FUNDING ORGANIZATION	RESEARCH ORGANIZATION	PROGRAM	DIRECTOR	CITY	COUNTRY	ABSTRACT
	MRC	MRC UNIT, THE GAMBIA	AETIOLOGY OF SEVERE PNEUMONIA IN GAMBIAN CHILDREN	HOWIE, S R C	FAJARA	GAMBIA	View
"Identification of the pathogens responsible for severe pneumonia in children is important for guiding treatment, identifying patterns of antimicrobial resistance, and planning prevention programmes. Conventional methods for determining the aetiology have significant limitations in sensitivity and specificity. There are difficulties both with obtaining appropriate clinical specimens and with analysing them meaningfully in the laboratory. Better methods for specimen collection and the identification of pathogens are urgently needed, therefore. We also need to be able to tell if an organism is causing disease or simply acting as a bystander.||This study is one of the severe pneumonia platform studies included in the QQ proposal. The objectives of this study are to |1. define the aetiology of severe pneumonia in Gambian children using the best available methods, |2. compare the utility of different specimen collection techniques, |3. assess the utility of molecular methods in determining aetiology |4. identify meaningful laboratory correlates of disease, and|5. To translate the findings of the study into practical approaches to improve diagnosis and treatment of patients||We will adopt a 4-dimensional strategy to the laboratory aspects of this challenge: |1. identification of all organisms present, |2. quantification of organisms, |3. measurement of organism markers of pathogenicity, and |4. measurement of markers of host-response to disease. |The ultimate aims of the work are to guide disease prevention work (including the development of vaccines) and to contribute to the development of practical diagnostic tools that will improve the outcome for children suffering from severe pneumonia and possibly other severe infections of childhood."
	MRC	MRC UNIT, THE GAMBIA	AN INVESTIGATION INTO THE EFFECTS OF PLASMODIUM FALCIPARUM INFECTION ON CELLULAR RESPONSES TO EPSTEIN BARR VIRUS	JAYASOORIYA, S	FAJARA	GAMBIA	View
The endemic form of Burkitts lymphoma (BL) discovered in Africa is strongly associated with Epstein Barr Virus (EBV) unlike its subsequently discovered sporadic and HIV associated counterparts. The endemic BL regions of Africa coincide with holoendemic malaria areas and have high prevalence rates of early EBV infection. EBV establishes a life-long latent infection in B-lymphocytes and is controlled by cytotoxic CD8 T cells. Previous studies suggest that P.falciparum infection suppresses the function of EBV-specific cytotoxic T cells. We aim to carry out the long overdue exploration of these cellular responses to EBV in order to confirm whether or not there is a true reduction in EBV specific T cell responses during acute malaria infections. This will be done by taking samples of blood from children with mild malaria before and after treatment and enumerating and comparing their immune responses to EBV at these time points. In order to determine if the immune response to EBV is recovered 28 days after treatment is started immune responses at this point will be compared to healthy control children.||If our findings confirm what we suspect in a second phase of the study we will explore in vitro mechanisms which could account for the loss of host cellular responses to EBV. Specifically we hope to explore whether the following play a role in impairing EBV specific T cell responses during acute malaria infection using a healthy adult donor in vitro system.||1) P falciparum induced T regulatory cells.|2) Soluble factors derived from Dendritic Cells (DCs) exposed to P.falciparum infected red blood cells||This project will describe the changes in EBV specific cellular immunity during acute malaria infection and dependent on these findings explore further mechanistic hypotheses to explain these. In doing so, it will aid understanding of the pathogenesis of endemic Burkitts lymphoma and the immunosuppressive properties of malaria.
	MRC	MRC UNIT, THE GAMBIA	CAN INDOOR RESIDUAL SPRAYING PROVIDE ADDITIONAL PROTECTION AGAINST CLINICAL MALARIA OVER CURRENT BEST PRACTICE?	JAWARA, M	FAJARA	GAMBIA	View
A 2 armed cluster-randomised controlled trial will be conducted to assess whether DDT IRS and LLINs combined provide better protection against clinical malaria in children, aged from 6-120 months, than LLINs alone. Clinical malaria will be recorded using passive case detection and exposure to malaria parasites assessed using light and exit traps followed by detection of Anopheles gambiae species and sporozoite infection. Susceptibility of vectors to insecticides will be measured phenotypically and by biomarkers for insecticide-resistance genes. A detailed economic assessment will be made to estimate the additional cost of averting a clinical case of malaria using IRS.
	MRC	MRC UNIT, THE GAMBIA	COMPARISON OF EXPRESSION PROFILES IN SPUTUM BETWEEN M. AFRICANUM AND M. TUBERCULOSIS	DE JONG, B	FAJARA	GAMBIA	View
"M. africanum, phylogenetically situated between M. tuberculosis and M. bovis in the M. tuberculosis complex, is an important cause of TB in West-Africa. Within the TB case contact studies, we genotyped isolates using spoligotyping and PCRs for lineage defining Large Sequence Polymorphisms. We correlate these genotypes with the clinical and immunological findings from TB cases and their household contacts (also see SCC 965).||Among 376 clinical isolates, 38% was identified as M. africanum and the rest as M. tuberculosis. Patients infected with M. africanum were more likely to be HIV positive (p=0.024), and more likely to be of Fula ethnicity (p=0.042). There was no difference in clinical or radiological findings between cases diseased with M. africanum relative to M. tuberculosis, and mortality was similar in those who were HIV negative and adherent to treatment (p=0.95). Among the 2495 contacts, transmission of M. africanum was similar to that of M. tuberculosis, as measured by Mantoux skin test (p=0.10). However, similarly to cases, contacts exposed to M. africanum were less likely to generate an IFNgamma response to ESAT-6 than those exposed to M. tuberculosis (p=0.024 for cases and p=0.004 for contacts). In addition, contacts of M. africanum were less likely to progress to disease during the 2 year follow-up (p=0.030).||The current proposal aims to compare the genome and the ex vivo transcriptome of M. africanum relative to M. tuberculosis in order to clarify the genetic underpinnings of these phenotypic differences.||The genomic comparison will be done in collaboration with the Wellcome Trust Sanger Centre which is in the process of sequencing one of our M. africanum isolates. Transcriptome comparisons will be conducted in parallel in collaboration with Stanford University (RT-qPCR for all 1200 operons of M. tuberculosis) and the Max Planck Institute (microarray after RNA-amplification).||So far, we have collected 39 of 60 sputum samples from TB patients directly in RNA fixative and extracted their RNA. Parallel samples are processed for DNA extraction and genotyping. The molecular work will be done by the Principal Investigator in the collaborating laboratories."
