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Malaria remains a major public health problem worldwide, with eradication efforts thwarted by drug and insecticide resistance and the lack of a broadly effective malaria vaccine. In continuously exposed communities, polyclonal infections are thought to reduce the risk of severe disease and promote the establishment of asymptomatic infections. We sought to investigate the relationship between the complexity of P. falciparum infection and underlying host adaptive immune responses in an area with high prevalence of asymptomatic parasitaemia in Cameroon. A cross-sectional study of 353 individuals aged 2 to 86 years (median age = 16 years) was conducted in five villages in the Centre Region of Cameroon. Plasmodium falciparum infection was detected by multiplex nested PCR in 316 samples, of which 278 were successfully genotyped. Of these, 60.1% (167/278) were polyclonal infections, the majority (80.2%) of which were from asymptomatic carriers. Host-parasite factors associated with polyclonal infection in the study population included peripheral blood parasite density, participant age and village of residence. The number of parasite clones per infected sample increased significantly with parasite density (r = 0.3912, p

BackgroundMalaria immuno-epidemiological data suggest that two key immunological processes, including anti-parasite and anti-disease immunity, may be implicated in the maintenance of asymptomatic infections. However, in highly exposed persons where the duration of chronic malaria infection could range from a few weeks to several years, the host immunological factors that determine the persistence of asymptomatic parasitemia remain poorly understood. This study therefore aimed to define the association between antibody or cytokine responses with asymptomatic malarial parasitemia amongst highly exposed individuals in Cameroon.MethodsAn initial cross-sectional study was carried out (week 1) and individuals were classified as having asymptomatic Plasmodium parasitemia with no fever (AS), symptomatic Plasmodium parasitemia with an axillary temperature ≥37.5°C (SY) and non-infected (NI) (no Plasmodium parasitemhia and a normal body temperature). Asymptomatic individuals were followed for 10 weeks before being treated with artemisinin-based combination treatment (ACT) and a final sample taken at week 16, 5 weeks after treatment. Those who continued to carry Plasmodium parasites with no measureable fever were defined as having chronic asymptomatic malaria. Antibody levels to P. falciparum schizont extract (SE), merozoite extract (ME), erythrocyte binding antigen-175 (EBA-175) and merozoite surface protein-1 (MSP-1) were quantified by ELISA. The growth inhibitory activities of circulating anti-parasite antibodies were quantified using the 3D7 laboratory strain of P. falciparum and a Sybr Green-I based parasite growth inhibition assay. Plasma levels of 38 cytokines were measured by Luminex technology.ResultsAmongst infected individuals, parasitemia significantly decreased with increased age although this decrease in parasitemia with age was only observed until age 20. Individuals older than 20 years of age had low parasitemia and this remained constant between the ages of 20 and 80. Although there was no difference in the magnitude of the antibody response between AS, SY and NI groups to any of the antigens tested, median levels of antibody against P. falciparum crude extracts and recombinant proteins EBA-175 and MSP-1 were significantly higher in those older than 20 years of age compared with the younger age group as might be expected from lower parasite levels. Parasite densities and P. falciparum clones fluctuated widely over the 10-week follow-up period in individuals with persistent asymptomatic parasitemia but collectively there was no change in the antibody levels over time within this group. Similar to antibody levels, many levels of the cytokines measured were stable over time. However, IL-10 levels decreased after treatment indicating that this cytokine was associated with parasite carriage in asymptomatic individuals. The ratio of IL-10 to pro-inflammatory cytokines, tumor necrosis factor-α (TNF-α) and lymphotoxin-α (LT-α) ratios were higher in the symptomatic group compared to the asymptomatic individuals but only in the younger (ConclusionTaken together, the above findings indicate that asymptomatic carriage of Plasmodium falciparum in children living in a hyperendemic area occurs independently of antibody responses, but is associated with induction of IL-10.

