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The antibody-dependent respiratory burst (ADRB) assay is a sensitive isoluminol-based chemiluminescence (CL) functional assay designed to assess the capacity of opsonizing antibodies against merozoites to induce neutrophil respiratory burst. ADRB was shown to measure protective immunity against malaria in endemic areas, but the assay needed further improvement to ensure better sensitivity and reproducibility. Here, we adjusted parameters such as the freezing–thawing procedure of merozoites, merozoites's concentration and the buffer solution's pH, and we used the improved assay to measure ADRB activity of 207 sera from 97 and 110 individuals living, respectively, in Dielmo and Ndiop villages with differing malaria endemicity. The improvement led to increased CL intensity and assay sensitivity, and a higher reproducibility. In both areas, ADRB activity correlated with malaria endemicity and individual's age discriminated groups with and without clinical malaria episodes, and significantly correlated with in vivo clinical protection from Plasmodium falciparum malaria. Our results demonstrate that the improved ADRB assay can be valuably used to assess acquired immunity during monitoring by control programmes and/or clinical trials.

Summary Merozoite surface proteins (MSPs) are critical for parasite invasion; they represent attractive targets for antibody-based protection against clinical malaria. To identify protection-associated target MSPs, the present study analysed antibody responses to whole merozoite extract (ME) and to defined MSP recombinant antigens in hospitalized patients from a low endemic urban area as a function of disease severity (mild versus cerebral malaria). Sera from 110 patients with confirmed severe cerebral malaria (CM) and 91 patients with mild malaria (MM) were analysed (mean age = 29 years) for total and subclass immunoglobulin (Ig)G to ME and total IgG to MSP1p19, MSP2, MSP3, MSP4 and MSP5 by enzyme-linked immunosorbent assay (ELISA). Functional antibody responses were evaluated using the antibody-dependent respiratory burst (ADRB) assay in a subset of sera. There was a trend towards higher IgG1 and IgG4 levels to ME in CM compared to MM; only ME IgM responses differed significantly between fatal and surviving CM patients. Increased prevalence of IgG to individual MSPs was found in the CM compared to the MM group, including significantly higher levels of IgG to MSP4 and MSP5 in the former. Sera from fatal (24·5%) versus surviving cases showed significantly lower IgG to MSP1p19 and MSP3 (P < 0·05). ADRB assay readouts correlated with high levels of anti-MSP IgG, and trended higher in sera from patients with surviving compared to fatal CM outcome (P = 0·07). These results document strong differential antibody responses to MSP antigens as targets of protective immunity against CM and in particular MSP1p19 and MSP3 as prognostic indicators.

Background Combined malaria control interventions have reduced mortality rate, number of clinical cases and parasite prevalence across Africa between 2000 and 2015. As a consequence, Plasmodium infections have become mostly asymptomatic and often sub-microscopic in many endemic areas. The present study aims to evaluate the contribution of asymptomatic sub-microscopic P. falciparum carriage on antibody responses against malaria antigens. Methods A total of 353 samples from a cross-sectional sampling conducted in Ndiop (Senegal) in 2016 were tested by qPCR and microscopy to determine parasite prevalence. Plasmodium falciparum positive samples were genotyped using msp-2 marker. The IgG seroprevalence against crude schizont antigen of a local P. falciparum strain, tested by ELISA, was compared to parasite prevalence. An age-matched, under 10 years, 10-15 and over 15 years cohort of 110 positive and negative qPCR samples were used to determine the impact of sub-microscopic carriage on IgG and IgM antibody responses against schizont extract and MSP3 recombinant antigen. Results The microscopic diagnosis was negative for all thick smears defining a study cohort of submicroscopic asymptomatic individuals with an overall parasite prevalence of 22.37% (79/353) by qPCR. Submicroscopic infections were associated with significant lower IgG responses in qPCR positive samples for individuals over 15 years of age, for both crude schizont (p=0.021) and MSP3 recombinant antigen (p=0.035). IgM responses were significantly higher (p=0.034) in children under 10 years, showing their susceptibility to primary infection. Above 15 years of age, a significant difference was found between parasite prevalence of 9.3% (33/353) and IgG seroprevalence of 23.8% (84/353) (p=0.01). The overall genetic diversity detected is characterized by a complexity of infection (COI) and a number of genotypes of 2.4 and 17, respectively.Conclusion Asymptomatic submicroscopic P. falciparum carriage is prevalent in the study area and is associated with immune tolerance to parasites in adults. The difference between parasite prevalence and IgG seroprevalence results from long-lived IgG in adults point out attention for elders in endemic areas as a stable parasite reservoir. In this context, mass molecular detection followed by treatment could be a valuable method for achieving elimination.

Background Ongoing efforts to fight Plasmodium falciparum malaria has reduced malaria in many areas, but new tools are needed to monitor further progress, including indicators of decreasing exposure to parasite infection. Sero-surveillance is considered promising to monitor exposure, transmission and immunity. Methods IgG responses to three antigen biomarkers were evaluated in a retrospective study involving: (i) surveys of 798 asymptomatic villagers from 2 Senegalese endemic settings conducted before 2002 and after the 2013 intensification of control measures, and (ii) in 105 symptomatic individuals from different settings in Côte d’Ivoire. Response to up to eight P. falciparum antigens, including recombinant MSP1p9 antigen and LSA141 peptide, were analysed using multiplex technology and responses to whole P. falciparum schizont extract (SE, local strain adapted to culture) were measured by ELISA. Results MSP1p9 and LSA141 IgG responses were shown to be relevant indicators monitoring immune status in the different study sites both from Côte d’Ivoire and Senegal. Between 2002 and 2013, individuals participating in both studies showed higher decline of sero-positivity in young ( 15 years: no decline to 15%) individuals from Dielmo and Ndiop. A mathematical sero-catalytic model from the complete Dielmo/Ndiop survey was used to reconstruct declining levels of sero-positivity in more detail, demonstrating that anti-SE seroprevalence levels most accurately reflected malaria exposure in the two villages. Conclusion For standard screening of population immune status at sites envisaging elimination, the use of ELISA-based assays targeting selected antigens can contribute to provide important epidemiologic surveillance data to aid malaria control programmes.