	MRC	MRC UNIT, THE GAMBIA	COMPARISON OF T LYMPHOCYTE KINETICS IN HIV-1 AND HIV-2 INFECTIONS	JAYE, A	FAJARA	GAMBIA	View
"HIV-2 infected individuals survive longer and have a more protracted stable CD4 T cell count than those infected with HIV-1. Studies in our laboratory have shown that both infections exhibit similar levels of cell mediated immune responses, when matched at high CD4 T cell count, but quality of effector functional responses are superior in HIV-2 infection, probably accounting for its lower viral replication. The difference in immunological and clinical course of HIV-1 and HIV-2 infections is likely to be related to the interaction of the virus and the survival of lymphocyte subpopulations. We hypothesise that, in HIV-2 infection, there is either less marked destruction of CD4 cells or an enhanced ability to replace lost CD4 cells. Destruction would be less marked if immune activation had less impact on proliferation rates; this would be expected to allow greater preservation of naove CD4 T cells. Alternatively, in HIV-2, lost cells may be replaced more efficiently. We will investigate this by comparing proliferation and disappearance rates of naove and memory T lymphocytes in 20 HIV-2 with 20 HIV-1 infected subjects, matched for CD4 count, and in healthy sero-negative controls. This will be performed by measuring the kinetics of incorporation of deuterium from orally administered deuterated glucose into DNA of lymphocyte subpopulations. We will relate such measurements to markers of activation (HLA-DR, CD38, CD69) and proliferation (nuclear antigen Ki67)."
	MRC	MRC UNIT, THE GAMBIA	CONJUNCTIVAL GENE EXPRESSION PROFILE BY MINI-OLIGO ARRAY FOR CYTOKINES & FIBROGENIC FACTORS IN SUBJECTS WITH TRICHIASIS	HOLLAND, M	FAJARA	GAMBIA	View
"Trachomatous Triachiasis is end result of severe conjunctival infections with C. trachomatis over many years. The underlying causes of disease are the immunologically driven responses which lead to conjunctival fibrosis. The relationship between the immune response and the fibrotic process is poorly defined in trachoma but in other infectious diseases the process is being unraveled. Understanding these processes offers the opportunity to develop an intervention which is based on blocking the development of pathology rather than one which focuses on sterilizing immunity which could itself lead to pathology. Using mini-Oligo DNA gene arrays for extracellular matrix proteins and adhesion molecules along with a cytokine array gives us the opportunity to test ~200 genes. By taking this focused approach we should identify the major genes and pathways involved in conjunctival fibrosis.||Methodology and Equipment: We will recruit subjects with trachomatous trichiasis (TT) and age, sex and location matched controls. From a sub-group of these subjects, 10 TT cases and 10 controls a conjunctival swab will be taken for the isolation of mRNA. All cases will be offered treatment under standard Gambian National Eye Care Programme (NECP) guidelines and protocols. TT surgery will conducted by local NECP community ophthalmic nurses. Arrays for Human Extracellular Matrix and adhesion molecules, Interleukins and Receptors and Th1/Th2/Th3 (GEArray Q superarray Series). Access to bioinformatic analysis software for reading and interpretation of the array is via the internet from Superarray.||Practical and Ethical considerations: Subjects with trichiasis will be referred for surgery conducted by trained NECP personnel. The presence of trachomatous scarring does not require specific treatment, but subjects are counselled according to NECP practice.||Results: Two low density gene arrays were used which covered 226 genes for extracellular matrix proteins/adhesion molecules and cytokines. This will be followed by quantitative gene expression by real-time RT-PCR on conjunctival RNA samples from a larger number of subjects with trichiasis and controls. Mini-gene array results identified that the transcription factor Jun B proto-oncogene (JunB) was up-regulated in the conjunctiva of TT subjects and implies a differential gene expression of downstream targets which this gene controls.||Conclusions: Interestingly, in JunB deficient mice, delayed wound healing and epidermal hyperproliferation is observed and in vitro experiments show that JunB is involved in the paracrine regulatory cytokine networks that control skin homeostasis and regeneration. The results of quantitative expression of JunB and a number of other genes known to be involved in other immune mediated fibrotic diseases (TNF, IL1?, IL-11, IL-11R?, IL-13 and IL-13R?2) will now be further investigated in immune mediated conjunctival scarring. Understanding the pathological process may lead to the possible application of a pharmacological intervention to the progression of conjunctival scarring."
	MRC	MRC UNIT, THE GAMBIA	DEVELOPMENT AND EVALUATION OF SEROTYPE-SPECIFIC PCRS FOR DETECTION OF STREPTOCOCCUS PNEUMONIA IN CLINICAL SPECIMENS	ANTONIO, M	FAJARA	GAMBIA	View
"Pneumococcal conjugate vaccines are efficacious against invasive pneumococcal disease in young children (Black 2000) and have an effect against carriage of pneumococci (Klugman 2001). There are reports of increased rates of colonization by non-vaccine serotypes after vaccination (Obaro, 1996; Sprat, 200) and concerns have been expressed about possible consequences of serotype replacement in vaccinated communities (Obaro, 2002). Long term effects of vaccines that project against some and not all of pneumococcal serotypes are still uncertain. Therefore, it is important to have continuing surveillance of pneumococcal serotypes from invasive and carriage sources. Current methods are insensitive for laboratory diagnosis of pneumococcal disease, particularly pneumonia and are grossly inadequate for studying colonization by multiple serotypes of Streptococcus pneumoniae. True carriage rates of multiple serotypes are yet not known because the methods for detecting multiple serotypes have relied mainly on picking several colonies directly from culture place for serotyping. We recently developed a two-step multiplex PCR assay, which is capable of distinguishing 9 pneumococcal serotypes commonly found in The Gambia. We now propose to (1) test strains of other bacterial species and other pneumococcal serotypes for confirmation of specificity and sensitivity of developed serotype specific primers and (2) do a formal evaluation of the PCR assay for direct detection of pneumococcal sterotypes in clinical specimens."
