Antibody responses within two leading Plasmodium vivax vaccine candidate antigens in three geographically diverse malaria-endemic regions of India.

BACKGROUND: Identifying highly immunogenic blood stage antigens which can work as target for naturally acquired antibodies in different eco-epidemiological settings is an important step for designing malaria vaccine. Blood stage proteins of Plasmodium vivax, apical membrane antigen-1 (PvAMA-1) and 19 kDa fragment of merozoite surface protein (PvMSP-119) are such promising vaccine candidate antigens. This study determined the naturally-acquired antibody response to PvAMA-1 and PvMSP-119 antigens in individuals living in three geographically diverse malaria endemic regions of India. METHODS: A total of 234 blood samples were collected from individuals living in three different eco-epidemiological settings, Chennai, Nadiad, and Rourkela of India. Indirect ELISA was performed to measure human IgG antibodies against recombinant PvAMA-1 and PvMSP-119 antigens. The difference in seroprevalence and factors associated with antibody responses at each site was statistically analysed. RESULTS: The overall seroprevalence was 40.6% for PvAMA-1 and 62.4% for PvMSP-119. Seroprevalence to PvAMA-1 was higher in Chennai (47%) followed by Nadiad (46.7%) and Rourkela (27.6%). For PvMSP-119, seroprevalence was higher in Chennai (80.3%) as compared to Nadiad (53.3%) and Rourkela (57.9%). Seroprevalence for both the antigens were found to be higher in Chennai where P. vivax is the dominant malaria species. In addition, heterogeneous antibody response was observed for PvAMA-1 and PvMSP-119 antigens at each of the study sites. Two factors, age and malaria positivity were significantly associated with seropositivity for both the antigens PvAMA-1 and PvMSP-119. CONCLUSION: These data suggest that natural acquired antibody response is higher for PvMSP-119 antigen as compared to PvAMA-1 antigen in individuals living in three geographically diverse malaria endemic regions in India. PvMSP-119 appears to be highly immunogenic in Indian population and has great potential as a malaria vaccine candidate. The differences in immune response against vaccine candidate antigens in different endemic settings should be taken into account for development of asexual stage based P. vivax malaria vaccine, which in turn can enhance malaria control efforts.

A Malaria-Resistant Phenotype with Immunological Correlates in a Tanzanian Birth Cohort Exposed to Intense Malaria Transmission.

AbstractMalaria incidence is highly heterogeneous even in areas of high transmission, although no conclusive evidence exists that innate or naturally acquired resistance can prevent infection over an extended period of time. This longitudinal study examined immunoparasitological evidence for a malaria-resistant phenotype in which children do not develop malaria despite an extended period of exposure to parasites. Within a birth cohort followed from 2002 to 2006 in Muheza, Tanzania, an area of intense transmission, children (N = 687) provided blood smears biweekly during infancy and monthly thereafter. Maternal and childhood characteristics were obtained, cord-blood cytokines were measured, and antibody responses were assayed as measures of stage-specific exposure. Sixty-three (9.2%) children had no blood smear-positive slides over 2 years of follow-up (range: 1-3.5 years) and were identified as malaria resistant. Malaria-resistant children were similar to other children with respect to completeness of follow-up and all maternal and childhood characteristics except residence area. Antibody seroprevalence was similar for two sporozoite antigens, but malaria-resistant children had a lower antibody seroprevalence to merozoite antigens merozoite surface protein 1 (5.4% versus 30.2%; P < 0.0001) and apical membrane antigen 1 (7.2% versus 33.3%; P < 0.0001). Malaria-resistant children had higher cytokine levels in cord blood, particularly interleukin-1ß. In summary, a subset of children living in an area of intense transmission was exposed to malaria parasites, but never developed patent parasitemia; this phenotype was associated with a distinct cytokine profile at birth and antibody profile during infancy. Further research with malaria-resistant children may identify mechanisms for naturally acquired immunity.

Antibody Responses to Plasmodium falciparum and Plasmodium vivax and Prospective Risk of Plasmodium spp. Infection Postpartum.

