

Simmons CP, Thwaites GE, Quyen NT, Chau TT, Mai PP, Dung NT, Stepniewska K, White NJ, Hien TT, Farrar J. 2005. The clinical benefit of adjunctive dexamethasone in tuberculous meningitis is not associated with measurable attenuation of peripheral or local immune responses. J Immunol, 175 (1), pp. 579-590. Read abstract

Outcome from tuberculous meningitis (TBM) is believed to be dependent on the severity of the intracerebral inflammatory response. We have recently shown that dexamethasone improved survival in adults with TBM and postulated that the clinical effect would be associated with a measurable systemic and intracerebral impact on immunological markers of inflammation. Prolonged inflammatory responses were detected in all TBM patients irrespective of treatment assignment (placebo or dexamethasone). The inflammatory response in the cerebrospinal fluid was characterized by a leukocytosis (predominantly CD3(+)CD4(+) T lymphocytes, phenotypically distinct from those in the peripheral blood), elevated concentrations of inflammatory and anti-inflammatory cytokines, chemokines, and evidence of prolonged blood-brain barrier dysfunction. Dexamethasone significantly modulated acute cerebrospinal fluid protein concentrations and marginally reduced IFN-gamma concentrations; other immunological and routine biochemical indices of inflammation were unaffected. Peripheral blood monocyte and T cell responses to Mycobacterium tuberculosis Ags were also unaffected. Dexamethasone does not appear to improve survival from TBM by attenuating immunological mediators of inflammation in the subarachnoid space or by suppressing peripheral T cell responses to mycobacterial Ags. These findings challenge previously held theories of corticosteroid action in this disease. An understanding of how dexamethasone acts in TBM may suggest novel and more effective treatment strategies. Hide abstract

Simmons CP, Dong T, Chau NV, Dung NT, Chau TN, Thao LETT, Dung NT, Hien TT, Rowland-Jones S, Farrar J. 2005. Early T-cell responses to dengue virus epitopes in Vietnamese adults with secondary dengue virus infections. J Virol, 79 (9), pp. 5665-5675. Read abstract | Read more

T-cell responses to dengue viruses may be important in both protective immunity and pathogenesis. This study of 48 Vietnamese adults with secondary dengue virus infections defined the breadth and magnitude of peripheral T-cell responses to 260 overlapping peptide antigens derived from a dengue virus serotype 2 (DV2) isolate. Forty-seven different peptides evoked significant gamma interferon enzyme-linked immunospot (ELISPOT) assay responses in 39 patients; of these, 34 peptides contained potentially novel T-cell epitopes. NS3 and particularly NS3200-324 were important T-cell targets. The breadth and magnitude of ELISPOT responses to DV2 peptides were independent of the infecting dengue virus serotype, suggesting that cross-reactive T cells dominate the acute response during secondary infection. Acute ELISPOT responses were weakly correlated with the extent of hemoconcentration in individual patients but not with the nadir of thrombocytopenia or overall clinical disease grade. NS3556-564 and Env414-422 were identified as novel HLA-A*24 and B*07-restricted CD8+ T-cell epitopes, respectively. Acute T-cell responses to natural variants of Env414-422 and NS3556-564 were largely cross-reactive and peaked during disease convalescence. The results highlight the importance of NS3 and cross-reactive T cells during acute secondary infection but suggest that the overall breadth and magnitude of the T-cell response is not significantly related to clinical disease grade. Hide abstract

Uren TK, Wijburg OL, Simmons C, Johansen FE, Brandtzaeg P, Strugnell RA. 2005. Vaccine-induced protection against gastrointestinal bacterial infections in the absence of secretory antibodies. Eur J Immunol, 35 (1), pp. 180-188. Read abstract | Read more

Secretory IgA (SIgA) is widely held to be responsible for the defense of the mucosae against pathogenics and other potentially harmful agents. In this study, polymeric Ig receptor (pIgR) knockout mice, which lack secretory antibodies (SAb), were used to investigate the role of vaccine-elicited SAb in protection against gastrointestinal bacterial infections. An essential role for specific SAb in protection against Vibrio cholerae was evident from experiments showing that vaccinated pIgR(-/-) mice, but not vaccinated C57BL/6 mice, were susceptible to cholera toxin challenge. Vaccination of C57BL/6 mice with Salmonella typhimurium elicited strong antigen-specific, mucosal responses, which blocked in vitro invasion of epithelia. However, vaccinated C57BL/6 and pIgR(-/-) mice were equally resistant to challenge infection with virulent S. typhimurium. Finally, we investigated the importance of SIgA in protection against recurrent infections with Citrobacter rodentium. Although higher numbers of bacteria were detected early after challenge infection in feces of vaccinated pIgR(-/-) mice compared with vaccinated C57BL/6 mice, both mouse strains showed complete clearance after 9 days. These results suggested that, in immune animals, SIgA is crucial for the protection of gastrointestinal surfaces against secreted bacterial toxins, may inhibit early colonization by C. rodentium, but is not essential for protection against re-infection with S. typhimurium or C. rodentium. Hide abstract