	MRC	MRC UNIT, THE GAMBIA	DIFFERENTIAL GENE EXPRESSION OF LEUKOCYTE SUBPOPULATIONS IN SEVERE AND MILD MALARIA	WALTHER, M	FAJARA	GAMBIA	View
Analysis of differential gene expression between mild and severe malaria provides insight into what genes influence the development of complicated disease, a poorly understood process. Microarray technology combined with sophisticated computational analysis tools and confirmatory molecular biology experiments are a powerful method of identifying pathways that determine poor outcome in disease and can thus be targeted for therapy. Preliminary work using microarray analysis in animal models of malaria has identified pathways putatively involved in the development of anemia or cerebral malaria.||Studies of Plasmodium falciparum infection in people have only begun to examine gene expression of parasites, or of peripheral blood mononuclear cells, and have not comprehensively examined differential gene expression between severe and mild malaria.||Using samples collected from our well-established platform recruiting children with severe and uncomplicated malaria, the aim of this study is to investigate global transcriptional differences between cases of severe and mild malaria, and to compare gene expression of major leukocyte subpopulations between these groups (CD4+, CD8+, CD14+) that will be isolated using magnetic bead-labelled antibodies. Genome-wide expression microarray analysis will be performed.||The goals of this study are two-fold to identify what genes and pathways are involved in the development of severe malaria and also what leukocyte populations may be responsible for the pathophysiological changes seen in severe disease.||We are currently in the process of optimizing depletion strategies for cell subsets and are exploring ways to improve the yield of high quality mRNA from these cell subsets. It is planned to recruit up to 10 samples of severe disease and 10 samples of uncomplicated disease for this study during the next malaria transmission season.||The arrays will be performed in collaboration with Prof Paul Kayes lab and Dr Naveed Aziz at the University of York.
	MRC	MRC UNIT, THE GAMBIA	DOUBLE BLIND PLACEBO-CONTROLLED RANDOMIZED TRIAL OF ISONIAZID FOR REVERSION OF POSITIVE IFNG ELISPOT IN TB CASE CONTACTS	ADETIFA, I	FAJARA	GAMBIA	View
"Current efforts to control the spread of tuberculosis are failing. An increasingly large number of new generation vaccines are being produced and a plan for assessing their ability to prevent disease and treat infection needs to be developed. The MRC Labs in The Gambia is well positioned to conduct safety and immunogenicity studies and also to conduct trials of the therapeutic effect of these vaccines in preventing disease in case contacts who are infected. This study is part one of a three-step plan to develop a reliable early surrogate marker of the therapeutic efficacy of new TB vaccines.The three-step plan is as follows:Evaluate the ability of isoniazid, known to be effective in the treatment of MTB infection, to revert the antigen-specific IFNg-ELISPOT in ESAT 6 and/or CFP-10 positive contacts of TB patients.Compare the ability of different combinations of a TB vaccine and isoniazid to revert the ELISPOT in a 4-arm randomized trial using:1) isoniazid alone, 2) a TB vaccine alone, 3) a combination of isoniazid and TB vaccine, and 4) placebo Compare, in a randomized trial, the ability of TB vaccine or vaccine plus isoniazid versus isoniazid alone to prevent the development of secondary disease.For this first step we will test the following hypothesis:Those receiving isoniazid have a significantly higher reversion rate of MTB-specific responses as measured with IFNg-ELISPOT assays compared to those who receive a placebo"
	MRC	MRC UNIT, THE GAMBIA	EFFICACY OF ZINC AS AN ADJUNCT THERAPY IN THE MANAGEMENT OF SEVERE PNEUMONIA AMONG GAMBIAN CHILDREN	HOWIE, S R C	FAJARA	GAMBIA	View
"Zinc is the most abundant trace metal inside most cells. It is a functionally essential component of more than 200 enzymes, pervading all metabolic pathways. The beneficial role of zinc in the maintenance and regeneration of pulmonary epithelial cells, cellular growth and differentiation, improvement of immune status, recovery from acute diarrhoea, and prevention of severe ALRI or pneumonia in children suggests the potential benefit of zinc in the treatment of pneumonia in children. Stunted children are at increased risk of zinc deficiency and severe infections including severe pneumonia. We will conduct a double blind randomized clinical trial of zinc as an adjunct treatment of severe or very severe pneumonia among stunted children. Four hundred and eighty stunted children 2 to 24 months of age presenting with severe or severe pneumonia will be randomized to receive zinc plus multivitamins or multivitamins only with antibiotics for treatment of severe or very severe pneumonia. Proportion of children failing to recover from severe pneumonia on day-5 post-enrolment or from very severe pneumonia on day-10 post enrolment will be compared between the groups. To confirm that stunted Gambian children are zinc deficient, we will continue zinc supplementation in a subgroup of 130 children for 6 months after recovery from severe or very severe pneumonia and measure the impact of zinc supplementation on linear growth and immune competence. If zinc is found to be efficacious as an adjunct treatment of severe or very severe pneumonia in chronically malnourished children, it will have far-reaching implication for child survival in many developing countries."