AbstractPostpartum women may have an altered susceptibility to Plasmodium falciparum and Plasmodium vivax. The relationship between naturally acquired malarial immunity and susceptibility to malaria postpartum is yet to be determined. IgG levels were measured against P. falciparum and P. vivax antigens from delivery in 201 postpartum and 201 nonpregnant controls over 12 weeks. Associations between time-varying antibody levels and time to first microscopically confirmed species-specific infection were determined by Cox regression. Associations between antibody levels and prospective risk of Plasmodium infection were similar in postpartum and control women. A 2-fold increase in P. falciparum antibody levels was associated with increased prospective risk of P. falciparum infection (hazard ratio [HR] range = 1.37-1.94). Antibody levels against most P. vivax antigens displayed no association with prospective risk of P. vivax infection (HR range = 1.02-1.05) with the exception of PvMSP119 antibodies that were weakly associated with prospective risk of P. vivax infection (HR = 1.14 (95% confidence interval = 1.02, 1.28) per 2-fold increase in levels). Associations between antibody levels and prospective risk of infection attenuated when adjusted for documented retrospective exposure. Serology may be a useful tool to predict and monitor women at increased risk of P. falciparum infection postpartum, particularly in the absence of a detailed history of retrospective infections.

Individuals living in a malaria-endemic area of Cameroon do not have an acquired antibody response to Plasmodium falciparum histidine-rich protein 2.

BACKGROUND: Diagnosis of Plasmodium falciparum is often based on detection of histidine-rich protein 2 (HRP2) in blood. Most HRP2-based assays have high sensitivity and specificity; however, authors have suggested that antibodies (Ab) to HRP2 could reduce assay sensitivity. This study sought to characterize the antibody response to HRP2 with respect to prevalence, class, subclass, affinity, and age distribution in Cameroonian children and adults residing in an area with high P. falciparum transmission. METHODS: Plasma samples from 181 Cameroonian children and adults who had been repeatedly exposed to P. falciparum and 112 samples from American adults who had never been exposed were tested for IgG Ab to HRP2. For comparison, Ab to the merozoite antigens MSP1, MSP2, MSP3 and the pregnancy-associated antigen VAR2CSA were measured using a multiplex bead-based assay. In addition, 81 plasma samples from slide-positive individuals were screened for IgM Ab to HRP2. RESULTS: As expected, children and adults had IgG Ab to MSP1, MSP2 and MSP3, antibody levels increased with age, and only women of child-bearing age had Ab to VAR2CSA; however, no convincing evidence was found that these individuals had an acquired antibody response to HRP2. That is, using two sources of recombinant HRP2, identical results were obtained when plasma from 110 Cameroonian adults and 112 US adults were screened for IgG Ab. Further studies showed that antibody prevalence and levels did not increase with age in Cameroonians between ages 5 and >80 years. Although a few samples from slide-positive Cameroonians had IgM values slightly above the American cut-off, it was unclear if the individuals had a true IgM response to HRP2 or if the values were due to non-specific binding from elevated immunoglobulin levels associated with infection. Data from prediction models showed a paucity of Class II T cell epitopes in HRP2. CONCLUSIONS: These data support the conclusion that most individuals in malaria-endemic areas do not produce an acquired humoral response to HRP2. The absence of Ab helps explain why HRP2-based assays are able to detect nanogram amounts of HRP2 and why HRP2 continues to circulate for a long time after parasite clearance.

Sparse serological evidence of Plasmodium vivax transmission in the Ouest and Sud-Est departments of Haiti.

BACKGROUND: Plasmodium vivax infections, while quite prevalent throughout South and Central America, are virtually non-existent in Haiti, where P. falciparum infections are detected in over 99% of malaria cases. Historically, few cases of P. vivax have been reported in Haiti; all of which were identified by microscopy and none were confirmed by molecular diagnostics. To further examine the transmission of P. vivax in Haiti, a cross-sectional seroepidemiological study was conducted. METHODS: Whole blood was collected from 814 community members and school children ranging in age between 2 and 80 years-of-age from four locations in the Ouest and Sud-Est Departments of Haiti. After separation of serum, samples were screened for antibodies toward P. vivax apical membrane antigen (AMA-1) and merozoite surface protein-119 (MSP-1) using an indirect enzyme-linked immunosorbent assay (ELISA). RESULTS: Of all participants screened, 4.42% (36/814) were seropositive for AMA-1, 4.55% (37/814) were seropositive for MSP-1, 7.99% (65/814) were seropositive to either antigen, and only 0.98% (7/814) were seropositive for both antigens. Seroconversion rates (SCR) for AMA-1, MSP-1, either AMA-1 or MSP-1, and for both AMA-1 and MSP-1 estimated from the cross-sectional seroprevalence indicated rates of P. vivax transmission of less than 1% per year. CONCLUSION: Given the lack of historical evidence of P. vivax infections on the island of Hispaniola, the sparse serological evidence of antibodies toward P. vivax identified in the current study further support the notion that the transmission of P. vivax malaria might be extremely low or even completely absent in Haiti.