Identification of parasite antigens targeted by immune effector mechanisms that confer protection against malaria is important for the design of a multi-component malaria vaccine. Here, the association of antibodies reacting with the Plasmodium falciparum merozoite surface protein-4 (MSP4) with protection against clinical malaria was investigated in a Senegalese community living in an area of moderate, seasonal malaria transmission. Blood samples were collected at the end of an 8-month long dry season without any recorded parasite transmission from 206 residents enrolled in a prospective follow-up study. Active daily clinical monitoring was implemented during the subsequent five months. Entomologic monitoring documented parasite transmission during the first three months of follow-up. Serum IgG levels were determined by ELISA against three MSP4 baculovirus-encoded recombinant protein constructs, namely the full-length MSP4p40, MSP4p30 devoid of a highly polymorphic sequence stretch and the conserved C-terminal EGF-containing MSP4p20, as well as against a merozoite crude extract. Community seroprevalence against all three constructs was quite high, the lowest being against MSP4p30. Seroprevalence and antibody levels against the three MSP4 constructs were age-dependent. IgG1 dominated the anti-MSP4p20 responses, while both IgG1 and IgG3 were observed against MSP4p40. Anti-MSP4 antibodies were associated with the antibody-dependent respiratory burst (ADRB) activity in a functional assay of merozoite phagocytosis by polymorphonuclear cells. Importantly, high antibody levels against each of the three MSP4 constructs at the end of the dry season were associated with reduced morbidity during the subsequent transmission season in an age-adjusted Poisson regression model (IRR = 0.65 [0.50-0.83], P < 0.001 for responses over the median values). These data are consistent with a protective role for the naturally acquired anti-MSP4 antibodies and support further development of MSP4 as a candidate component of malaria vaccine.

Malaria remains a major health problem in sub- Saharan African countries despite substantial decreases in morbidity and mortality due to sustained control programs. Vaccines candidates were mainly tested in rural endemic setting; however increasing proportion of the population is living in urban area. Evaluation of the qualitative or quantitative immune responses to key targets of anti-Plasmodium immunity requires further investigation in urban area. In a cohort of 144 patients with mild malaria living in Dakar, we analyzed IgG responses against target antigens of P. falciparum: CSP, LSA-3NR2 and GLURP by ELISA. A mean age of 15 yrs (4-65 yrs) was found and patients were separated in 59 adults (

Background. Management of clinical malaria requires the development of reliable diagnostic methods and efficient biomarkers for follow-up of patients. Protection is partly based on IgG responses to parasite antigens exposed at the surface of infected erythrocytes (iRBCs). These IgG responses appeared low during clinical infection, particularly in severe disease.Methods. We analyzed the IgG binding capacity to the surface of live erythrocytes infected by knob positive FCR3 strain. Sera from 69 cerebral malaria (CM) and 72 mild malaria (MM) cases were analyzed by ELISA for IgG responses to five antigens from iRBC and by flow cytometry for IgG binding as expressed in labeling index ratio (LIR). The relationship between IgG levels, LIR, parasitemia, age, and the clinical outcomes was evaluated.Results. We found a significant decrease of LIR in adult CM fatal cases compared to surviving patients (p=0.019). In MM, LIRs were correlated to IgG anti-iRBC and anti-PfEMP3/5 levels. In CM, no correlation was found between LIR, IgG levels, and parasitemia.Conclusion. The IgG binding assay was able to discriminate outcome of cerebral malaria cases and it deserves further development as a potential functional-associated assay for symptomatic malaria analysis.

International audience; Background: Serological markers are potentially useful tools for monitoring the progress of malaria control programs , but a better understanding of antibody response dynamics is necessary. The use of a magnetic bead-based immunoassay (MBA) is advantageous compared to ELISA, due to its multiplexing capacity, but limited information is available on the standardization and validation of this assay. Methods: Several parameters for multiplex testing of antibodies to Plasmodium antigens were analysed using a set of 4 antigens and 98 sera from Senegalese rural asymptomatic and urban symptomatic individuals. The 4 antigens included Plasmodium falciparum CSP and PfAMA1 peptides, recombinant P. falciparum MSP4p20 and a Plasmodium malariae CSP (PmCSP) peptide. Comparisons with ELISA were done using MSP4p20 and whole schizont extract (SE) antigens.Results: The use of fewer beads (1000 beads per well instead of 2000) and 5 µg of antigen per 10 6 bead were validated as lower amounts. The use of a carrier protein (BSA) was shown to be critical when using peptides and the effect of a 24 h delayed measures was evaluated (5-25% signal decrease). Analysis of Ab responses showed almost equally high levels and prevalence in all transmission settings. Clear distinctions between rural and urban malaria were noted using PmCSP and SE antigens.Conclusions: This study underlines the importance of further optimization of the MBA technique and highlights the interest of using multistage/multispecies antigens for surveillance of malaria in endemic settings.

Background Evaluation of local Plasmodium falciparum malaria transmission has been investigated previously using the reversible catalytic model based on prevalence of antibody responses to single antigen to estimate seroconversion rates. High correlations were observed between seroconversion rates and entomological inoculation rates (EIR). However, in this model, the effects of malaria control interventions and clinical episodes on serological measurements were not assessed. This study monitors the use of antibody responses to P. falciparum crude extracts for assessing malaria transmission, compares seroconversion rates estimated from longitudinal data to those derived from cross-sectional surveys and investigates the effects of malaria control interventions on these measures in an area of declining malaria transmission. In addition, the validity of this model was evaluated by comparison with the alternative model. Methods Five cross-sectional surveys were carried out at the end of the wet season in Dielmo, a malaria-endemic Senegalese rural area in 2000, 2002, 2008, 2010 and 2012. Antibodies against schizonts crude extract of a local P. falciparum strain adapted to culture (Pf 07/03) were measured by ELISA. Age-specific seroprevalence model was used both for cross-sectional surveys and longitudinal data (combined data of all surveys). Results A total of 1504 plasma samples obtained through several years follow-up of 350 subjects was used in this study. Seroconversion rates based on P. falciparum schizonts crude extract were estimated for each cross-sectional survey and were found strongly correlated with EIR. High variability between SCRs from cross-sectional and longitudinal surveys was observed. In longitudinal studies, the alternative catalytic reversible model adjusted better with serological data than the catalytic model. Clinical malaria attacks and malaria control interventions were found to have significant effect on seroconversion. Discussion The results of the study suggested that crude extract was a good serological tool that could be used to assess the level of malaria exposure in areas where malaria transmission is declining. However, additional parameters such as clinical malaria and malaria control interventions must be taken into account for determining serological measurements for more accuracy in transmission assessment. Electronic supplementary material The online version of this article (doi:10.1186/s12936-017-2052-0) contains supplementary material, which is available to authorized users.

International audience; Recent control scale-up has reduced malaria in many areas but new tools are needed to monitor further progress, including indicators of decreasing exposure to parasite infection. Although serology is considered a promising approach in this regard, the serological impact of control interventions has been so far studied using indirect quantification of exposure. Cohort surveys concomitantly recording entomological and malariometric indices have been conducted in two Senegalese settings where supervised control intensification implemented in 2006 shifted malaria from historically holoendemic in Dielmo and mesoendemic in Ndiop to hypoendemic in both settings by 2013. We analyse here serological signatures of declining transmission using archived blood samples. Responses against ten pre-erythrocytic and erythrocytic antigens from Plasmodium falciparum and P. malariae alongside an Anopheles gambiae salivary gland antigen were analysed. Cross-sectional surveys conducted before (2002) and after (2013) control intensification showed a major impact of control intensification in both settings. The age-associated prevalence, magnitude and breadth of the IgG responses to all antigens were village-specific in 2002. In 2013, remarkably similar patterns were observed in both villages, with marginal responses against all parasite antigens in the 0-5y children and reduced responses in all previously seropositive age groups. Waning of humoral responses of individuals who were immune at the time of control intensification was studied from 2006 to 2013 using yearly samplings. Longitudinal data were analysed using the Cochran-Armittage trend test and an age-related reversible catalytic conversion model. This showed that the antigen-specific antibody declines were more rapid in older children than adults. There was a strong association of antibody decline with the declining entomological inoculation rate. We thus identified serological markers of declining exposure to malaria parasites that should help future monitoring of progress towards malaria elimination.