	MRC	MRC UNIT, THE GAMBIA	ERYTHROCYTE INVASION AND MEROZOITE LIGAND GENE EXPRESSION IN SEVERE AND MILD PLASMODIUM FALCIPARUM MALARIA	CONWAY, D	FAJARA	GAMBIA	View
"Invasion of erythrocytes by the malaria parasite Plasmodium falciparum is central to malaria parasite replication and virulence. It is a multistep process involving several interactions between human receptors and parasite ligands. The erythrocytic stage of infection is responsible for the pathology associated with malaria, so a vaccine-induced response that inhibited invasion would prevent disease. The known ligands used by P. falciparum merozoites to invade erythrocytes have been identified in culture-adapted laboratory isolates. The receptors vary in expression of erythrocyte binding antigenic (EBA) proteins and reticulocyte binding protein homologues (Rh). Their relative importance, and thus prioritization as vaccine candidates, is uncertain because cultured isolates may vary in their dependency on particular ligand-receptor interactions from clinical isolates (i.e. they differentially use alternative pathways of invasion). Up to day, very few studies have been conducted on invasion by isolates collected freshly from patients.||In the context of a case-control study comparing severe and uncomplicated cases of malaria in Gambian children, parasite invasion phenotypes and schizont stage transcript expression profiles of the eight eba and Rh protein coding genes without internal stop codons were determined for 163 clinical isolates cultured ex vivo in The Gambia. There was extensive diversity in ability to invade erythrocytes treated with neuraminidase, trypsin or chymotrypsin, and severe malaria isolates were less restricted by trypsin treatment than mild malaria isolates (P = 0.015). Expression profiles of the eba and Rh genes showed distinct clusters indicating coordinated alternative transcription. The most divergent of five major clusters was dominated by Rh2b with virtually no eba175 or eba140 (which were dominant in the other four clusters). There were significant correlations between particular transcripts and parasitemia (Rh5 was positively and eba140 negatively correlated, P < 0.001 for each) and age of patients (eba181 was positively and eba175 negatively correlated, P < 0.01 for each), but not with invasion phenotypes or severity of malaria. Severe and mild malaria isolates were also evenly represented across the different expression clusters."
	MRC	MRC UNIT, THE GAMBIA	EVALUATING DIFFERENT INDICES FOR MONITORING TEMPORAL & SPATIAL CHANGES IN MALARIA TRANSMISSION & DISEASE IN GAMBIA	ODURO, A	FAJARA	GAMBIA
	MRC	MRC UNIT, THE GAMBIA	GAMBIA HEPATITIS INTERVENTION STUDY	MENDY, M	FAJARA	GAMBIA
	MRC	MRC UNIT, THE GAMBIA	INTERACTION BETWEEN LIVE AND KILLED VACCINES: EFFECT OF DTP COMBINED VACCINE ON T CELL MEMORY AFTER MEASLES VACCINATION	FLANAGAN, K L	FAJARA	GAMBIA	View
"The introduction of routine vaccination in infants in the form of the expanded program on immunisation (EPI) has had an enormous impact on childhood morbidity and mortality, and millions of doses of vaccines are administered each year. However, the current EPI schedules were recommended >20 years ago, and the WHO Strategic Advisory Group of Experts agree that it may be time to review the scientific evidence and revise current immunization schedules. In the developing world routine vaccines are frequently missed or administered in the wrong order, but there is increasing evidence that the order in which live and killed vaccines are administered is important. Specifically, live vaccines (e.g. measles vaccine (MV)) provide beneficial effects other than protecting against the target pathogen, whereas co-administration of killed vaccines (e.g. diphtheria, tetanus, pertussis (DTP)) seems to negate these beneficial effects leading to increased all cause mortality. Moreover, in most of these studies boys and girls seem to behave differently with survival risks / benefits favouring one sex more than the other. The aim of this proposal is to investigate the immunological basis behind these observations.||300 children have been enrolled into a prospective randomised study to either receive their EPI vaccines according to Gambian guidelines; have the third dose of DTP, which is normally given at 4 months, delayed and administered with measles vaccine at 9 months of age; or receive DTP3 alone at 9 months. Boys and girls are randomised separately for later analysis by sex. Memory responses are being tested at 4 weeks and 9 months after the 9 month vaccines to measles vaccine specific and unrelated antigens. Children will then be challenged with MV at 18 months of age in order to mimic a measles virus infection, and 4 week post challenge effector responses will be analysed.|The results will be used to identify markers that might be associated with poor outcomes for use as a practical tool for testing in field studies, including samples from an ongoing large scale randomised trial of DTP / MV interactions in Guinea Bissau. Our study will contribute to the scientific framework for recommending safe EPI practices, and may have direct effects in documenting that DTP should not be given with or after MV. The results will also inform us about potential interactions that might be applied to other vaccine combinations, which is particularly important given the number of new vaccines that are currently being developed."
	MRC	MRC UNIT, THE GAMBIA	INVASIVENESS OF BACTERIA PATHOGENS IN GAMBIAN CHILDREN	ANTONIO, M	FAJARA	GAMBIA	View
"The ability to distinguish accurately between different strains within a bacterial species is key to disease treatment, epidemiological surveillance and to vaccine development. Until recently the methods that were used to characterise bacterial isolates were unsatisfactory as they indexed genetic variation that was difficult to compare between laboratories. A new approach; multilocus sequence typing (MLST) unambiguously characterises isolates of bacterial pathogens using the nucleotide sequences of internal fragments of seven house-keeping genes scattered roughly equally around the circular bacterial chromosome. Most MLST studies thus far have focused on bacteria isolated in the USA and Europe although most bacterial disease occurs in the developing world; hence a considerable portion of the global diversity of bacterial pathogens is unexplored. This is crucial in terms of future vaccine development in Africa as one needs to know what types of strains or serotypes to target in a vaccine, because different geographical regions may harbour different clonal groupings and different clonal groupings may predominate at different periods of time. We therefore propose to undertake a detailed MLST study on bacterial isolates and determine which bacterial clones are more likely to cause invasive disease in The Gambia. We will then compare strains causing disproportionately more invasive disease with strains less likely associated with invasive disease and with global isolates at the genetic level."