Serological evidence of vector and parasite exposure in Southern Ghana: the dynamics of malaria transmission intensity.

BACKGROUND: Seroepidemiology provides robust estimates for tracking malaria transmission when intensity is low and useful when there is no baseline entomological data. Serological evidence of exposure to malaria vectors and parasite contribute to our understanding of the risk of pathogen transmission, and facilitates implementation of targeted interventions. Ab to Anopheles gambiae salivary peptide (gSG6-P1) and merozoite surface protein one (MSP-1(19)) reflect human exposure to malaria vectors and parasites. This study estimated malaria transmission dynamics using serological evidence of vector and parasite exposure in southern Ghana. METHODS: Total IgG responses to both antigens in an age stratified cohort (<5, 5-14, >14) were measured from South-eastern Ghana. 295 randomly selected sera were analyzed from archived samples belonging to a cohort study that were followed at 3 consecutive survey months (n = 885); February, May and August 2009. Temporal variations in seroprevalence of both antigens as well as differences between the age-stratified cohorts were determined by χ (2) test with p < 0.05 statistically significant. Non-parametric repeated ANOVA - Friedman's test was used to test differences in antibody levels. Seroprevalence data were fitted to reversible catalytic model to estimate sero-conversion rates. RESULTS: Whereas parasite prevalence was generally low 2.4%, 2.7% and 2.4% with no apparent trends with season, seroprevalence to both gSG6-P1 and MSP1(19) were high (59%, 50.9%, 52.2%) and 57.6%, 52.3% and 43.6% in respective order from Feb. to August. Repeated measures ANOVA showed differences in median antibody levels across surveys with specific significant differences between February and May but not August by post hoc Dunn's multiple comparison tests for gSG6-P1. For MSP1(19), no differences were observed in antibody levels between February and May but a significant decline was observed from May to August. Seroconversion rates for gSG6-P1 increased by 1.5 folds from February to August and 3 folds for MSP1(19). CONCLUSION: Data suggests exposure to infectious bites may be declining whereas mosquito bites remains high. Sustained malaria control efforts and surveillance are needed to drive malaria further down and to prevent catastrophic rebound. Operational factors for scaling up have been discussed.

Detection of human malaria using recombinant Plasmodium knowlesi merozoire surface protein-1 (MSP-119) expressed in Escherichia coli.

Malaria remains one of the world's most important infectious diseases and is responsible for enormous mortality and morbidity. Human infection with Plasmodium knowlesi is widely distributed in Southeast Asia. Merozoite surface protein-119 (MSP-119), which plays an important role in protective immunity against asexual blood stage malaria parasites, appears as a leading immunogenic antigen of Plasmodium sp. We evaluated the sensitivity and specificity of recombinant P. knowlesi MSP-119 (rMSP-119) for detection of malarial infection. rMSP-119 was expressed in Escherichia coli expression system and the purified rMSP-119 was evaluated with malaria, non-malaria and healthy human serum samples (n = 215) in immunoblots. The sensitivity of rMSP-119 for detection of P. knowlesi, Plasmodium falciparum, Plasmodium vivax and Plasmodium ovale infection was 95.5%, 75.0%, 85.7% and 100%, respectively. rMSP-119 did not react with all the non-malaria and healthy donor sera, which represents 100% specificity. The rMSP-119 could be used as a potential antigen in serodiagnosis of malarial infection in humans.

Serological measures of malaria transmission in Haiti: comparison of longitudinal and cross-sectional methods.