Petrovska L, Aspinall RJ, Barber L, Clare S, Simmons CP, Stratford R, Khan SA, Lemoine NR, Frankel G, Holden DW, Dougan G. 2004. Salmonella enterica serovar Typhimurium interaction with dendritic cells: impact of the sifA gene. Cell Microbiol, 6 (11), pp. 1071-1084. Read abstract | Read more

Salmonella enterica serovar Typhimurium (S. Typhimurium) and several mutant derivatives were able to enter efficiently murine bone marrow-derived dendritic cells using mechanisms predominantly independent of the Salmonella pathogenicity island 1 type III secretion system. The levels of intracellular bacteria did not increase significantly over many hours after invasion. Using fluid endocytic tracers and other markers, S. Typhimurium-containing vacuoles (SCVs) were physically distinguishable from early endocytic compartments. Fifty to eighty per cent of SCVs harbouring wild-type S. Typhimurium or aroA, invH and ssaV mutant derivatives were associated with late endosome markers. In contrast, S. Typhimurium sifA was shown to escape the SCVs into the cytosol of infected dendritic cells. S. Typhimurium aroC sifA was more efficient than S. Typhimurium aroC in delivering a eukaryotic promoter-driven green fluorescent protein reporter gene for expression in dendritic cells. In contrast, S. Typhimurium aroC sifA did not detectably increase the efficiency of MHC class I presentation of the model antigen ovalbumin to T cells compared to a similar aroC derivative. Mice infected with the S. Typhimurium aroC sifA expressing ovalbumin did not develop detectably enhanced levels of cytotoxic T cell or interferon-gamma production compared to S. Typhimurium aroC derivatives. Hide abstract

Clare S, Goldin R, Hale C, Aspinall R, Simmons C, Mastroeni P, Dougan G. 2003. Intracellular adhesion molecule 1 plays a key role in acquired immunity to salmonellosis. Infect Immun, 71 (10), pp. 5881-5891. Read abstract | Read more

This study investigated the role of intracellular adhesion molecule 1 (ICAM-1) during Salmonella enterica serovar Typhimurium infection of mice. We show that ICAM-1 is expressed in and around granulomas on day 4 of infection in wild-type mice. However, when naive ICAM-1(-/-) mice were challenged with a sublethal dose of serovar Typhimurium, there were no detectable differences in systemic bacterial burden over the first 9 days of infection compared to wild-type control mice. When mice were immunized with the S. enterica serovar Typhimurium vaccine strain SL2361 and then challenged with the virulent S. enterica serovar Typhimurium strain C5, 100% of the ICAM-1(-/-) mice succumbed to infection, compared to 30% of wild-type mice. T-cell responses, as measured by activation via interleukin-2 production, as well as antibody responses were comparable in the ICAM-1(-/-) and wild-type mice. Following challenge, counts in organs were significantly higher in the ICAM-1(-/-) mice, and histological examination of organs showed pathological differences. Strain SL3261-immunized wild-type mice had cellular infiltrate and normal granuloma formation in the liver and spleen on days 5 and 10 after challenge with strain C5. ICAM-1(-/-) mice had a similar infiltrate on day 5, whereas on day 10 the infiltrate was more widespread and there were fewer macrophages associated with the granulomas. High circulating levels of tumor necrosis factor alpha and gamma interferon, as well as a high burden of strain C5 in the blood, accompanied the differences in histopathology. In this study we show that ICAM-1 plays a critical role during rechallenge of immunized mice with virulent S. enterica serovar Typhimurium. Hide abstract

Simmons CP, Clare S, Ghaem-Maghami M, Uren TK, Rankin J, Huett A, Goldin R, Lewis DJ et al. 2003. Central role for B lymphocytes and CD4+ T cells in immunity to infection by the attaching and effacing pathogen Citrobacter rodentium. Infect Immun, 71 (9), pp. 5077-5086. Read abstract | Read more