	MRC	MRC UNIT, THE GAMBIA	INVESTIGATING THE POTENTIAL ROLE OF HEME OXYGENASE 1 IN P. FALCIPARUM MALARIA	WALTHER, M	FAJARA	GAMBIA	View
"In order to promote parasite clearance without inducing immunopathology malaria-induced immune effector mechanisms need to be tightly regulated.||Work in murine malaria models has demonstrated a protective role for the enzyme hemoxigenase 1 (HO-1) in the pathogenesis of severe malaria. To study the role of HO-1 with regard to disease outcome in P.falciparum patients, we have enrolled 274 Gambian children with severe malaria (cases) or uncomplicated malaria (controls) into a study for detailed immunological and molecular analysis, using 9 colour flow-cytometry, RT-PCR, HO-1 ELISA, detection of free heme and Western blot for HO-1 detection.||Data are currently being analysed."
	MRC	MRC UNIT, THE GAMBIA	KIR AND HLA CLASS I GENETIC VARIATIONS IN SEVERE MALARIA CAUSED BY PLASMODIUM FALCIPARUM	YINDOM, L	FAJARA	GAMBIA	View
"Mortality resulting from malaria infection is very high in children under 5 years old in the developing world. Following infection with Plasmodium falciparum some people remain asymptomatic while others develop either mild or severe form of malaria. The reasons behind this diverse clinical presentation in malaria endemic regions are still not well understood but are thought to be influenced by both parasite and host genetic factors.|In this study, we aim to investigate the role of two highly polymorphic regions of the human genome (Human Leukocyte Antigen (HLA) and Killer Immunoglobulin-like Receptors (KIR)) in severe malaria caused by Plasmodium falciparum.|KIR genotyping was done by PCR-SSP (Sequence specific priming) techniques while the HLA typing was performed by sequencing exons 2 and 3 of HLA class I loci on human chromosome 6.|KIR data has been generated from 321 malaria cases and 315 control subjects. Preliminary analyses suggest that inhibitory KIR genes are present in most samples (frequencies ranging from 87-100% except for 2DL2 (76%) and 2DL5 (59%)). The frequencies of activating KIR genes are significantly lower compared to their corresponding inhibitory counterparts ranging from 10 to 46% with KIR3DS1 being the lowest (10.2%).|HLA typing is in progress. Association of individual KIR genes with disease severity is currently being analysed. HLA-KIR interactions in modulating the outcome of infection will be explored."
	MRC	MRC UNIT, THE GAMBIA	LONG TERM VACCINE EFFICACY OF HEPATITIS B VACCINATION OF KENEBA - MANDUAR RESIDENTS	MENDY, M	FAJARA	GAMBIA	View
"Baseline surveys of Hepatitis B virus (HBV) infection among the population of Keneba, Manduar and Kantong Kunda were completed in 1974, 1980 and 1984. In 1984, all non-immune children under the age of 5 years were vaccinated against HBV, and since then all children born in the villages have been vaccinated and their data entered into a database, which now contains details on over 2000 individuals. This long standing cohort of HBV vaccinated children has since been followed up in 1989, 1993, and 1998 to determine the duration of protective antibody responses induced by infant vaccination, and to determine vaccine efficacy against infection and against carriage. Another survey round to study long term efficacy of HBV vaccination is now due. Concurrently carriers of HBV have also been regularly followed up. Primary aims of this study are: 1) to assess vaccine efficacy against break through infection (anti-Bc present) and against chronic carriage; 2) to assess the persistence of vaccine induced antibody levels (anti-HBs) related to time since vaccination and to peak antibody levels; 3) to explore the spatial relationship between the pressure of infection in a household (by assessing duration of residence with a chronic carrier) and clustering of break through infection; and 4) to explore the genetic determinants of immune responses in relation to antibody responses and to breakthrough infection."
	MRC	MRC UNIT, THE GAMBIA	MALARIA SPECIFIC MEMORY B CELLS IN MALARIA EXPOSED INDIVIDUALS - QUANTIFYING & MONITORING THE KINETICS & DEVELOPMENT	NOGARO, S	FAJARA	GAMBIA	View
Monitoring the kinetics & development of malaria specific memory B cells in malaria exposed individuals |Maintaining immunological memory to Plasmodium falciparum infections is essential, as it remains the only natural form of protection in malaria endemic areas. The beneficial role of malaria specific antibodies has long been established & is involved notably in, inhibiting parasite replication, parasite invasion & the sequestration of the parasite, thereby reducing pathology in the host. However, the half-life of anti-malarial antibodies is short, compared to protective immune responses to other pathogens, suggesting that immunological memory to malaria may be short lived.|Until now, technical resources were inadequate to quantify malaria antigen specific memory B cells accurately. However, recent improvements to techniques, such as B cell ELISpots, allow us to address this question. In this scope, an age-stratified cross-sectional survey was carried out in Brefet, Foni District, in May 2009 (SCC 1158) to quantify malaria specific memory B cells in individuals exposed to malaria. Preliminary findings suggest that detectable amounts of malaria specific memory B cells are the same for all age groups, however the percentage of responders increases with age. We hypothesize that younger children are less able to mount a durable memory response to malaria than older children, & speculate that younger children produce predominantly short-lived plasma cells in response to malaria, whereas older children are able to mount a response dominated by long-lived plasma cells.|We therefore would like to monitor the frequency & kinetics of malaria specific memory B cells after a known confirmed infection using B cell ELISpots. Therefore, we propose to recruit a cohort of children from patients who are enrolled in the ongoing case control study comparing severe cases with uncomplicated cases (SCC 1077, SCC 1143) during the 2009 transmission season, & to follow them up for 6 months. After consent has been obtained & in line with the Ethics Committee guidelines, a volume of blood approved for the respective age groups will be requested from study participants at day 0, day 7, day 28, day 56 & every two months thereafter until summer 2010.||Quantifying malaria specific memory B cells in individuals exposed to malaria|Maintaining immunological memory to Plasmodium falciparum infections is essential, as it remains the only natural form of protection in malaria endemic areas. The beneficial role of malaria specific antibodies has long been established & is involved notably in, inhibiting parasite replication, parasite invasion & the sequestration of the parasite, thereby reducing pathology in the host. However, the half-life of anti-malarial antibodies is short compared to protective immune responses to other pathogens, suggesting that immunological memory to malaria may be short lived.|Until now, technical resources were inadequate to quantify malaria antigen specific memory B cells accurately. However, recent improvements to techniques, such as B cell ELISpots, has allowed us to address this question. After an initial pilot phase, we carried out an age-stratified cross-sectional survey in Brefet (Foni district). In line with the Ethics Committee guidelines, a volume of blood approved for the respective age groups was requested from study participants & ELISAs & ELISpots experiments were performed in parallel, assessing memory B cell & antibody responses specific to up to five blood-stage malarial antigens. During the transmission season, participants were followed up for clinical episodes of malaria, & at the end of the transmission season participants were screened for evidence of recent malaria exposure. Patients with evidence of recent malaria exposure were asked to donate a second blood sample alongside a similar number of age-matched controls, selected from participants who did not suffer from malaria during the observation period.