BACKGROUND: Efforts to monitor malaria transmission increasingly use cross-sectional surveys to estimate transmission intensity from seroprevalence data using malarial antibodies. To date, seroconversion rates estimated from cross-sectional surveys have not been compared to rates estimated in prospective cohorts. Our objective was to compare seroconversion rates estimated in a prospective cohort with those from a cross-sectional survey in a low-transmission population. METHODS AND FINDINGS: The analysis included two studies from Haiti: a prospective cohort of 142 children ages ≤ 11 years followed for up to 9 years, and a concurrent cross-sectional survey of 383 individuals ages 0-90 years old. From all individuals, we analyzed 1,154 blood spot specimens for the malaria antibody MSP-1(19) using a multiplex bead antigen assay. We classified individuals as positive for malaria using a cutoff derived from the mean plus 3 standard deviations in antibody responses from a negative control set of unexposed individuals. We estimated prospective seroconversion rates from the longitudinal cohort based on 13 incident seroconversions among 646 person-years at risk. We also estimated seroconversion rates from the cross-sectional survey using a reversible catalytic model fit with maximum likelihood. We found the two approaches provided consistent results: the seroconversion rate for ages ≤ 11 years was 0.020 (0.010, 0.032) estimated prospectively versus 0.023 (0.001, 0.052) in the cross-sectional survey. CONCLUSIONS: The estimation of seroconversion rates using cross-sectional data is a widespread and generalizable problem for many infectious diseases that can be measured using antibody titers. The consistency between these two estimates lends credibility to model-based estimates of malaria seroconversion rates using cross-sectional surveys. This study also demonstrates the utility of including malaria antibody measures in multiplex assays alongside targets for vaccine coverage and other neglected tropical diseases, which together could comprise an integrated, large-scale serological surveillance platform.

Avaliação de marcadores moleculares na determinação da diversidade clonal nas infecções causadas por Plasmodium vivax

A malária é causada por parasitos do gênero Plasmodium, sendo P. vivax a espécie mais amplamente distribuída no mundo. Em áreas endêmicas, é frequente a coinfecção em um mesmo indivíduo por diferentes variantes de P. vivax,caracterizando uma infecção múltipla. Nestas infecções, diferentes proporções das variantes podem ser relevantes na competição entre elas, na virulência e na resistência às drogas. O objetivo deste trabalho foi de avaliar a eficiência dos marcadores moleculares na identificação de infecções múltiplas por P.vivax. Desta forma, foram selecionados seis marcadores (MS06, MS07, MSP-3α, MSP-1B2,MSP-1B10 e MN21) caracterizados por polimorfismos de tamanho analisados por eletroforese capilar. Para cumprir este objetivo foram usadas três estratégias metodológicas. Inicialmente dois diferentes alelos amplificados de cada loci foram clonados em plasmídeos para o estabelecimento de infecções múltiplas artificiais.

Na segunda estratégia foram realizadas misturas artificiais de DNAs de pacientes com parasitemia e níveis de leucócitos semelhantes. E por fim, realizou-se a genotipagem de isolados de P. vivax de 47 pacientes da Amazônia Brasileira, sendo 22 pacientes com infecção múltipla previamente identificada. Ao simularmos infecções múltiplas artificiais com diferentes proporções de cada clone ou DNA de paciente verificamos que: (1) para os MS e TR, alelos menores foram preferencialmente amplificados por PCR; (2) para ambos os blocos da MSP-1,ocorreu titulação nos resultados; (3) para MSP-3α não houve amplificação preferencial do alelo menor; (4) a utilização do critério de altura mínima de » da do pico predominante para a detecção dos alelos raros foi mais eficiente na detecção da multiplicidade de infecção. Na genotipagem de isolados de campo os marcadores MS06, MS07, MSP-1B10, MSP-3α e MN21 foram suficientes para detecção de100% das infecções múltiplas em pacientes. Assim, estamos propondo um painel demarcadores neutros (MS06, MS07 e MN21) e não neutros (MSP-3α e MSP-1B10) na detecção de infecções múltiplas utilizando o critério menos estringente para detecção dos alelos raros.