Citrobacter rodentium, an attaching-effacing bacterial pathogen, establishes an acute infection of the murine colonic epithelium and induces a mild colitis in immunocompetent mice. This study describes the role of T-cell subsets and B lymphocytes in immunity to C. rodentium. C57Bl/6 mice orally infected with C. rodentium resolved infection within 3 to 4 weeks. Conversely, systemic and colonic tissues of RAG1(-/-) mice orally infected with C. rodentium contained high and sustained pathogen loads, and in the colon this resulted in a severe colitis. C57Bl/6 mice depleted of CD4(+) T cells, but not CD8(+) T cells, were highly susceptible to infection and also developed severe colitis. Mice depleted of CD4(+) T cells also had diminished immunoglobulin G (IgG) and IgA antibody responses to two C. rodentium virulence-associated determinants, i.e., EspA and intimin, despite having a massively increased pathogen burden. Mice with an intact T-cell compartment, but lacking B cells ( micro MT mice), were highly susceptible to C. rodentium infection. Systemic immunity, but not mucosal immunity, could be restored by adoptive transfer of convalescent immune sera to infected micro MT mice. Adoptive transfer of immune B cells, but not naïve B cells, provided highly variable immunity to recipient micro MT mice. The results suggest that B-cell-mediated immune responses are central to resolution of a C. rodentium infection but that the mechanism through which this occurs requires further investigation. These data are relevant to understanding immunity to enteric attaching and effacing bacterial pathogens of humans. Hide abstract

Mundy R, Pickard D, Wilson RK, Simmons CP, Dougan G, Frankel G. 2003. Identification of a novel type IV pilus gene cluster required for gastrointestinal colonization of Citrobacter rodentium. Mol Microbiol, 48 (3), pp. 795-809. Read abstract | Read more

Citrobacter rodentium is used as an in vivo model system for clinically significant enteric pathogens such as enterohaemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC). These pathogens all colonize the lumen side of the host gastrointestinal tract via attaching and effacing (A/E) lesion formation. In order to identify genes required for the colonization of A/E-forming pathogens, a library of signature-tagged transposon mutants of C. rodentium was constructed and screened in mice. Of the 576 mutants tested, 14 were attenuated in their ability to colonize the descending colon. Of these, eight mapped to the locus of enterocyte effacement (LEE), which is required for the formation of A/E lesions, underlying the importance of this mechanism for pathogenesis. Another mutant, P5H2, was found to have a transposon insertion in an open reading frame that has strong similarity to type IV pilus nucleotide-binding proteins. The region flanking the transposon insertion was sequenced, identifying a cluster of 12 genes that encode the first described pilus of C. rodentium (named colonization factor Citrobacter, CFC). The proteins encoded by cfc genes have identity to proteins of the type IV COF pilus of enterotoxigenic E. coli (ETEC), the toxin co-regulated pilus of Vibrio cholerae and the bundle-forming pilus of EPEC. A non-polar mutation in cfcI, complementation of this strain with wild-type cfcI and complementation of strain P5H2 with wild-type cfcH confirmed that these genes are required for colonization of the gastrointestinal tract by C. rodentium. Thus, CFC provides a convenient model to study type IV pilus-mediated pathogen-host interactions under physiological conditions in the natural colonic environment. Hide abstract

MacDonald TT, Frankel G, Dougan G, Goncalves NS, Simmons C. 2003. Host defences to Citrobacter rodentium. Int J Med Microbiol, 293 (1), pp. 87-93. Read abstract | Read more

Citrobacter rodentium is a natural non-invasive bacterial pathogen which infects the distal colon of mice. It uses the same molecular mechanisms of type III secretion as human enteropathogenic and enterohemorrhagic Escherichia coli to colonise the epithelial cells of the gut and is therefore an ideal model to study host-bacterial pathogen interactions in vivo. Infection elicits mucosal inflammation with similarities to inflammatory bowel disease, and so it is a readily accessible model to investigate the relationship between inflammation and anti-bacterial immunity in the gut. Hide abstract

Petrovska L, Lopes L, Simmons CP, Pizza M, Dougan G, Chain BM. 2003. Modulation of dendritic cell endocytosis and antigen processing pathways by Escherichia coli heat-labile enterotoxin and mutant derivatives. Vaccine, 21 (13-14), pp. 1445-1454. Read abstract | Read more

Escherichia coli heat-labile enterotoxin (LT) is known to be a potent adjuvant of both the mucosal and systemic immune systems but the mechanism of action leading to adjuvant activity remains incompletely understood. This study investigates the action of LT and LT mutants with impaired enzymatic activity, on the function of dendritic cells. Wild-type LT and LTR72, which retains some ADP ribosyltransferase activity, induced a selective increase in cell surface expression of B7.1, and a selective decrease of CD40 expression on mouse bone marrow derived dendritic cells. LTK63 and LT-B had no obvious effect on the expression of these antigens on similar dendritic cells. LT-treated dendritic cells also showed a profoundly impaired ability to present protein antigen (ovalbumin) to cognate T cells, although this effect was not observed with non-toxic LT mutants. LT and LTR72-treated cells showed a slower rate of receptor-mediated endocytosis as measured by flow cytometric analysis of uptake of fluorescently labelled dextran. Furthermore, confocal microscopy showed changes in the intracellular distribution of endocytosed molecules, and of the class II containing acidic antigen processing compartments. This response of dendritic cells to toxin is likely to play an important role in determining the adjuvant activity of these molecules. Hide abstract