	MRC	MRC UNIT, THE GAMBIA	MOLECULAR METHODS FOR ANTI-MALARIAL DRUG RESISTANCE SURVEILLANCE: EVOLUTION OF CQ/PYRIMETHAMINE-SULPHADOXINE RESISTANCE	NWAKANMA, D	FAJARA	GAMBIA	View
"The resistance of Plasmodium falciparum to the cheap and affordable anti-malarial drugs, Chloroquine (CQ) and Pyrimethamine-Sulphadoxine (SP), has led to adoption of Artemisinin-based combination therapy (ACT), as first-line treatment for malaria. To protect these new anti-malarial drugs, a system to monitor the emergence and spread of drug resistance is needed. Although the standard method for determining drug efficacy is by measuring the clinical responses of patients to drug treatment, molecular markers have been recognised as potentially powerful tools for surveillance of anti-malarial drug resistance and could provide early warning signals of impending drug failure as genotypic resistance precedes parasitological and clinical resistance. Routine screening of parasite isolates from sentinel sites for drug resistance should be a major component of any malaria surveillance system. In preparation for the establishment of prospective drug resistance surveillance in The Gambia, we are analyzing archived blood samples collected in cross sectional malaria surveys conducted in the Farafenni area of The Gambia spanning a period of over twenty years from 1984 to 2008. Using both classical restriction analysis of PCR amplified gene products (PCR-RFLP) and a real-time PCR assay that offers the advantage of increased assay throughput, parasite DNA isolated from malaria-positive blood samples collected in 1984, 1988, 1991, 2001, 2004 and 2008 (~ 100 from each time point) will be genotyped at loci associated with resistance to the anti-malarial drugs Chloroquine (CQ) and Sulphadoxine-Pyrimethamine (SP). Preliminary results show progressive increase in frequency of alleles of different parasite genes that cause Chloroquine and Sulphadoxine-Pyrimethamine resistance. Over this time, the Pfcrt allele 76T (encoding threonine at amino acid position 76) has increased from undetectable levels to near fixation. We are also investigating the alleles of the parasite gene Pfcrt in more detail to reveal if the resistance is from the same genetic source as has been described elsewhere in Africa, by determining the complete coding sequence and if the flanking microsatellite loci have the same alleles that mark the common resistance haplotype. Results will be analysed to determine the rate of change in allele frequencies and estimate the selective coefficients over this historical time. The project should provide important information on the previous evolution of anti-malarial drug resistance in this region and the practicalities of establishing high throughput assays for molecular genotyping of drug resistance as part of a malaria surveillance system."
	MRC	MRC UNIT, THE GAMBIA	NEUTRALIZING ANTIBODY RESPONSES IN HIV-2	DE SILVA, T	FAJARA	GAMBIA	View
"Twenty-five years after the beginning of the AIDS pandemic, gaps in knowledge limit the development of an effective vaccine. Understanding the molecular basis of a broad and potent neutralizing antibody (NAb) response in natural HIV infection is a gap that if filled could provide critical information relevant to HIV vaccine design. Exploring a potential role for protective humoral immunity in the majority of HIV-2 infected individuals who behave as long term non-progressors warrants further study, as does the possibility that in some HIV-2 infected individuals NAb responses may be cross-reactive and protect against subsequent HIV-1 infection. In contrast to HIV-1, potent NAb responses in HIV-2 were described in early reports and recent confirmatory findings using newer technology are very encouraging. Thus the primary objectives of this research are to:||1) Compare autologous NAb responses in HIV-1 and HIV-2 infected individuals with asymptomatic HIV infection and preserved CD4 counts; with HIV-2 infected individuals stratified into those who progress to advanced immunosuppression and those who remain long term non-progressors.||2) Compare HIV-1 cross-neutralizing and enhancing antibody responses in HIV-2 infected patients who subsequently go on to acquire HIV-1 coinfection, with those who remain HIV-2 mono-infected, matched as best possible for HIV-1 exposure."