Avaliação de marcadores moleculares na determinação da diversidade clonal nas infecções causadas por Plasmodium vivax / Evaluation of molecular markers to determine the clonal diversity in infections caused by Plasmodium vivax

A malária é causada por parasitos do gênero Plasmodium, sendo P. vivax a espécie mais amplamente distribuída no mundo. Em áreas endêmicas, é frequente a coinfecção em um mesmo indivíduo por diferentes variantes de P. vivax,caracterizando uma infecção múltipla. Nestas infecções, diferentes proporções das variantes podem ser relevantes na competição entre elas, na virulência e na resistência às drogas. O objetivo deste trabalho foi de avaliar a eficiência dos marcadores moleculares na identificação de infecções múltiplas por P.vivax. Desta forma, foram selecionados seis marcadores (MS06, MS07, MSP-3α, MSP-1B2,MSP-1B10 e MN21) caracterizados por polimorfismos de tamanho analisados por eletroforese capilar. Para cumprir este objetivo foram usadas três estratégias metodológicas. Inicialmente dois diferentes alelos amplificados de cada loci foram clonados em plasmídeos para o estabelecimento de infecções múltiplas artificiais...

Detection of etiological agents of malaria in howler monkeys from Atlantic Forests, rescued in regions of São Paulo city, Brazil.

BACKGROUND: In some states of the Brazilian extra-Amazonian region, such as the Atlantic Forest area, autochthonous human cases of malaria were related to simian malarias and vice versa. METHODS: To verify the presence of Plasmodium, 50 blood samples of howler monkeys (Alouatta guariba clamitans) rescued from the Metropolitan Region of Saõ Paulo city, where the Atlantic Forest is present, were analyzed. The samples were submitted to microscopy (thin and thick blood smears), enzyme-linked immunosorbent assays (ELISA), indirect immunofluorescent assay (IFA), and polymerase chain reaction (PCR). RESULTS: Only one smear showed forms reminiscent of Plasmodium vivax. In ELISA, the frequencies of antibodies against synthetic peptides corresponding to circumsporozoite protein of P. vivax VK210 'classic' (Pvc), P. vivax VK247, human P. vivax-like (Pvk and Pvl), P. malariae/P. brasilianum (Pm), and P. falciparum (Pf) were 24.0% (12/50) for Pvc, 8.0% (04/50) for Pvk, 6.0% (03/50) for Pvl, 24.0% (12/50) for Pm, and 28.0% (14/50) for Pf, while the frequency of antibodies against PvMSP119 recombinant proteins was 42.0% (21/50). No serum reacted against PfMSP1-19. In IFA,the seropositivity of antibodies against asexual forms of P. malariae was 31.3% (15/48). We utilized three PCR protocols to develop a molecular consensus (positive results in, at least, two protocols). The frequency of Plasmodium infections detected by PCR was 18.0% (09/50) for P. vivax, 4.0% (02/50) for P. malariae, and 76.0% (38/50) of samples were negative. The molecular consensus was not seen in 4.0% (02/50) of samples. CONCLUSIONS: These results suggest that a possible interaction between human and simian malaria coming from a zoonotic cycle cannot be discarded because simians that live in the areas of the Atlantic Forest could play a role as a reservoir for Plasmodium.

Phase Ia clinical evaluation of the Plasmodium falciparum blood-stage antigen MSP1 in ChAd63 and MVA vaccine vectors.

Efficacy trials of antibody-inducing protein-in-adjuvant vaccines targeting the blood-stage Plasmodium falciparum malaria parasite have so far shown disappointing results. The induction of cell-mediated responses in conjunction with antibody responses is thought to be one alternative strategy that could achieve protective efficacy in humans. Here, we prepared chimpanzee adenovirus 63 (ChAd63) and modified vaccinia virus Ankara (MVA) replication-deficient vectors encoding the well-studied P. falciparum blood-stage malaria antigen merozoite surface protein 1 (MSP1). A phase Ia clinical trial was conducted in healthy adults of a ChAd63-MVA MSP1 heterologous prime-boost immunization regime. The vaccine was safe and generally well tolerated. Fewer systemic adverse events (AEs) were observed following ChAd63 MSP1 than MVA MSP1 administration. Exceptionally strong T-cell responses were induced, and these displayed a mixed of CD4(+) and CD8(+) phenotype. Substantial MSP1-specific serum immunoglobulin G (IgG) antibody responses were also induced, which were capable of recognizing native parasite antigen, but these did not reach titers sufficient to neutralize P. falciparum parasites in vitro. This viral vectored vaccine regime is thus a leading approach for the induction of strong cellular and humoral immunogenicity against difficult disease targets in humans. Further studies are required to assess whether this strategy can achieve protective efficacy against blood-stage malaria infection.