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 In low-malaria–transmission areas of Madagascar, annual parasite incidence (API) from routine data has been used to target indoor residual spraying at subdistrict commune level. To assess validity of this approach, we conducted school-based serological surveys and health facility (HF) data quality assessments in 7 districts to compare API to gold-standard commune-level serological measures. Methods At 2 primary schools in each of 93 communes, 60 students were randomly selected with parents and teachers. Capillary blood was drawn for rapid diagnostic tests (RDTs) and serology. Multiplex bead-based immunoassays to detect antibodies to 5 Plasmodium falciparum antigens were conducted, and finite mixture models used to characterize seronegative and seropositive populations. Reversible catalytic models generated commune-level annual seroconversion rates (SCRs). HF register data were abstracted to assess completeness and accuracy. Results RDT positivity from 12 770 samples was 0.5%. Seroprevalence to tested antigens ranged from 17.9% (MSP-1) to 59.7% (PF13). Median commune-level SCR was 0.0108 (range, 0.001–0.075). Compared to SCRs, API identified 71% (95% confidence interval, 51%–87%) of the 30% highest-transmission communes; sensitivity declined at lower levels. Routine data accuracy did not substantially affect API performance. Conclusions API performs reasonably well at identifying higher-transmission communes but sensitivity declined at lower transmission levels.

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.

Background Malaria in Senegal is due essentially to infections by Plasmodium falciparum and, to a lesser extent to Plasmodium malariae and Plasmodium ovale. By the use of molecular methods, detection of Plasmodium vivax has been recently reported in the region of Kedougou, raising the question of appraisal of its potential prevalence in this setting. Methods A retrospective serological study was carried out using 188 samples taken from 2010 to 2011 in a longitudinal school survey during which 48 asymptomatic children (9–11 years) were recruited. Four collections of samples collected during two successive dry and rainy seasons were analysed for antibody responses to P. vivax and P. falciparum. Recombinant P. falciparum and P. vivax MSP1 antigens and total P. falciparum schizont lysate from African 07/03 strain (adapted to culture) were used for ELISA. Nested PCR amplification was used for molecular detection of P. vivax. Results A surprising high prevalence of IgG responses against P. vivax MSP1 was evidenced with 53% of positive samples and 58% of the individuals that were found positive to this antigen. There was 77% of responders to P. falciparum outlined by 63% of positive samples. Prevalence of responders did not differ as function of seasons. Levels of antibodies to P. falciparum fluctuated with significant increasing between dry and rainy season (P

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.

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.

International audience; Background: Multiplex assays are currently used to facilitate the evaluation of antibody (Ab) responses to multiple Plasmodium falciparum antigens from large field-based epidemiological studies. The present study aimed at (i) optimizing parameters of a novel cost-effective, compact and reliable magnetic bead-based multiplex immunoassay (MBA) carried out with the MAGPIX ®-Luminex system and (ii) comparing the results with those obtained using standard ELISA technology. Methods: Several MBA parameters including antigen amount for coupling, plasma dilution, type of plates, buffers, washing procedure to minimize bead loss, and bead quantity for testing were optimized. Antibody responses to two recombinant and two peptidic P. falciparum antigens, one Anopheles gambiae salivary gland peptide gSG6 and Bovine Serum Albumin as negative control were tested by MBA and ELISA using sera from 14 villagers from an hyper-endemic Senegalese village. Results: The MBA procedures were developed to reflect responses observed in the standard ELISA protocols used in previous studies. Using the finalized MBA protocol, a strong significant positive correlation (P

This article demonstrate the usefulness of primate models such as african green monkeys for relevant preclinical work on vaccines; International audience; The human immunodeficiency virus type 1 (HIV-1) Tat is a key protein playing a major role in the infectivity of the virus. Thus, HIV-Tat based vaccines have been proposed as an attractive option to treat AIDS. Recently, we have shown that the recombinant detoxified adenylate cyclase (CyaA) from Bordetella pertussis carrying HIV-Tat (CyaA-E5-Tat), targets dendritic cells (DCs) and induces specific Th1 polarized and neutralizing antibody responses in mice. To further explore the potentialities of this prototype vaccine for human use, we analyzed the CyaA-E5-Tat induced antibody responses in non-human primates and established the biological characteristics of these antibodies. African Green Monkeys (AGM) were immunized with CyaA-E5-Tat in the presence or in the absence of alum adjuvant. First, we showed that the anti-CyaA antibodies induced by such immunization does not interfere with the binding of CyaA-E5-Tat to its receptor at the DC surface, the alphaMbeta2 integrin. Monkeys immunized with CyaA-E5-Tat, with or without alum, produced anti-Tat antibodies that mainly recognized the N-terminal domain of the Tat protein. Importantly, all sera obtained after three immunizations displayed the capacity to bind to Tat and neutralize its transactivating function in vitro. Finally, in the absence of alum, CyaA-E5-Tat, induced Th1 Tat specific T cell responses. These findings reveal that CyaA-E5-Tat is efficiently delivered in non-human primates and had a significant impact on the generation of neutralizing anti-Tat antibodies. These observations are, thus, encouraging for the use of the CyaA vector in human and also suggest that CyaA-E5-Tat might be a useful tool to decipher the biological characteristic of such antibodies.