	MRC	MRC UNIT, THE GAMBIA	OXYGEN IN UNDER-5 PNEUMONIA: A PARTICIPATORY HEALTH NEEDS ASSESSMENT IN PREPARATION FOR AN INTERVENTION TRIAL	HOWIE, S R C	FAJARA	GAMBIA	View
"Acute lower respiratory infections (ALRI) account for over 2 million deaths in children under the age of 5 years globally every year. Despite efforts directed at prevention through vaccination, effective case-management will remain a key strategy in reducing childhood mortality from ALRI. While much work has been done to determine the best antibiotic treatment for ALRI, little work has been done on vital supportive treatments. Oxygen therapy has the most potential to save lives of all supportive treatments and is firmly on the WHO agenda for ALRI research. There is not enough oxygen and it is not getting to the right children. Failed solutions to the oxygen problem are not hard to find. The key to the effectiveness of oxygen therapy will be getting enough oxygen to the right children in the real context of a resource-poor setting. The best solutions are likely to be found through a process that involves key stakeholders in rationally assessing need, determining appropriate solutions and implementing these. We have undertaken a situational analysis in The Gambia and an options analysis identifying best current solutions. However, effective delivery will require overcoming key technical barriers by technology development to enable supply to be maintained even where there is poor electricity and maintenance capacity, as is often the case in The Gambia and elsewhere in the developing world. Within a broad Health Needs Assessment framework this project is developing solutions that will be applicable not only in The Gambia but in many parts of the developing world. We propose a modular oxygen supply system that would serve two beds and up to 5 children, operating reliably even if power supplies are erratic and requiring little or no maintenance over 5 years. The individual elements of such a system will not be new but the combination and application will be innovative.||Outputs: ||Beyond good intentions: lessons on equipment donation from an African hospital. |Stephen RC Howie, Sarah E Hill, Momodou Sanneh, Malick Njie, Philip C Hill, Kim Mulholland, Richard A Adegbola. Bulletin World Health Organization; 2008, 86(1):52-6 ||Oxygen for severe pneumonia in The Gambia, West Africa: a situational analysis. Sarah E Hill, Ousman Njie, Momodou Sanneh, Mariatou Jallow, David Peel, Malick Njie, Martin Weber, Philip C Hill, Richard A Adegbola, Stephen RC Howie. Int J Tuberc Lung Dis; 2009 May;13(5):587-93||Meeting oxygen needs in Africa: an options analysis from The Gambia. Stephen RC Howie, Sarah Hill, Augustine Ebonyi, Gautam Krishnan, Ousman Njie, Momodou Sanneh, Mariatou Jallow, Warren Stevens, Kevin Taylor, Martin W Weber, Pamela Collier Njai, Mary Tapgun, Tumani Corrah, Kim Mulholland , David Peel, Malick Njie, Philip C Hill, Richard A Adegbola. Bulletin World Health Organization; 2009 (in press, accepted 6 April 2009; e-published ahead of print 24 August 2009 doi: 10.2471/BLT.08.058370, http://www.who.int/bulletin/volumes/87/08-058370.pdf )||Oxygen Concentrators for Use in Tropical Countries: A Survey. David Peel, Stephen RC Howie. Journal of Clinical Engineering; 2009 (in press, accepted 6 August 2009)"
	MRC	MRC UNIT, THE GAMBIA	REGULATORY T CELLS AND BCG IMMUNOGENICITY IN EARLY LIFE	FLANAGAN, K L	FAJARA	GAMBIA	View
"BCG is the only available vaccine against Mycobacterium tuberculosis (TB) yet its protective efficacy is highly variable for unknown reasons. The most widely accepted explanation is that natural immunity to atypical mycobacteria renders BCG less effective. Twenty two percent of 3 month old Gambian infants exhibited a positive tuberculin skin test (TST) without BCG vaccination, suggesting priming by environmental mycobacteria. Recently, specialised regulatory T cells (Tregs) have been shown to control immune responses to self / foreign antigens. We hypothesise that Tregs are induced by exposure to environmental mycobacteria and attenuate the immunogenicity of BCG. 200 babies are being recruited at birth and randomised to receive BCG vaccination either immediately (n=100) or at 4.5 months (n=100). We are examining immunological responses to various mycobacterial and M tuberculosis specific antigens at birth, 4.5 and 9 months of age by flow cytometry, cytokine secretion and gene expression. All participants have a TST at 4.5 months to examine for reactivity which might be explained by environmental mycobacterial exposure. Each child is followed up monthly during the trial for morbidity and anthropometric assessment and then every three months until 3 years of age.We will assess whether the immune response to BCG is attenuated in those with pre-exposure to environmental mycobacteria, and whether Treg levels are related to the quality of the immune response to BCG. This study aims to create a better understanding of the mechanisms responsible for the poor efficacy of BCG in the developing world and should help in the design of strategies to increase the protective efficacy of BCG. Moreover, further insights into the role of Tregs in the development of TB specific immunity in early life may provide novel approaches for the development of the newer generation TB vaccines that are being developed throughout the world. One vaccine approach has been to prime with BCG and boost with a recombinant TB antigen, and our findings are likely to be particularly applicable to those taking this TB vaccine approach."
	MRC	MRC UNIT, THE GAMBIA	RISK FACTORS FOR SEVERE PNEUMONIA IN GAMBIAN CHILDREN	HOWIE, S R C	FAJARA	GAMBIA	View
"Pneumonia is one of the most important causes of serious illness among children in The Gambia. We are doing research into what causes it and how it can be better prevented and treated. The successful prevention of severe pneumonia will require a detailed understanding of modifiable risk factors to guide international policy on prevention and treatment. Despite the proven efficacy of the conjugate pneumococcal vaccine in this setting, its introduction into routine use would be expected to relieve only a minority of the burden of disease. A central question is why some children with pneumonia progress to severe pneumonia. There is a reasonable body of evidence regarding risk factors for pneumonia, but more limited data on severe pneumonia. As proposed in the quinquennial plan we will undertake a case-control study using the severe pneumonia platform to study what we believe may be three key risk factors for severe pneumonia: indoor air pollution from cooking smoke, bedsharing and poor weaning practices. Three hundred cases of children with severe pneumonia will be compared to non-severe pneumonia controls and healthy community controls over a period of 2 years. All three risk factors have potentially simple and practical corresponding interventions that could be taken to trial, and this is the long term goal."
	MRC	MRC UNIT, THE GAMBIA	TB CASE CONTACT STUDY (TBCC)	CONWAY, D	FAJARA	GAMBIA	View
"To date, studies investigating factors associated with development of TB have involved the comparison of TB cases with healthy, tuberculin skin test positive community controls. A problem with this approach is that it cannot be established whether the presence of factors, particularly serological and immunological, represents the cause or outcome of clinical disease. Prospective collection of samples from TB infected contacts prior to the development of clinical symptoms is required to investigate this and is a primary purpose of the proposed project. Blood samples and information will be collected from household contacts soon after exposure to sputum-positive TB index cases. Serum, peripheral blood mononuclear cells (PBMCs), RNA and DNA would be prepared and stored, 2 year follow up would then be performed to identify those that develop TB and selected samples assessed retrospectively. The proposed longitudinal study and sample collection is based on, and will extend some of the data collection of the existing TBGENENV study (SCC no 728). As per TBGENENV, the longitudinal cohort and sample collection will facilitate descriptive epidemiological and other case control studies to investigate genetic, host (immunological) and environment (household) factors associated with TB. It is envisaged that the identification of immunological or serological factors or indicators could lead to the development of prognostics and diagnostics for clinical TB. Finally the sample collection will facilitate vaccine candidate assessment and trials by providing baseline data and epidemiological information about TB in The Gambia. The TBGENENV design for collecting blood samples and information by questionnaire and observation from TB index cases, household contacts and external household controls will be followed. The major changes will be to increase the number of contacts recruited and the volumes of blood taken so that immune response assays using PBMCs can be performed and progressing and non-progressing contacts compared."