Thwaites GE, Simmons CP, Than Ha Quyen N, Thi Hong Chau T, Phuong Mai P, Thi Dung N, Hoan Phu N, White NP, Tinh Hien T, Farrar JJ. 2003. Pathophysiology and prognosis in vietnamese adults with tuberculous meningitis. J Infect Dis, 188 (8), pp. 1105-1115. Read abstract | Read more

The pathogenesis of tuberculous meningitis remains unclear, and there are few data describing the kinetics of the immune response during the course of its treatment. We measured concentrations of pro- and anti-inflammatory cytokines in serial blood and cerebrospinal fluid (CSF) samples from 21 adults who were being treated for tuberculous meningitis. CSF concentrations of soluble tumor necrosis factor-alpha receptors and of matrix metalloprotein-9 and its tissue inhibitor were also measured, and blood-brain barrier permeability was assessed by the albumin and IgG partition indices. CSF concentrations of lactate, interleukin-8, and interferon-gamma were high before treatment and then decreased rapidly with antituberculosis chemotherapy. However, significant immune activation and blood-brain barrier dysfunction were still apparent after 60 days of treatment. Death was associated with high initial CSF concentrations of lactate, low numbers of white blood cells, in particular neutrophils, and low CSF glucose levels. Hide abstract

Reece S, Simmons CP, Fitzhenry RJ, Ghaem-Maghami M, Mundy R, Hale C, Matthews S, Dougan G, Phillips AD, Frankel G. 2002. Tyrosine residues at the immunoglobulin-C-type lectin inter-domain boundary of intimin are not involved in Tir-binding but implicated in colonisation of the host. Microbes Infect, 4 (14), pp. 1389-1399. Read abstract | Read more

Intimin is an outer membrane adhesion molecule involved in bacterial adhesion to intestinal epithelium by several human and animal enteric pathogens, including enteropathogenic and enterohaemorrhagic Escherichia coli and Citrobacter rodentium. Intimin binds to the translocated intimin receptor, Tir, which is delivered to the plasma membrane of the host cell by a type III protein translocation system. Intimin is also implicated in binding to a host cell-encoded intimin receptor (Hir). The receptor-binding activity of intimin resides within the carboxy terminus 280 amino acids (Int280) of the polypeptide. Structural analysis of this region revealed two immunoglobulin-like domains, the second of which forms a number of contacts with the distal C-type lectin-like module. Specific orientation differences at this inter-domain boundary, which consists of several tyrosine residues, were detected between the crystal and solution structures. In this study, we determined the influence of site-directed mutagenesis of each of four tyrosine residues on intimin-Tir interactions and on intimin-mediated intimate attachment. The mutant intimins were also studied using a variety of in vitro and in vivo infection models. The results show that three of the four Tyr, although not essential for A/E lesion formation in vitro, are required for efficient colonisation of the mouse host following oral challenge. Hide abstract

Reece S, Simmons CP, Fitzhenry RJ, Batchelor M, Hale C, Matthews S, Phillips AD, Dougan G, Frankel G. 2002. Mutagenesis of conserved tryptophan residues within the receptor-binding domain of intimin: influence on binding activity and virulence. Microbiology, 148 (Pt 3), pp. 657-665. Read abstract

Intimate bacterial adhesion to intestinal epithelium is a pathogenic mechanism shared by several human and animal enteric pathogens, including enteropathogenic and enterohaemorrhagic Escherichia coli and Citrobacter rodentium. The proteins directly involved in this process are the outer-membrane adhesion molecule intimin and the translocated intimin receptor, Tir. The receptor-binding activity of intimin resides within the carboxy terminus 280 aa (Int280) of the polypeptide. Four tryptophan residues, W117/776, W136/795, W222/881 and W240/899, are conserved within different Int280 molecules that otherwise show considerable sequence variation. In this study the influence of site-directed mutagenesis of each of the four tryptophan residues on intimin-Tir interactions and on intimin-mediated intimate attachment was determined. The mutant intimins were also studied using a variety of in vitro and in vivo infection models. The results show that all the substitutions modulated intimin activity, although some mutations had more profound effects than others. Hide abstract

Simmons CP, Goncalves NS, Ghaem-Maghami M, Bajaj-Elliott M, Clare S, Neves B, Frankel G, Dougan G, MacDonald TT. 2002. Impaired resistance and enhanced pathology during infection with a noninvasive, attaching-effacing enteric bacterial pathogen, Citrobacter rodentium, in mice lacking IL-12 or IFN-gamma. J Immunol, 168 (4), pp. 1804-1812. Read abstract