Background Coordinated scaled-up malaria control interventions have substantially contributed to the dramatic decrease of malaria-related morbidity and mortality in several endemic countries, including Senegal. However, the impacts of a given malaria control intervention on vector and parasite populations, acquired immunity, and disease burden remain very poorly documented largely due to the lack of continuous surveys. This study took advantage of the sera bank established as part of the Dielmo longitudinal project to investigate the dynamics of IgG antibody responses that accompanied the epidemiological changes resulting from malaria control interventions. Schizonts crude extract of a local strain of Plasmodium falciparum (Pfsch07/03) was used in ELISA to measure and compare seroprevalence and magnitude of IgG antibody responses from 2000 to 2012. Results The prevalence of Pfsch07/03 IgG antibody responses progressively decreased from 97.25% in 2000 to 57.3% in 2012. The prevalence of Pfsch07/03 antibodies categorized between three different age groups (15 years) revealed increased seroprevalence with age ranging from 47.19 to 62.67 and 89.45%, respectively in (15 years) old age groups. A marked drop in seroprevalence was observed after 2008 and was significant in the younger (15 years (p = 1.00). Conclusions The study revealed a substantial contribution of all malaria control interventions to the decrease of IgG antibodies responses to Pfsch07/03 throughout prevention of human-mosquitos contacts, or reduction of parasite biomass. The present study demonstrates the wider potential of sero-epidemiological analysis in monitoring changes in malaria transmission resulting from a given malaria control intervention.

International audience; Background: Advances in malaria control have reduced the burden of disease resulting from exposure to parasite infections. The consequences on naturally acquired immunity are unclear. A magnetic bead‑based immunoassay (MBA) to assess antibody levels in populations living in endemic areas was previously evaluated. In this study, the effect of clinical attacks on immunity was analysed in three sentinel sites of Ivory Coast. Methods: Recombinant proteins or peptides derived from liver or blood stage antigens of Plasmodium falciparum (CSP, LSA1 41 , LSA3, SALSA, PF13‑DBL1α 1 , GLURP, AMA1, MSP1p19, MSP4p20), the CSP of Plasmodium malariae and the salivary glands antigen of Anopheles gambiae (gSG6) were covalently linked to a colour‑coded microsphere (Luminex ™ beads) for the multiplex assay. ELISA was used for whole parasite extract antigen. Blood samples (n = 94) of patients consulting for symptomatic malaria attacks and living in three different malaria endemic settings (rural and periurban) were analysed. Results: Highly variable seroprevalence of antibody responses against parasite antigens was found ranging from 3 (gSG6) to 97 % (MSP4p20). A marked prevalence and significantly higher level of antibodies was found in patients from the rural site (Korhogo), those harbouring the lowest level of parasitaemia. The use of whole schizont extract could not discriminate immunity level, contrary to parasite‑derived recombinant proteins or peptides. Prevalence of responders to LSA1 41 and levels of antibodies to PF13 were significantly different between the three settings. Moreover, the post‑treatment clearance of parasites was clearly associated with a significantly higher level of antibody response for almost 50 % of the parasite antigens tested.Conclusion: The multiplex MBA‑Magpix technology assay provides an accurate high throughput monitoring of parasite‑specific antibodies during symptomatic malaria. The levels of antibody responses may provide a risk criterion with respect to the degree of parasitic infection. Additionally, they can be used as an indicator in the implementation of malaria prevention and local control strategies.

Background Identification of plasmodial antigens targeted by protective immune mechanisms is important for malaria vaccine development. Among functional assays, the neutrophil antibody-dependent respiratory burst (ADRB) induced by opsonized Plasmodium falciparum merozoites has been correlated with acquired immunity to clinical malaria in endemic areas, but the target merozoite antigens are unknown. Here, the contribution of antibodies to the conserved C-terminal domain of the P. falciparum merozoite surface protein-1 (PfMSP1p19) in mediating ADRB was investigated in sera from individuals living in two Senegalese villages with differing malaria endemicity. Methods Anti-PfMSP1p19 antibody levels in sera from 233 villagers were investigated and the involvement of anti-PfMSP1p19 antibodies in ADRB was explored in a subset of samples using (1) isogenic P. falciparum parasite clones expressing P. falciparum or Plasmodium chabaudi MSP1p19; (2) PfMSP1p19-coated plaque ADRB; and, (3) ADRB triggering using sera depleted from PfMSP1p19 antibodies by absorption onto the baculovirus recombinant antigen. Results ADRB activity correlated with anti-PfMSP1p19 IgG levels (P

Pro‐inflammatory cytokines induced by glycosylphosphatidylinositols (GPIs) of Plasmodium falciparum contribute to malaria pathogenesis and hence, the naturally acquired anti‐GPI antibody thought to provide protection against severe malaria (SM) by neutralizing the stimulatory activity of GPIs. In previous studies, the anti‐GPI antibody levels increased with age in parallel with the development of acquired immunity, and high levels of anti‐GPI antibodies were associated with mild malaria (MM) cases. In the present study, the relationship between the levels of pro‐inflammatory cytokines and anti‐GPI IgG antibody responses, parasitemia, and the clinical outcomes were evaluated in SM and mild malaria (MM) patients. Sera from a total of 110 SM and 72 MM cases after excluding of ineligible patients were analyzed for the levels of anti‐GPI antibodies, IgG subclasses, and cytokine responses by ELISA. While the total anti‐GPI antibody levels were similar in overall SM and MM groups, they were significantly higher in surviving SM patients than in fatal SM cases. In the case of cytokines, the TNF‐α and IL‐6 levels were significantly higher in SM compared to MM, whereas the IL‐10 levels were similar in both groups. The data presented here demonstrate that high levels of the circulatory pro‐inflammatory, TNF‐α, and IL‐6, are indicators of malaria severity, whereas anti‐inflammatory cytokine IL‐10 level does not differentiate SM and MM cases. Further, among SM patients, relatively low levels of anti‐GPI antibodies are indicators of fatal outcomes compared to survivors, suggesting that anti‐GPI antibodies provide some level of protection against SM fatality.