Antibodies to Plasmodium falciparum erythrocyte-binding antigen-175 are associated with protection from clinical malaria.

BACKGROUND: Antibodies to blood-stage Plasmodium falciparum antigens have been associated with protection against clinical malaria in some studies but not others. Many of these studies have not assessed whether high-titer antibodies are associated with protection and have not adjusted for differences in malaria exposure. METHODS: The presence of high-titer antibodies to apical membrane antigen-1, erythrocyte-binding antigen-175 (EBA-175), and merozoite surface protein-119 (MSP-119) was assessed in 87 children living in a malaria holoendemic area of Kenya. The children were prospectively assessed during 1 year for clinical malaria. RESULTS: In unadjusted analyses, high-titer antibodies to MSP-119, but not EBA-175 or apical membrane antigen-1, were associated with protection from clinical malaria. However, after adjustment for exposure, only high-titer antibodies to EBA-175 were associated with protection from clinical malaria (hazard ratio, 0.48; 95% confidence interval [CI], 0.24, 0.95; P = 0.03), and with reduced episodes of clinical malaria (incidence rate ratio, 0.50; 95% CI, 0.31, 0.81; P = 0.005). A trend toward increased protection from clinical malaria in children was seen with antibodies to both EBA-175 and MSP-119 (hazard ratio, 0.26; 95% CI, 0.03, 1.94; P = 0.18). CONCLUSIONS: High-titer antibodies to EBA-175 are associated with protection from clinical malaria in children in a malaria holoendemic area of Kenya. Accurate estimates of antibody-associated protection from clinical malaria require adjustment for malaria exposure.

Human cord blood CD4+CD25hi regulatory T cells suppress prenatally acquired T cell responses to Plasmodium falciparum antigens.

In malaria endemic regions, a fetus is often exposed in utero to Plasmodium falciparum blood-stage Ags. In some newborns, this can result in the induction of immune suppression. We have previously shown these modulated immune responses to persist postnatally, with a subsequent increase in a child's susceptibility to infection. To test the hypothesis that this immune suppression is partially mediated by malaria-specific regulatory T cells (T(regs)) in utero, cord blood mononuclear cells (CBMC) were obtained from 44 Kenyan newborns of women with and without malaria at delivery. CD4(+)CD25(lo) T cells and CD4(+)CD25(hi) FOXP3(+) cells (T(regs)) were enriched from CBMC. T(reg) frequency and HLA-DR expression on T(regs) were significantly greater for Kenyan as compared with North American CBMC (p < 0.01). CBMC/CD4(+) T cells cultured with P. falciparum blood-stage Ags induced production of IFN-γ, IL-13, IL-10, and/or IL-5 in 50% of samples. Partial depletion of CD25(hi) cells augmented the Ag-driven IFN-γ production in 69% of subjects with malaria-specific responses and revealed additional Ag-reactive lymphocytes in previously unresponsive individuals (n = 3). Addition of T(regs) to CD4(+)CD25(lo) cells suppressed spontaneous and malaria Ag-driven production of IFN-γ in a dose-dependent fashion, until production was completely inhibited in most subjects. In contrast, T(regs) only partially suppressed malaria-induced Th2 cytokines. IL-10 or TGF-ß did not mediate this suppression. Thus, prenatal exposure to malaria blood-stage Ags induces T(regs) that primarily suppress Th1-type recall responses to P. falciparum blood-stage Ags. Persistence of these T(regs) postnatally could modify a child's susceptibility to malaria infection and disease.

Suppressive and additive effects in protection mediated by combinations of monoclonal antibodies specific for merozoite surface protein 1 of Plasmodium yoelii.