Mice infected with Citrobacter rodentium represent an excellent model in which to examine immune defenses against an attaching-effacing enteric bacterial pathogen. Colonic tissue from mice infected with C. rodentium harbors increased transcripts for IL-12 and IFN-gamma and displays mucosal pathology compared with uninfected controls. In this study, the role of IL-12 and IFN-gamma in host defense and mucosal injury during C. rodentium infection was examined using gene knockout mice. IL-12p40(-/-) and IFN-gamma(-/-) mice were significantly more susceptible to mucosal and gut-derived systemic C. rodentium infection. In particular, a proportion of IL-12p40(-/-) mice died during infection. Analysis of the gut mucosa of IL-12p40(-/-) mice revealed an influx of CD4(+) T cells and a local IFN-gamma response. Infected IL-12p40(-/-) and IFN-gamma(-/-) mice also mounted anti-Citrobacter serum and gut-associated IgA responses and strongly expressed inducible NO synthase (iNOS) in mucosal tissue, despite diminished serum nitrite/nitrate levels. However, iNOS does not detectably contribute to host defense against C. rodentium, as iNOS(-/-) mice were not more susceptible to infection. However, C57BL/6 mice infected with C. rodentium up-regulated expression of the mouse beta-defensin (mBD)-1 and mBD-3 in colonic tissue. In contrast, expression of mBD-3, but not mBD-1, was significantly attenuated during infection of IL-12- and IFN-gamma-deficient mice, suggesting mBD-3 may contribute to host defense. These studies are among the first to examine mechanisms of host resistance to an attaching-effacing pathogen and show an important role for IL-12 and IFN-gamma in limiting bacterial infection of the colonic epithelium. Hide abstract

Gonçalves NS, Ghaem-Maghami M, Monteleone G, Frankel G, Dougan G, Lewis DJ, Simmons CP, MacDonald TT. 2001. Critical role for tumor necrosis factor alpha in controlling the number of lumenal pathogenic bacteria and immunopathology in infectious colitis. Infect Immun, 69 (11), pp. 6651-6659. Read abstract | Read more

Infection of mice with the intestinal bacterial pathogen Citrobacter rodentium results in colonic mucosal hyperplasia and a local Th1 inflammatory response similar to that seen in mouse models of inflammatory bowel disease. In these latter models, and in patients with Crohn's disease, neutralization of tumor necrosis factor alpha (TNF-alpha) is of therapeutic benefit. Since there is no information on the role of TNF-alpha in either immunity to noninvasive bacterial pathogens or on the role of TNF-alpha in the immunopathology of infectious colitis, we investigated C. rodentium infection in TNFRp55(-/-) mice. In TNFRp55(-/-) mice, there were higher colonic bacterial burdens, but the organisms were cleared at the same rate as C57BL/6 mice, showing that TNF-alpha is not needed for protective antibacterial immunity. The most striking feature of infection in TNFRp55(-/-) mice, however, was the markedly enhanced pathology, with increased mucosal weight and thickness, increased T-cell infiltrate, and a markedly greater mucosal Th1 response. Interleukin-12 p40 transcripts were markedly elevated in C. rodentium-infected TNFRp55(-/-) mice, and this was associated with enhanced mucosal STAT4 phosphorylation. TNF-alpha is not obligatory for protective immunity to C. rodentium in mice; however, it appears to play some role in downregulating mucosal pathology and Th1 immune responses. Hide abstract

Ghaem-Maghami M, Simmons CP, Daniell S, Pizza M, Lewis D, Frankel G, Dougan G. 2001. Intimin-specific immune responses prevent bacterial colonization by the attaching-effacing pathogen Citrobacter rodentium. Infect Immun, 69 (9), pp. 5597-5605. Read abstract | Read more

The formation of attaching and effacing (A/E) lesions on gut enterocytes is central to the pathogenesis of enterohemorrhagic (EHEC) Escherichia coli, enteropathogenic E. coli (EPEC), and the rodent pathogen Citrobacter rodentium. Genes encoding A/E lesion formation map to a chromosomal pathogenicity island termed the locus of enterocyte effacement (LEE). Here we show that the LEE-encoded proteins EspA, EspB, Tir, and intimin are the targets of long-lived humoral immune responses in C. rodentium-infected mice. Mice infected with C. rodentium developed robust acquired immunity and were resistant to reinfection with wild-type C. rodentium or a C. rodentium derivative, DBS255(pCVD438), which expressed intimin derived from EPEC strain E2348/69. The receptor-binding domain of intimin polypeptides is located within the carboxy-terminal 280 amino acids (Int280). Mucosal and systemic vaccination regimens using enterotoxin-based adjuvants were employed to elicit immune responses to recombinant Int280alpha from EPEC strain E2348/69. Mice vaccinated subcutaneously with Int280alpha, in the absence of adjuvant, were significantly more resistant to oral challenge with DBS255(pCVD438) but not with wild-type C. rodentium. This type-specific immunity could not be overcome by employing an exposed, highly conserved domain of intimin (Int388-667) as a vaccine. These results show that anti-intimin immune responses can modulate the outcome of a C. rodentium infection and support the use of intimin as a component of a type-specific EPEC or EHEC vaccine. Hide abstract