As a result of damaging endothelial cells (ECs), Mycobacterium leprae triggers the production of antibodies (Abs). These anti-EC Abs (AECAs) can be divided into two types. The first type nonspecifically reacts with components of the cytosol (CY) and can be detected by enzyme-linked immunosorbent assay (ELISA). The second specifically reacts with the EC membrane (MB) and requires fluorescence-activated cell sorter (FACS) analysis to be detected. The presence of both types of AECAs was determined in 68 leprosy patients. The ELISA was positive for 35 of them but also for 30 of 34 malaria patients and 17 of 50 healthy African controls. However, whereas FACS analysis showed MB reactivity in only three malaria patients and four controls, this reactivity was found in 27 leprosy patients, more of those having the lepromatous than the tuberculoid form. Specificity for MB, which we failed to absorb by incubation with CY lysates, predominated over that for CY in leprosy, unlike malaria, where the EC reactivity was restricted to the CY. Western blot analysis and two-dimensional electrophoresis revealed that calreticulin, vimentin, tubulin, and heat shock protein 70 were targeted by AECAs from leprosy patients, but other proteins remained unidentified. These auto-Abs, but not those from malaria patients, did activate ECs, as indicated by the E-selectin and intercellular adhesion molecule 1 upregulation, and/or induced them into apoptosis, as documented by four different methods. Our findings suggest that, in some but not all leprosy patients, AECAs may play a role in pathogenesis.

Sequestration of the mature Plasmodium falciparum forms complicates detection, quantification and molecular analysis of human infections. Whether the circulating parasites represent all or only a subset of co-infecting genotypes is unclear. We have investigated this issue and compared placenta and peripheral blood msp1 and msp2 genotypes in 58 women delivering with an ICT-positive placenta in Guediawaye, Senegal. Most placenta (91%) and blood samples (98%) were multiply infected. Multiplicity of infection was positively correlated in both tissues. However, the placental and circulating genotype profiles differed markedly. Only 10% of matched peripheral blood/placenta samples had identical genotypes, whereas 74% had only partially concordant genotypes, with some alleles detected in both tissues, together with additional allele(s) detected in one tissue only. Eight women (14%) had totally discordant placental and peripheral blood genotypes. Thus, in the vast majority of cases, some sequestered genotypes remain hidden, undetected in the peripheral circulation, indicating that analysis of peripheral parasites generates a partial picture of a P. falciparum infection.

SummaryAnticardiolipin (ACA), anti-β2 glycoprotein I (β2GPI), and antiprothrombin antibodies of IgG and IgM classes were quantitated by enzyme-linked immunosorbent assays in 176 untreated leprosy patients across the histopathological spectrum. Positivity rates ranged from 21% (IgG ACA) to 30% (IgM anti-prothrombin) versus 4% in healthy controls (p

Antigen reactive B cells in tonsil specimens from teenagers from a region moderately exposed to P. falciparum were capable of being differentiated in vitro and producing specific immunoglobulin (Ig)G in up to 33% of individual experiments. Mononuclear cells or purified (s)gamma+ CD19+ B cells from peripheral blood or tonsil specimens from P falciparum-immune Senegalese subjects produced antigen-specific IgG upon appropriate stimulation in vitro. One fraction of this IgG was produced de novo by differentiated B cells and another fraction was likely bound on the surface of circulating or resident CD19+ sgamma+ B cells which were found in significantly greater numbers in individuals from rural Senegal as compared to nonimmune European controls. This study further documents the baseline levels of in vitro driven anti-P. falciparum IgG antibody production by mononuclear cells from blood and tonsils in immune populations exposed to P. falciparum differentially. Furthermore, this study demonstrates the relevance and potential utility of tonsils as a source of B lymphocytes to characterize further specific antibody responses to P. falciparum antigens in immune populations.

We have reported previously that the recombinant Glutathione S-transferase GTR23, induced protection after immunisation of naive or previously exposed Saimiri monkeys. We investigated the immunogenicity of carrier-free R23 repeats in pre-exposed animals in two adjuvant formulations. Three of five monkeys immunised with alum-formulated repeats and one of two animals immunised with the Polyalphaolefine formulation produced high titres of cytophilic antibodies with a primary type kinetics, indicating that the anti-P. falciparum antibodies present on the day of challenge were R23-specific. The four responders in R23-specific antibodies were protected against a challenge infection with the virulent FUP/SP strain. The other three animals failed to respond to immunisation and experienced an infection that required drug treatment. Unlike the other three animals that experienced an infection requiring drug treatment. These experiments support further development of the R23 repeats as a vaccine candidate.

The C-terminal 19 000 MW fragment of merozoite surface protein-1 (MSP119) is one of the most promising candidate antigens for a malaria vaccine. Baculovirus recombinant Plasmodium falciparum MSP119 has been used to define conditions for the in vitro production of specific antibodies by purified human blood B cells in a culture system where T-cell signals were provided by the engagement of CD40 molecules and exogenous cytokines. MSP119 preferentially induced surface immunoglobulin G (IgG) -positive (sgamma+) B lymphocytes from P. falciparum-immune donors to differentiate and produce antigen-specific IgG. In contrast, naïve B cells or cells from non-immune donors could not be induced to secrete parasite-specific IgG in vitro. Although IgG secretion was obtained in the absence of exogenous cytokines, it was dependent on B-cell-derived interleukin-10 (IL-10) and/or B-cell factor(s) under the control of IL-10, since IgG levels were significantly decreased in the presence of neutralizing anti-IL-10 antibodies. These results demonstrate at the cellular level that a single malaria vaccine candidate polypeptide can direct parasite-specific antibody production mediated by the secretion of potentiating factors.

This study reports on T-cell proliferative responses to the 19-kDa C-terminal domain of the Plasmodium falciparum merozoite surface protein (MSP1(19)). Three different recombinant proteins were used: an Escherichia coli product expressing the first EGF-like domain and Saccharomyces cerevisiae and baculovirus/insect-cell-produced proteins containing both EGF-like domains, the latter protein being produced with or without N-glycosylation. Cell donors were P. falciparum-immune adults with no recent history of clinical malaria and recruited from three Senegalese settings with different epidemiological parasite transmission. Each mononuclear-blood-cell preparation was stimulated with a range of concentrations of the three proteins. Most subjects' mononuclear cells were reactive to at least one protein, but significant differences in lymphoproliferation were seen between the settings and within individual cultures depending on the protein source and concentration. Importantly, lymphoproliferation indices correlated inversely with the intensity of P. falciparum malaria transmission. When purified T lymphocytes were cultured in the presence of MSP1(19) plus autologous monocytes, B lymphocytes or a proposed CD1+ dendritic-cell population as costimulatory cells, significant differences were observed depending on the individual's previous exposure to parasites. This study shows that the stimulation of lymphocyte proliferation in vitro with MSP1(19) depends on several factors, including epidemiological conditions and protein preparations.

This study shows markedly different isotype distributions of antibodies to asexual blood stages of Plasmodium falciparum and to merozoite surface protein 1 in clinically immune Senegalese adults depending on the study site. The relationships between immunoglobulin M (IgM) and IgG and between IgG3 and IgG1 antibodies differed in settings where transmission is perennial compared to settings where it is seasonal. This suggests a role for antibody class and/or subclass production and utilization in the regulation of protective immunity to such antigens.