	MRC	MRC UNIT, THE GAMBIA	THE ROLE OF REGULATORY T CELLS IN THE PATHOGENESIS OF HIV-2 INFECTION	JAYE, A	FAJARA	GAMBIA	View
"A virus-specific CD8+ T lymphocyte response early in the course of viral infections is essential for the successful control of viral replication, and a maintained response is needed for continued viral control. However, HIV infection is characterized by an aberrant immune activation and paradoxically immune suppression also occurs from early infection. Ultimately HIV causes progressive immune dysfunction and death. The majority of HIV-2 infected individuals have low or undetectable viral load and are regarded as Long-term non-progressors (LTNP). T regulatory cells (T reg) are a subset of CD4+ T cells with immunosuppressive properties and these cells may play a critical role in counteracting immunopathology resulting from persistent high level of immune stimulation from chronic viral infections. There is a need to elucidate if T regs play a protective role by dampening immune activation or have a role in accentuating infection by suppressing immune control. An understanding of the balance between T reg and effector T cell functions in HIV-2 infection would greatly assist the development of an HIV vaccine. We postulate that in HIV-2 subjects with a normal CD4 count and a low viral load (LTNPs) the physiological balance between regulatory T cell activity and T cell activation is maintained whereas in both HIV-1 or HIV-2 patients with progressive disease and a high viral load this is not the case. In the first instance we will evaluate the numbers and function of regulatory T-cells in a cross-sectional study of HIV-1 and HIV-2 individuals with high CD4 T cell count and then characterize both Tregs and effector T cell phenotypes in HIV-2 LTNPs."
	MRC	MRC UNIT, THE GAMBIA	TRANSMISSIBILITY OF MYCOBACTERIUM AFRICANUM COMPARED WITH THE 'CAMEROON' STRAIN OF M TUBERCULOSIS	DE JONG, B	FAJARA	GAMBIA	View
"Within the Tuberculosis Case contact (TBCC) study at MRC in the Gambia, we identified Mycobacterium africanum as an important cause of TB with 38% prevalence among smear positive TB patients. We identified several differences between M. africanum and M. tuberculosis sensu stricto, including an association with HIV infection, and a lower IFNg response among cases and contacts infected with M. africanum. ||The present study aims at clarifying further differences, by continued mycobacterial genotyping within the next Case Contact study, the Isoniazid Reversion Study (IRS). These include the hypothesis that the Cameroon strain, a subspecies of M. tuberculosis sensu stricto which in Cameroon appears to have replaced M. africanum over the last 30 years, is more readily transmissible than M. africanum. The Cameroon strain was found at low prevalence (3%) in the TBCC study, and continued genotyping of Case Contact isolates allows us to study this and other strains in more detail.||For genotyping, we use spoligotyping as well as PCR for strain defining Large Sequence Polymorphisms .||So far, 376 isolates of TB patients have been genotyped (both TBCC and IRS Case Contact studies), and these patients have in total 2495 household contacts. IRS isolates continue to include 38% M. africanum. We identified one group of isolates within M. tuberculosis sensu stricto which has only sporadically been identified elsewhere and may be named the Gambian family. When comparing tuberculin skin test positivity rates, as a measure of transmission, by strain for those strains with information on ? 100 contacts, we did not identify a significant difference between strains. However, the rate of primary progression within the 2 year follow-up differed between contacts of different strains, and was higher for contacts of Beijing family isolates and of strain RD174, and lower for contacts exposed to M. africanum. This fits with other findings on the Beijing and RD174 strains, which identified respectively a stronger immunosuppressive effect and a deletion in the DosR latency regulon.||In summary, this ongoing genotyping work has defined the molecular epidemiology of TB in the Gambia and highlighted the high prevalence of M. africanum in the region. Important phenotypic differences between M. africanum and M. tuberculosis, from the current and prior Case Contact Study, include an association with HIV infection and attenuated ESAT-6 immunogenicity. In addition, M. africanum infection appears to progress to disease at lower rates than M. tuberculosis. Genomic studies that aim to explain these differences may aid in our understanding of immunity to TB."
	MRC	MRC UNIT, THE GAMBIA	TRIAL ASSESSING IMPACT OF VACCINATION WITH A PNEUMOCOCCAL CONJUGATE VACCINE ON NASOPHARYNGEAL CARRIAGE OF PNEUMOCOCCI	ROCA, A	FAJARA	GAMBIA	View
"Streptococcus pneumoniae is responsible for many deaths in young children in developing countries. The development of pneumococcal polysaccharide/protein conjugate vaccines offers a possible way of preventing these infections and results from initial trials are encouraging. It is likely that Prevenar R, a seven-valent pneumococcal conjugate vaccine, will soon be introduced into childhood immunization programme in The Gambia. Pneumococci of serotypes 1 or 5 are important causes of invasive disease in The Gambia but are found infrequently in the nasopharynx and are not contained in Prevenar R. Could introduction of Prevenar R enhance their ability to establish themselves in the nasopharynx and subsequently to cause invasive disease? For these reasons it is essential to determine whether the introduction of Prevenar R into a community where type 1 and type 5 pneumococci are important causes of invasive disease will result in an increase in the nasopharyngeal carriage rate of these and other pneumococcal serotypes, not contained in this vaccine. In order to study the effects of maximum immune pressure of the kind that will be seen only several years after routine use of the vaccine we propose to study the effects of vaccination of a whole community as well as that of vaccinating just the infant population."