Simmons CP, Clare S, Dougan G. 2001. Understanding mucosal responsiveness: lessons from enteric bacterial pathogens. Semin Immunol, 13 (3), pp. 201-209. Read abstract | Read more

Mucosal immune responses must discriminate between commensal flora within the lumen and potential pathogens. These responses are highly adapted to induce protection without excessive inflammation. The balances that regulate mucosal immune and inflammatory responses have to be understood if effective mucosal immunity is to be induced through local immunization. This review will summarize some of the lessons learnt from studies of antigens derived from enteric bacterial pathogens and discuss how the gastrointestinal epithelia can 'fight back' when it encounters pathogens. Hide abstract

Chiesa MD, Martensen PM, Simmons C, Porakishvili N, Justesen J, Dougan G, Roitt IM, Delves PJ, Lund T. 2001. Refocusing of B-cell responses following a single amino acid substitution in an antigen. Immunology, 103 (2), pp. 172-178. Read abstract | Read more

Intranasal immunization of BALB/c strain mice was carried out using baculovirus-derived human chorionic gonadotrophin (hCG) beta-chain, together with Escherichia coli heat-labile enterotoxin. Gonadotrophin-reactive immunoglobulin A (IgA) was induced in a remote mucosal site, the lung, in addition to a systemic IgG response. The extensive sequence homology with luteinizing hormone (LH) results in the production of LH cross-reactive antibodies when holo-hCG is used as an immunogen. In contrast to wild-type hCGbeta, a mutated hCGbeta-chain containing an arginine to glutamic acid substitution at position 68 did not induce the production of antibodies which cross-react with LH. Furthermore, the epitopes utilized in the B-cell response to the mutated hCGbeta shifted away from the immunodominant region of the parent wild-type molecule towards epitopes within the normally weakly immunogenic C terminus. This shift in epitope usage was also seen following intramuscular immunization of rabbits. Thus, a single amino acid change, which does not disrupt the overall structure of the molecule, refocuses the immune response away from a disadvantageous cross-reactive epitope region and towards a normally weakly immunogenic but antigen-unique area. Similar mutational strategies for epitope-refocusing may be applicable to other vaccine candidate molecules. Hide abstract

Reece S, Simmons CP, Fitzhenry RJ, Matthews S, Phillips AD, Dougan G, Frankel G. 2001. Site-directed mutagenesis of intimin alpha modulates intimin-mediated tissue tropism and host specificity. Mol Microbiol, 40 (1), pp. 86-98. Read abstract | Read more

The hallmark of enteropathogenic (EPEC) and enterohaemorrhagic (EHEC) Escherchia coli adhesion to host cells is intimate attachment leading to the formation of distinctive 'attaching and effacing' lesions. This event is mediated, in part, by binding of the bacterial adhesion molecule intimin to a second bacterial protein, Tir, delivered by a type III secretion system into the host cell plasma membrane. The receptor-binding activity of intimin is localized to the C-terminal 280 amino acids (Int280) and at least five distinct intimin types (alpha, beta, gamma, delta and epsilon) have been identified thus far. In addition to binding to Tir, intimin can also bind to a component encoded by the host. The consequence of latter intimin-binding activity may determine tissue tropism and host specificity. In this study we selected three amino acids in intimin, which are implicated in Tir binding, for site-directed mutagenesis. We used the yeast two-hybrid system and gel overlays to study intimin-Tir protein interaction. In addition, the biological consequences of the mutagenesis was tested using a number of infection models (cultured epithelial cells, human intestinal explants and a mouse model). We report that while an I237/897A substitution (positions numbered according to Int280alpha/whole intimin alpha) in intimin alpha did not have any affect on its biological activity, a T255/914A substitution attenuated intimin activity in vivo. In contrast, the mutation V252/911A affected tissue targeting in the human intestinal explant model and attenuated the biological activity of intimin in the mouse model. This study provides the first clues of the molecular basis of how intimin mediates tissue tropism and host specificity. Hide abstract

Simmons CP, Ghaem-Magami M, Petrovska L, Lopes L, Chain BM, Williams NA, Dougan G. 2001. Immunomodulation using bacterial enterotoxins. Scand J Immunol, 53 (3), pp. 218-226. Read abstract | Read more

Immunologic unresponsiveness (tolerance) is a key feature of the mucosal immune system, and deliberate vaccination by a mucosal route can effectively induce immune suppression. However, some bacterial-derived proteins, e.g. cholera toxin and the heat labile toxin of Escherichia coli, are immunogenic and immunomodulatory at mucosal surfaces and can effectively adjuvant immune responses to codelivered bystander antigens. This review summarizes some of the structural and biological characteristics of these toxins and provides examples of how these properties have been exploited for tolerance induction and mucosal vaccine development. Hide abstract

Goncalves N, Simmons CP, Ghaem-Maghami M, Monteleone G, Bajaj-Elliott M, Goldin R, Frankel G, Dougan G, MacDonald TT. 2001. The role of IL-12 and IFN-gamma in immunity to Citrobacter rodentium infection GUT, 48 pp. A75-A75.