We have previously shown that Plasmodium falciparum recombinant antigens PfEB200, R23, and Pfi72 consistently inhibit opsonization of infected red blood cells by protective hyperimmune Saimiri sera, indicating that they present target epitopes involved in the phagocytosis of infected red blood cells. We report here an analysis of the immune response elicited in naive squirrel monkeys injected with the individual recombinant antigens or with a mixture of the three antigens combined with a synthetic peptide. In the three administration protocols investigated, there was no evidence for the production of antibody contributing to the phagocytosis of infected red blood cells, contrasting with the increase of opsonizing antibodies elicited by these antigens in monkeys with a prior (> or = 500 days) experience with malaria infection. However, the recombinant antigens were highly immunogenic, inducing specific antibody responses to P. falciparum and to the recombinant antigens. When the monkeys immunized with the antigen combination were challenged with blood-stage parasites, there was substantial protection: three of seven immunized animals self-cured and two others experienced a delayed peak of parasitemia. Taken together with our previous findings, these results suggest that PfEB200, R23, and Pfi72 constitute interesting vaccine candidates, and show that the presence of antibodies promoting phagocytosis of infected red blood cells is not a prerequisite for protection after immunization with these antigens in the Saimiri model.

Since the identification of the HIV virus, important advances have been achieved in the definition of potential subunit vaccines. We investigated the immunogenicity of a recombinant gp160 antigen and of two gp41 peptides from HIV-1LAI associated with seven different adjuvant formulations in squirrel monkeys. All animals were immunized twice with gp160 and then with a gp41 peptide using the same formulation. All adjuvants used led to a subsequent antibody response against gp160, and 55% of the animals immunized developed anti-gp160 antibodies that could neutralize the virus in vitro. Specific anti-gp41 antibody response was also observed. Results obtained underlined the key role of the adjuvant formulation in the antibody response against a given part of the immunogen, and indicate that such immunogenicity-related investigation can be carried out conveniently in the squirrel monkey Saimiri sciureus.

We have previously shown that Plasmodium falciparum recombinant antigens PfEB200, R23, and Pfi72 inhibit opsonization of infected erythrocytes by hyperimmune Saimiri sera, indicating that they contain target epitopes involved in the phagocytosis of infected erythrocytes. We have investigated in this study the immune response of Saimiri monkeys with previous experience of malaria infections (preimmune monkeys) after injection of these recombinant antigens, administered alone or simultaneously. The humoral response to the recombinant antigens was monitored by radioimmunoassay, and the response to P. falciparum blood stages was assayed by immunofluorescence. The relative proportion of protective versus nonprotective immunoglobulin subtypes was investigated by using 3A2/G6 and 3E4/H8 monoclonal antibodies, and the capacity of the antisera to promote in vitro phagocytosis of infected erythrocytes was evaluated. The antigens evoked in most cases a secondary-type antibody response, resulting in important increases in antigen-specific antibody titers and concomitantly in anti-P. falciparum titers. The ratio of 3A2/G6 to 3E4/H8 immunoglobulin subtypes varied with the immunogen used. Opsonizing antibodies were boosted in several animals, the most promising combination being the mixture of PfEB200 and R23 that induced long-lasting production in five of five animals. The detectable opsonizing activity appearing after immunization of the animals was antigen specific, as it was lost after adsorption of the recombinant antigens. The challenge of the animals with blood stage parasites confirmed previous findings showing a correlation between the presence of detectable opsonizing antibodies in serum and protection.

The squirrel monkey Saimiri sciureus is an experimental host for a range of human pathogens, and for the assessment of vaccine candidate antigens and vaccine strategies. This experimental host is thus particularly suitable for the follow-up of humoral responses. To understand some of the mechanisms that underlie the defense against experimental pathogens, there is a need of basic knowledge on cellular immune effectors also. The authors report here their experience in characterizing squirrel monkey blood T and B lymphocytes, and in studying in vitro induced activation and proliferation of T and B cells. Particular emphasis is given to the in vitro differentiation of squirrel monkey B cells into immunoglobulin secreting cells, with respect to Plasmodium falciparum antigens.

Different ways to improve antibody (Ab) responses following immunizations with selected antigens (TT and HSVgD) were investigated, and thus new adjuvant formulations and carrier molecules in a non-human primate experimental host, the squirrel monkey Saimiri sciureus, were assayed. Both quantitative and qualitative humoral responses were determined by means of radio-immunoassays using monoclonal Ab directed at Saimiri IgG. First, the adjuvanticity of the Syntex (SAF-1) adjuvant and of five new adjuvant formulations were assessed towards the selected Ag. This indicated that all the adjuvants induced similar antigen-specific Ab responses, although the adjuvants could modify to some extent the pattern of the qualitative Ab response. Second, we evaluated an adjuvant-free vaccine approach using a synthetic Ag from the circumsporozoite protein of Plasmodium falciparum as immunogen, this Ag being coupled to purified protein derivative (PPD) or to a recombinant heat shock protein from Mycobacterium tuberculosis. These constructs led to good antibody responses as well as an excellent memory effect. Bacille Calmette-Guérin (BCG) priming was required in conjunction with PPD as a carrier molecule to allow homogeneous Ab responses, whereas the heat shock protein construct gave a less homogeneous Ab response regardless of whether a BCG priming was done. We, in addition, discuss the relevance of Saimiri monkeys as experimental models for studies directed at evaluating the immunogenicity of further vaccine candidates.

The effect of several adjuvants and of adjuvant doses on the quantitative and qualitative antibody response to tetanus toxoid (TT) and a recombinant herpes simplex virus peptide (HSVgD) was evaluated in the New World monkey Saimiri sciureus. All adjuvant formulations were effective in inducing a strong antibody response to these antigens. The qualitative antibody response, as defined by monoclonal antibodies 3A2/G6 and 4G3/B5, was determined. Only 3A2/G6+ antibodies were induced after immunization with HSVgD irrespective of the adjuvant used and after immunization with TT alone. However, both types of antibodies were induced against TT in the presence of the adjuvants tested. These data suggest that both adjuvant and antigens can intervene in driving the quality of the antibody response induced after immunization. Strong antibody response to TT was induced at any adjuvant dose tested, suggesting that lower doses are as effective as higher doses. These findings can have implications on the design of future trials of vaccine candidate constructs in monkeys and eventually in human beings.