Summary: Drug resistance to Plasmodium falciparum contributes to major health problems in central Africa and, as a consequence, poverty. We have analyzed the efficacy of three currently available antimalarial drugs to treat symptomatic, uncomplicated P. falciparum malaria in semiimmune adults living in Bangui, Central Republic of Africa. 210 consecutive individuals were enrolled in the survey, of which 45 were excluded. Those having received dihydroartemisin proved significantly less parasitemic than those having received quinine per os or sulfadoxin-pyrimethamin (χ2 = 16.93 ; p < 0.05), and 75 % recovered in two days compared to 57 and 44 %, respectively. The 25 % who did not recover benefited from a second cure with dihydroartemisin, which proved 100 % efficient. The most accurate protocol remains to be established by analyzing clinical and parasitological data and taking into account the economics of the country.

International audience; Antibodies to Plasmodium falciparum C-terminal merozoite surface protein 1 (PfMSP-1p19) have been correlated with protection against malaria, but this association may apply to many merozoite antigens. To address this question, we conducted a prospective serological study of 205 individuals in an active 5-month clinical survey in a Senegalese village where malaria is mesoendemic. Before the 2000 rainy season, antibody responses specific for recombinant baculovirus PfMSP-1p19 or merozoite extracts were compared with 2 in vitro functional antibody activities (inhibition of parasite grown and erythrocyte invasion) and with the number of clinical episodes during 5 months of follow-up. Antibody levels to PfMSP-1p19 and merozoite extract correlated, respectively, with erythrocyte invasion and parasite growth inhibition. Although antibody levels to both antigen preparations were associated with age, functional parameters were not. High levels of anti-PfMSP-1p19 immunoglobulin G were associated with reduced malaria in an age-adjusted multivariate analysis. These results support baculovirus PfMSP-1p19-based vaccine development.

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

Summary Background A better understanding of the effect of malaria control interventions on vector and parasite populations, acquired immunity, and burden of the disease is needed to guide strategies to eliminate malaria from highly endemic areas. We monitored and analysed the changes in malaria epidemiology in a village community in Senegal, west Africa, over 22 years. Methods Between 1990 and 2012, we did a prospective longitudinal study of the inhabitants of Dielmo, Senegal, to identify all episodes of fever and investigate the relation between malaria host, vector, and parasite. Our study included daily medical surveillance with systematic parasite detection in individuals with fever. We measured parasite prevalence four times a year with cross-sectional surveys. We monitored malaria transmission monthly with night collection of mosquitoes. Malaria treatment changed over the years, from quinine (1990–94), to chloroquine (1995–2003), amodiaquine plus sulfadoxine-pyrimethamine (2003–06), and finally artesunate plus amodiaquine (2006–12). Insecticide-treated nets (ITNs) were introduced in 2008. Findings We monitored 776 villagers aged 0–101 years for 2 378 150 person-days of follow-up. Entomological inoculation rate ranged from 142·5 infected bites per person per year in 1990 to 482·6 in 2000, and 7·6 in 2012. Parasite prevalence in children declined from 87% in 1990 to 0·3 % in 2012. In adults, it declined from 58% to 0·3%. We recorded 23 546 fever episodes during the study, including 8243 clinical attacks caused by Plasmodium falciparum , 290 by Plasmodium malariae , and 219 by Plasmodium ovale . Three deaths were directly attributable to malaria, and two to severe adverse events of antimalarial drugs. The incidence of malaria attacks ranged from 1·50 attacks per person-year in 1990 to 2·63 in 2000, and to only 0·046 in 2012. The greatest changes were associated with the replacement of chloroquine and the introduction of ITNs. Interpretation Malaria control policies combining prompt treatment of clinical attacks and deployment of ITNs can nearly eliminate parasite carriage and greatly reduce the burden of malaria in populations exposed to intense perennial malaria transmission. The choice of drugs seems crucial. Rapid decline of clinical immunity allows rapid detection and treatment of novel infections and thus has a key role in sustaining effectiveness of combining artemisinin-based combination therapy and ITNs despite increasing pyrethroid resistance. Funding Pasteur Institutes of Dakar and Paris, Institut de Recherche pour le Developpement, and French Ministry of Cooperation.

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.