Goncalves N, Simmons CP, Ghaem-Maghami M, Monteleone G, Frankel G, Dougan G, MacDonald TT. 2001. Increased susceptibility of TNFRp55 deficient mice to Citrobacter rodentium (mouse EPEC) infection GUT, 48 pp. A75-A75.

Simmons CP, Hussell T, Sparer T, Walzl G, Openshaw P, Dougan G. 2001. Mucosal delivery of a respiratory syncytial virus CTL peptide with enterotoxin-based adjuvants elicits protective, immunopathogenic, and immunoregulatory antiviral CD8+ T cell responses. J Immunol, 166 (2), pp. 1106-1113. Read abstract

In an effort to develop a safe and effective vaccine against respiratory syncytial virus (RSV), we used Escherichia coli heat-labile toxin (LT), and LTK63 (an LT mutant devoid of ADP-ribosyltransferase activity) to elicit murine CD8(+) CTL responses to an intranasally codelivered CTL peptide from the second matrix protein (M2) of RSV. M2(82-90)-specific CD8(+) T cells were detected by IFN-gamma enzyme-linked immunospot and (51)Cr release assay in local and systemic lymph nodes, and their induction was dependent on the use of a mucosal adjuvant. CTL elicited by peptide immunization afforded protection against RSV challenge, but also enhanced weight loss. CTL-mediated viral clearance was not dependent on IFN-gamma since depletion using specific mAb during RSV challenge did not affect cellular recruitment or viral clearance. Depletion of IFN-gamma did, however, reduce the concentration of TNF detected in lung homogenates of challenged mice and largely prevented the weight loss associated with CTL-mediated viral clearance. Mice primed with the attachment glycoprotein (G) develop lung eosinophilia after intranasal RSV challenge. Mucosal peptide vaccination reduced pulmonary eosinophilia in mice subsequently immunized with G and challenged with RSV. These studies emphasize that protective and immunoregulatory CD8(+) CTL responses can be mucosally elicited using enterotoxin-based mucosal adjuvants but that resistance against viral infection may be accompanied by enhanced disease. Hide abstract

Dougan G, Ghaem-Maghami M, Pickard D, Frankel G, Douce G, Clare S, Dunstan S, Simmons C. 2000. The immune responses to bacterial antigens encountered in vivo at mucosal surfaces. Philos Trans R Soc Lond B Biol Sci, 355 (1397), pp. 705-712. Read abstract | Read more

Mammals have evolved a sophisticated immune system for handling antigens encountered at their mucosal surfaces. The way in which mucosally delivered antigens are handled influences our ability to design effective mucosal vaccines. Live attenuated derivatives of pathogens are one route towards the development of mucosal vaccines. However, some molecules, described as mucosal immunogens, are inherently immunogenic at mucosal surfaces. Studies on mucosal immunogens may facilitate the identification of common characteristics that contribute to mucosal immunogenicity and aid the development of novel, non-living mucosal vaccines and immunostimulators. Hide abstract

Goncalves NS, Ghaemmaghami M, Frankel G, Simmons C, Dougan G, MacDonald TT. 2000. Increased bacterial burden and inflammation in TNF receptor knockout mouse infected with Citrobacter rodentium (mouse EPEC). GASTROENTEROLOGY, 118 (4), pp. A574-A574.

Mastroeni P, Simmons C, Fowler R, Hormaeche CE, Dougan G. 2000. Igh-6(-/-) (B-cell-deficient) mice fail to mount solid acquired resistance to oral challenge with virulent Salmonella enterica serovar typhimurium and show impaired Th1 T-cell responses to Salmonella antigens. Infect Immun, 68 (1), pp. 46-53. Read abstract | Read more

In the present study we evaluated the role of B cells in acquired immunity to Salmonella infection by using gene-targeted B-cell-deficient innately susceptible mice on a C57BL/6 background (Igh-6(-/-)). Igh-6(-/-) mice immunized with a live, attenuated aroA Salmonella enterica serovar Typhimurium vaccine strain showed impaired long-term acquired resistance against the virulent serovar Typhimurium strain C5. Igh-6(-/-) mice were able to control a primary infection and to clear the inoculum from the reticuloendothelial system. However, Igh-6(-/-) mice, unlike Igh-6(+/+) C57BL/6 controls, did not survive an oral challenge with strain C5 at 4 months after vaccination. Transfer of immune serum did not restore resistance in Igh-6(-/-) mice. Total splenocytes and purified CD4(+) T cells obtained from Igh-6(-/-) mice 4 months after vaccination showed reduced ability to release Th1-type cytokines (interleukin 2 and gamma interferon) upon in vitro restimulation with serovar Typhimurium soluble cell extracts compared to cells obtained from Igh-6(+/+) C57BL/6 control mice. Therefore, the impaired resistance to oral challenge with virulent serovar Typhimurium observed in B-cell-deficient mice, which cannot be restored by passive transfer of Salmonella-immune serum, may be in part due to a reduced serovar Typhimurium-specific T-cell response following primary immunization. Hide abstract