Blood B lymphocytes obtained from Plasmodium falciparum-immune Saimiri monkeys were assayed for their in vitro differentiation in immunoglobulin-secreting cells upon restimulation with P. falciparum-parasitized Saimiri red blood cells. Selected culture conditions enabled appropriately stimulated blood B cells to secrete 3F11/G10+ IgG, detected in the supernatants by means of a dot immunobinding assay. Primed blood B lymphocytes from P. falciparum-immune Saimiri monkeys were thus able to secrete IgG when restimulated by parasitized red blood cells in the presence of T cell- and monocyte-derived cytokines (recombinant human cytokines). These primed blood B cells, which were able to differentiate, were shown to secrete antibodies reactive with P. falciparum-infected red blood cells, as detected by means of an indirect immunofluorescence assay, and reactive with P. falciparum-infected red blood cell extracts, as detected by means of Western blot analysis. Furthermore, due to the possibility of discriminating between IgG subtypes in the squirrel monkey (3F11/G10+::3A2/G6+ IgG [associated with protection against the blood stages of P. falciparum] vs. 3F11/G10+::3E4/H8+ IgG [usually not functionally associated with protection]), we have attempted to estimate the respective proportions of each IgG subtype. In defined culture conditions, Saimiri monkey blood B cells preferentially secrete 3F11/G10+::3E4/H8+ IgG in response to parasitized red blood cells. We therefore discuss the conditions that would render this assay suitable for the selection, among P. falciparum blood stage antigens, of those that have major B-cell epitopes.

When used as a polyclonal activator of blood immunocompetent cells in the squirrel monkey, pokeweed mitogen (PWM) failed to induce lymphoproliferation unless either autologous or human serum was added to the culture medium, instead of a serum assumed to be deprived of Ig such as foetal bovine serum. We thus attempted to delineate activation processes initiated by PWM and which required both monocytes and native Ig molecules. For instance, the physical presence of live monocytes within lymphocyte suspensions vs. paraformaldehyde-fixed monocytes or products of monocytic origin was an absolute prerequisite for inducing squirrel monkey lymphocytes to proliferate in the presence of PWM. In addition, a combination of monocytes and native Ig were further shown to induce lymphocytes to proliferate in the presence of PWM. We therefore questioned whether Fc gamma R on the surface of monocytes vs. Fc gamma R expressed by other cells were involved in the initiation of PWM-driven lymphoproliferation. PWM may represent a useful tool for studies of Fc gamma R-mediated immunological processes.

International audience; BACKGROUND: Effective vaccines to combat malaria are urgently needed, but have proved elusive in the absence of validated correlates of natural immunity. Repeated blood stage infections induce antibodies considered to be the main arbiters of protection from pathology, but their essential functions have remained speculative. METHODOLOGY/PRINCIPAL FINDINGS: This study evaluated antibody dependent respiratory burst (ADRB) activity in polymorphonuclear neutrophils (PMN) induced by Plasmodium falciparum merozoites and antibodies in the sera of two different African endemic populations, and investigated its association with naturally acquired clinical protection. Respiratory bursts by freshly isolated PMN were quantified by chemiluminescence readout in the presence of isoluminol, which preferentially detects extra-cellular reactive oxygen species (ROS). Using a standardized, high throughput protocol, 230 sera were analyzed from individuals of all age groups living in meso- (Ndiop) or holo-endemic (Dielmo) Senegalese villages, and enrolled in a cross-sectional prospective study with intensive follow-up. Statistical significance was determined using non-parametric tests and Poisson regression models. The most important finding was that PMN ADRB activity was correlated with acquired clinical protection from malaria in both high and low transmission areas (P = 0.006 and 0.036 respectively). Strikingly, individuals in Dielmo with dichotomized high ADRB indexes were seventeen fold less susceptible to malaria attacks (P = 0.006). Complementary results showed that ADRB activity was (i) dependent on intact merozoites and IgG opsonins, but not parasitized erythrocytes, or complement, (ii) correlated with merozoite specific cytophilic IgG1 and IgG3 antibody titers (P

BackgroundThe time necessary for malaria parasite to re-appear in the blood following treatment (re-infection time) is an indirect method for evaluating the immune defences operating against pre-erythrocytic and early erythrocytic malaria stages. Few longitudinal data are available in populations in whom malaria transmission level had also been measured.MethodsOne hundred and ten individuals from the village of Ndiop (Senegal), aged between one and 72 years, were cured of malaria by quinine (25 mg/day oral Quinimax™ in three equal daily doses, for seven days). Thereafter, thick blood films were examined to detect the reappearance ofPlasmodium falciparumevery week, for 11 weeks after treatment. Malaria transmission was simultaneously measured weekly by night collection of biting mosquitoes.ResultsMalaria transmission was on average 15.3 infective bites per person during the 77 days follow up. The median reappearance time for the whole study population was 46.8 days, whereas individuals would have received an average one infective bite every 5 days. At the end of the follow-up, after 77 days, 103 of the 110 individuals (93.6%; CI 95% [89.0–98.2]) had been re-infected withP. falciparum. The median reappearance time ('re-positivation') was longer in subjects with patent parasitaemia at enrolment than in parasitologically-negative individuals (58 days vs. 45.9; p = 0.03) and in adults > 30 years than in younger subjects (58.6 days vs. 42.7; p = 0.0002). In a multivariate Cox PH model controlling for the sickle cell trait, G6PD deficiency and the type of habitat, the presence of parasitaemia at enrolment and age ≥ 30 years were independently predictive of a reduced risk of re-infection (PH = 0.5 [95% CI: 0.3–0.9] and 0.4; [95% CI: 0.2–0.6] respectively).ConclusionResults indicate the existence of a substantial resistance to sporozoites inoculations, but which was ultimately overcome in almost every individual after 2 1/2 months of natural challenges. Such a study design and the results obtained suggest that, despite a small sample size, this approach can contribute to assess the impact of intervention methods, such as the efficacy vector-control measures or of malaria pre-erythrocytic stages vaccines.

A comment on the paper entitled: Flow cytometric analysis on reactivity of human T lymphocyte-specific and cytokine-receptor-specific antibodies with peripheral blood mononuclear cells of chimpanzee (Pan troglodytes), rhesus macaque (Macaca mulatto), and squirrel monkey (Saimiri sciureus). Ozwara et al., J Med Primatol 26:164–167, 1997.

International audience; Glycosylphosphatidylinositol (GPI) membrane anchors of Plasmodium falciparum surface proteins are thought to be important factors contributing to malaria pathogenesis, and anti-GPI antibodies have been suggested to provide protection by neutralizing the toxic activity of GPIs. In this study, IgG responses against P. falciparum GPIs and a baculovirus recombinant MSP1p19 antigen were evaluated in two distinct groups of 70 patients each, who were hospitalized with malaria. Anti-GPI IgGs were significantly lower in patients hospitalized with confirmed cerebral malaria compared to those with mild malaria (P < 0.01) but did not discriminate for fatal outcome. In contrast, a specific marker of the anti-parasite immunity, as monitored by the anti-MSP1p19 IgG response, was similar in both cerebral and mild malaria individuals, although it was significantly lower in a subgroup with fatal outcomes. These results are consistent with a potential anti-toxin role for anti-GPI antibodies associated with protection against cerebral malaria.

We recently developed an efficient strategy based on a fully synthetic dendrimeric carbohydrate display (multiple antigenic glycopeptide; MAG) to induce anticarbohydrate antibody responses for therapeutic vaccination against cancer. Here, we show the superior efficacy of the MAG strategy over the traditional keyhole limpet hemocyanin glycoconjugate to elicit an anticarbohydrate IgG response against the tumor-associated Tn antigen. We highlight the influence of the aglyconic carrier elements of such a tumor antigen for their recognition by the immune system. Finally, we additionally developed the MAG system by introducing promiscuous HLA-restricted T-helper epitopes and performed its immunological evaluation in nonhuman primates. MAG:Tn vaccines induced in all of the animals strong tumor-specific anti-Tn antibodies that can mediate antibody-dependent cell cytotoxicity against human tumor. Therefore, the preclinical evaluation of the MAG:Tn vaccine demonstrates that it represents a safe and highly promising immunotherapeutic molecularly defined tool for targeting breast, colon, and prostate cancers that express the carbohydrate Tn antigen.

To investigate the potential for use of a well-established strain of Plasmodium falciparum as a reference strain for infected red blood cell (IRBC) surface reactivity, we monitored the binding of specific immunoglobulin G (IgG) from immune individuals to the reference Knob-positive FCR3 strain by flow cytometry. To permit interassay comparison for 162 plasma samples drawn after the rainy season, a labeling index (LI) was defined as the percentage of labeled parasites multiplied by the mean peak intensity. An LI ratio (LIR) was then calculated as the LI of the sample divided by the LI of the control. LIRs were calculated for individuals living in Dielmo and Ndiop, two Senegalese villages where P. falciparum is transmitted holoendemically and mesoendemically, respectively. The incidence (persons with an LIR of >3) observed in Dielmo was lower than that observed in Ndiop. Significantly higher LIRs were observed (i) for samples from Ndiop than for samples from Dielmo ( P < 0.01) and (ii) in Ndiop, in subjects with hemoglobin AS (HbAS) than in those with hemoglobin AA ( P = 0.03). No correlation with the cumulative age-associated immune status of the villagers was evidenced, contrary to antibody (Ab) responses against conserved IRBC-associated antigen (Ag) measured by enzyme-linked immunosorbent assay. These results are consistent with the notions that protection in HbAS individuals may relate to an increased IgG response to IRBC membrane Ags and that cell surface reactivity parallels IgG responses even though it is in itself a distinct indicator of the anti- P. falciparum Ab response. Measures of IgG binding to live IRBC are thus relevant for the functional screening of conserved IRBC-associated Ags that contribute to parasite destruction in vivo, as these Ags might be included in a multitarget vaccine.

International audience; Antibodies are characteristically induced in many parasitic infection processes. The class and subclass of the antibody response is instrumental because each isotype has a distinct biological function. It is thus crucially important for an infected individual to mount the most appropriate secondary antibody response--that is the response that has the best chance of clearing the infection and/or controlling disease. This represents a fundamental of vaccine strategies. Immuno-epidemiological surveys and in vitro models of antibody production have helped to understand some of the goals which should be achieved when designing antiparasitic vaccines.

SUMMARY This study has evaluated the individual control of isotype production and the influence of external signals that can be experimentally provided in vitro, in antibody responses to two different recombinant Schistosoma antigens (Sh28GST and TPx-1). Peripheral blood mononuclear cells or enriched B cell fractions obtained from S. haematobium infected Senegalese adults were induced to terminal differentiation in vitro. The production of antibody to either antigen was donor-dependent and for each donor it was antigen-dependent. Differentiation to IgG1 and IgG3 production, and possibly IgA, specific to these conserved parasite antigens could be regulated differentially in vitro. Exogenous IL-2 and IL-10 or IL-10 and TGF-β led to the production of specific IgG3 or IgG1 and/or IgA, respectively. This is the first report on such experimentally induced differential regulation of antigen-specific IgG1 and IgG3. This may have implications in designing protocols for protein based-vaccinations aiming at eliciting antibody responses of certain protective-type isotypes.

an example of the power of re-infection studies for preclinical analysys of vaccine candidates against P falciparum; International audience; Plasmodium falciparum expresses many antigens, which elicit various immune responses in exposed individuals, but no simple surrogate marker for protection has yet been developed. In this prospective survey, we looked for immune responses predictive of protection at various stages of progression from parasite inoculation to onset of disease. We studied 110 Senegalese volunteers from an area in which malaria is mesoendemic after they had received eradication therapy. We evaluated 4 protection-related outcomes (reappearance of parasitemia, duration of asymptomatic carriage, time to first clinical episode, and incidence of clinical episodes) in terms of levels of immunoglobulin G (IgG) against 3 crude parasite extracts and 5 conserved antigens during a 5-month period. Kaplan-Meier estimates and age-adjusted regression models showed these 4 outcomes to be associated with different patterns of IgG response to PfEMP3-cl5 (derived from P. falciparum erythrocyte membrane protein 3), PfEB200, MSP-1(19) (derived from merozoite surface protein-1), [NANP]10, infected red blood cell membrane, and merozoite and schizont extracts. It should, therefore, be possible to develop surrogate markers for each end point on the basis of IgG response to a limited number of conserved antigens.

International audience; Immunity against the blood stage of Plasmodium falciparum malaria is associated with protective-type antibodies of certain classes and subclasses. Field studies have demonstrated the differential regulation of various IgG subclasses depending on the dynamics of parasite transmission and on the immune status of the individuals tested. The intrinsic properties of each IgG subclass has a crucial role in protection, both because immunoglobulin levels are dependent on their production and clearance from blood and because antibodies are actively used for parasite clearance. In vitro models using B cells obtained from P. falciparum-immune adults have enabled study of the production of various antibody subclasses depending on the individual and on the antigens used. Ex vivo and in vitro observations from immune donors have helped to extend our understanding of the development and regulation of the antibody response and to design more effective vaccine strategies.

This study examined the evolution of immunoglobulin (Ig) G1 and IgG3 antibodies against the asexual stage Plasmodium falciparum protein, MSP1(19), before and after a heavy malaria transmission period in clinically immune Senegalese subjects living under different epidemiological conditions. Plasma was tested for antibodies to a yeast-produced, recombinant PfMSP1(19) antigen (the Q-KNG allelic variant) that has previously been demonstrated to react with IgG1, IgG3, or both in the majority of these people. Anti-P. falciparum antibodies of the IgG1 and IgG3 subclasses, previously reported to be associated with protection, were shown to evolve independently one from another after the transmission period in both settings. These results suggest differential regulation of MSP1(19)-specific IgG1 and IgG3. The precise role of these antibody isotypes in maintaining malaria immunity remains to be determined.