Simmons CP, Mastroeni P, Fowler R, Ghaem-maghami M, Lycke N, Pizza M, Rappuoli R, Dougan G. 1999. MHC class I-restricted cytotoxic lymphocyte responses induced by enterotoxin-based mucosal adjuvants. J Immunol, 163 (12), pp. 6502-6510. Read abstract

The ability of enterotoxin-based mucosal adjuvants to induce CD8+ MHC class I-restricted CTL responses to a codelivered bystander Ag was examined. Escherichia coli heat-labile toxin (LT), or derivatives of LT carrying mutations in the A subunit (LTR72, LTK63), were tested in parallel with cholera toxin (CT) or a fusion protein consisting of the A1 subunit of CT fused to the Ig binding domain of Staphylococcus aureus protein A (called CTA1-DD). Intranasal (i.n.) immunization of C57BL/6 mice with CT, CTA1-DD, LT, LTR72, LTK63, but not rLT-B, elicited MHC class I-restricted CD8+ T cell responses to coadministered OVA or the OVA CTL peptide SIINFEKL (OVA257-264). CT, LT, and LTR72 also induced CTL responses to OVA after s.c. or oral coimmunization whereas LTK63 only activated responses after s.c. coimmunization. rLT-B was unable to adjuvant CTL responses to OVA or OVA257-264 administered by any route. Mice treated with an anti-CD4 mAb to deplete CD4+ T cells mounted significant OVA-specific CTL responses after i.n. coadministration of LT with OVA or OVA257-264. Both 51Cr release assays and IFN-gamma enzyme-linked immunospot assays indicated that IFN-gamma-/- and IL-12 p40-/- gene knockout mice developed CTL responses equivalent to those detected in normal C57BL/6 mice. The results highlight the versatility of toxin-based adjuvants and suggest that LT potentiates CTL responses independently of IL-12 and IFN-gamma and probably by a mechanism unrelated to cross-priming. Hide abstract

Mastroeni P, Bowe F, Cahill R, Simmons C, Dougan G. 1999. Vaccines against gut pathogens. Gut, 45 (5), pp. 633-635. | Read more

Dunstan SJ, Simmons CP, Strugnell RA. 1999. Use of in vivo-regulated promoters to deliver antigens from attenuated Salmonella enterica var. Typhimurium. Infect Immun, 67 (10), pp. 5133-5141. Read abstract

This study describes the construction and analysis of three in vivo-inducible promoter expression plasmids, containing pnirB, ppagC, and pkatG, for the delivery of foreign antigens in the DeltaaroAD mutant of Salmonella enterica var. Typhimurium (hereafter referred to as S. typhimurium). The reporter genes encoding beta-galactosidase and firefly luciferase were used to assess the comparative levels of promoter activity in S. typhimurium in vitro in response to different induction stimuli and in vivo in immunized mice. It was determined that the ppagC construct directed the expression of more beta-galactosidase and luciferase in S. typhimurium than the pnirB and pkatG constructs, both in vitro and in vivo. The gene encoding the C fragment of tetanus toxin was expressed in the aroAD mutant of S. typhimurium (BRD509) under the control of the three promoters. Mice orally immunized with attenuated S. typhimurium expressing C fragment under control of the pagC promoter [BRD509(pKK/ppagC/C frag)] mounted the highest tetanus toxoid-specific serum antibody response. Levels of luciferase expression in vivo and C-fragment expression in vitro from the pagC promoter appeared to be equivalent to if not lower than the levels of expression detected with the constitutive trc promoter. However, mice immunized with BRD509(pKK/ppagC/C frag) induced significantly higher levels of tetanus toxoid-specific antibody than BRD509(pKK/C frag)-immunized mice, suggesting that the specific location of foreign antigen expression may be important for immunogenicity. Mutagenesis of the ribosome binding sites (RBS) in the three promoter/C fragment expression plasmids was also performed. Despite optimization of the RBS in the three different promoter elements, the expression levels in vivo and overall immunogenicity of C fragment when delivered to mice by attenuated S. typhimurium were not affected. These studies suggest that in vivo-inducible promoters may give rise to enhanced immunogenicity and increase the efficacy of S. typhimurium as a vaccine vector. Hide abstract

Simmons CP, Farrar J, Chau VVC, Wills B. Dengue New England Journal of Medicine, 15 pp. 1423-1432.

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