Cytophilic antibodies (Abs) play a critical role in protection againstPlasmodium falciparumblood stages, yet little is known about the parameters regulating production of these Abs. We used an in vitro culture system to study the subclass distribution of antigen (Ag)-specific immunoglobulin G (IgG) produced by peripheral blood mononuclear cells (PBMCs) from individuals exposed toP. falciparumor unexposed individuals. PBMCs, cultivated with or without cytokines and exogenous CD40/CD40L signals, were stimulated with a crude parasite extract, recombinant vaccine candidates derived from conserved Ags (19-kDa C terminus of merozoite surface protein 1 [MSP119], R23, and PfEB200), or recombinant Ags derived from the polymorphic Ags MSP1 block 2 and MSP2. NoP. falciparum-specific Ab production was detected in PBMCs from unexposed individuals. PBMCs from donors exposed frequently toP. falciparuminfections produced multiple IgG subclasses when they were stimulated with the parasite extract but usually only one IgG subclass when they were stimulated with a recombinant Ag. Optimal Ab production required addition of interleukin-2 (IL-2) and IL-10 for all antigenic preparations. The IgG subclass distribution was both donor and Ag dependent and was only minimally influenced by the exogenous cytokine environment. In vitro IgG production and subclass distribution correlated with plasma Abs to some Ags (MSP119, R23, and MSP2) but not others (PfEB200 and the three MSP1 block 2-derived Ags). Data presented here suggest that intrinsic properties of the protein Ag itself play a major role in determining the subclass of the Ab response, which has important implications for rational design of vaccine delivery.

Cord blood B cells obtained from neonates of healthy Senegalese mothers were assayed in vitro for their capacity to fully differentiate and secrete immunoglobulins (Ig) of various classes and subclasses. Stimulation of mononuclear cells with SAC particles or anti-μ antibodies in the presence of IL-4, or with IL-2 and IL-10 induced a strong production of IgG, provided that an additional CD40/CD40L signal was present, in contrast to adult cell cultures. Cord blood mononuclear cells differentially stimulated with various cytokines in order to lead to Ig heavy chain switching and production of the various classes/subclasses consistently produced IgG1, IgG3, IgG4, IgE and IgA. This system has been applied to immune cells from African neonates that have not been extensively studied previously. Estimation of Ig production as OD ratios could be applied to cultures where cord blood B cells are stimulated with defined antigens of human pathogens to which the fetus immune system was primed in utero.

International audience; The recombinant R23, PfEB200, and GST-5 antigens derive from conserved antigens associated with the Plasmodium falciparum-infected erythrocyte membrane. They were identified as targets of protective antibodies in the Saimiri sciureus model. We have assessed here the humoral response to these antigens in humans. Cross-sectional surveys were conducted in two Senegalese villages with different levels of endemicity. The prevalence of specific IgG and IgM was similar and influenced by age in both localities. The anti-R23 antibodies decreased after the rainy season, particularly in the children less than ten years old. The anti-PfEB200 response did not show significant seasonal variation. The anti-GST-5 response increased in both the less-than 10-year-old and the greater-than 10-year-old groups after the rainy season in Dielmo, but only in the Ndiop villagers who were more than 10-years-old. Thus, antigen-specific seasonal variations of antibody levels were influenced differently by age in both villages. The isotype distribution was antigen-specific and differed for both seasons.

International audience; Yellow fever (YF) vaccine using the 17D strain of YF attenuated virus has been produced at the Institut Pasteur in Dakar since 1962. Until now, the stabilised YF had an expiry date of utilization of two years from the end of the lot control process under storage at +4 degrees C. We conducted a stability study to assess the three full year validity of this preparation, when correctly stored at +4 degrees C to optimise the conditions of production, storage and availability of such a vaccine. The activity of 19 consecutive batches of vaccines kept for three years at +4 degrees C was compared to that of the same batches that were kept three years at -20 degrees C. Using the in vitro microculture method, we found that three-year storage at +4 degrees C induced a higher loss of activity than storage at -20 degrees C or than the accelerated degradation test of vaccines kept for 14 days at 37 degrees C. Whatever the conditions of storage, in all cases decreases in activity were below the WHO's requirements, i.e., < 1 log PFU/dose, and residual activity of the selected batches was over 1000 mouse LD50 per dose. We demonstrated that the 17D YF vaccine produced in Dakar has a shelf-life of three years and that its required potency was maintained at +4 degrees C, after reconstitution with saline diluent, following three-year storage at +4 degrees C.

Antigens exposed at the surface of Plasmodium falciparum parasitized red blood cells (pRBCs) represent potential targets for protective antibodies involved in opsonization and immune phagocytosis of pRBCs. We measured the recognition of parasitized red blood cell membrane associated antigens by IgG in the plasma of clinically immune individuals by flow cytometry and ELISA. The plasmas were selected on the basis of preexisting IgG antibodies to pRBC membrane associated recombinant proteins. In every plasma sample IgG could bind the surface of live pRBCs in flow cytometry. In addition, there was a significant correlation between the level of IgG recognition of live pRBCs and of pRBC membrane ghost proteins or major identified antigens by ELISA. Flow cytometry thus represents a technique suitable to test for the accessibility and potential functionality of IgG antibodies directed to antigens expressed by the surface of pRBCs.

A baculovirus recombinant antigen corresponding to the C-terminal 19 000 MW fragment of Plasmodium falciparum merozoite surface protein 1 (MSP119), has been used to prime T cells from individuals with no previous exposure to malaria, to provide help for the induction of a parasite specific antibody response in vitro. Although MSP119 alone could induce a small but detectable T-cell response, which included interleukin-4 (IL-4) secretion, this response was significantly increased by the presence of IL-2. In addition, IL-4 was shown to synergize with IL-2 for the induction of antigen-specific T-cell responses. If interferon-gamma (IFN-gamma), IL-12, or neutralizing anti-IL-4 antibody was present at the time of priming, the T-cell responses were abolished. Parasite-specific immunoglobulin G (IgG) could be detected after secondary restimulation with MSP119, IL-10 and anti-CD40 monoclonal antibody in cultures containing MSP119 primed T cells, autologous B cells, IL-2 and IL-4. No antibody was secreted in the absence of primed T cells in this B-cell culture assay. These data show that recombinant MSP119, a leading malaria vaccine candidate, can prime non-immune human lymphocytes under defined in vitro experimental conditions, which include regulatory cytokines and/or other costimulatory molecules. This is a complementary approach for exploring immunogenic mechanisms of potential vaccine candidates such as P. falciparum antigens in humans.

Protective immunity to P . falciparum blood stage infection is thought to be dependent on IgG antibodies, although the mechanisms that underlie such immunity are not clearly understood. One of the antigens thought to be involved in this protective response is MSP1. The present study has examined the levels and distribution of IgG (and IgM) antibodies to the C-terminal 19 kDa fragment of MSP1 in plasma from P . falciparum immune adult Senegalese and the capacity of the peripheral blood mononuclear cells from these patients to either proliferate or secrete IFN-γ, IL-10 or IL-4 in vitro in response to this antigen. Specific antibodies were found in 74% of individuals’ plasma; 44% of mononuclear cells proved capable of proliferating in vitro and IFN-γ, IL-10 and IL-4 were detected in 37, 23 and 0% of culture supernatants, respectively. No significant association was found between the presence of antibodies and immune cell reactivity under the culture conditions used. This study emphasizes the complexity of the mechanisms responsible for the sustained production of potentially protective antibodies in response to proposed T-cell dependent P. falciparum blood stage antigens.