BACKGROUND: The merozoite surface protein (MSP)-1 is a target antigen of protective immunity and a malaria vaccine candidate. The nature of this protective immune response warrants further investigation: although specific antibody is thought to play a major role, the mechanisms of protection are still unclear. Monoclonal antibodies (mAbs) specific for the C-terminus of MSP-1 from Plasmodium yoelii have been shown previously to provide protection against challenge infection when administered by passive immunization to mice. Three protective mAbs were re-examined and, in particular, the effect of combinations of antibodies on the protection provided was studied. It was found that a combination of two antibodies can either provide additive protective effects or result in a suppression of protection. In this report the importance of antibody subclass and epitope specificity in the outcome of these passive immunization experiments are discussed. METHODS: The minimum protective dose (MPD) for each mAb was determined, and then combinations of antibody at their MPD were investigated for their ability to control parasitaemia and promote survival in groups of mice. Mice were inoculated over three days with the MPD and challenged with a blood stage infection of the virulent P. yoelii 17 XL. The resultant parasitaemia was assessed daily on Giemsa-stained blood films. Following the infection the presence of MSP-1 specific antibodies in the sera was monitored, and the proliferative responses of cells in the spleen of protected mice were measured. RESULTS: Combining antibodies resulted in either an additive effect on protection, with reduced peak parasitaemia and better survival, or resulted in a suppression of protection over that achieved by a single antibody alone. An additive effect was observed when B6 and F5 that have the same isotype and similar fine specificity, were combined. However, a combination of mAb D3, an IgG2a, with either B6 or F5 (both IgG3) suppressed protection, an effect that may have been due to the combination of different isotypes or to the different fine specificity of the antibodies. CONCLUSIONS: These results suggest that a combination of protective antibodies with either the same or different isotypes can produce either an additive or a suppressive effect in passive immunization. This phenomenon may be important in better understanding immunity in this experimental mouse model of malaria.

Immunophoretic rapid diagnostic tests as a source of immunoglobulins for estimating malaria sero-prevalence and transmission intensity.

BACKGROUND: Sero-epidemiological methods are being developed as a tool for rapid assessment of malaria transmission intensity. Simple blood collection methods for use in field settings will make this more feasible. This paper describes validation of such a method, by analysing immunoglobulins from blood retained within immunophoretic rapid diagnostic tests (RDTs) for Plasmodium falciparum. RDTs are now widely used for the diagnosis of malaria and estimation of parasite rates, and this method represents a further use for these devices in malaria control. METHODS: Immunoglobulins eluted from RDTs, designed to detect parasite histidine rich protein-2 (HRP-2), were analysed by indirect ELISA for IgG recognizing the P. falciparum blood stage antigens merozoite surface protein-1(19) (MSP-1(19)) and apical membrane antigen-1 (AMA-1). Optimal storage conditions for RDTs were evaluated by comparing antibody responses from RDTs stored in dry or humid conditions at 4 degrees C or at ambient temperature (with or without air-conditioning) for 7, 31 or 70 days. Antibody levels estimated using 3,700 RDT samples from attendees at health facilities in North-eastern Tanzania were compared with contemporaneously collected filter paper blood spots (FPBS) and used to estimate seroconversion rates. RESULTS: Storage of RDTs at 4 degrees C was optimal for immunoglobulin recovery but short-term storage at ambient temperatures did not substantially affect anti-malarial IgG levels. Results from RDTs were comparable with those from FPBSs, for both antigens. RDT-generated titres tended to be slightly higher than those generated from FPBSs, possibly due to greater recovery of immunoglobulins from RDTs compared to filter paper. Importantly, however, RDT-based seroconversion rates, and hence serological estimates of malaria transmission intensity, agreed closely with those from FPBSs. CONCLUSION: RDTs represent a practical option for collecting blood for sero-epidemiological surveys, with potential cost and logistical advantages over filter paper and other blood collection methods. RDT-based seroepidemiology can be incorporated into routine monitoring of malaria endemicity, providing information to supplement parasite prevalence rates and generating rapid, robust assessment of malaria transmission intensity at minimal extra cost.

Fine specificity of neonatal lymphocytes to an abundant malaria blood-stage antigen: epitope mapping of Plasmodium falciparum MSP1(33).

Cord blood T cells have been reported to respond to a variety of exogenous Ags, including environmental allergens and various viruses and parasites, as demonstrated by enhanced proliferation and cytokine secretion. This finding is evidence that Ags in the maternal environment transplacentally prime and result in fetal development of memory T cells. Some studies suggest these neonatal T cell responses may arise by nonspecific activation of T cells that express TCRs with low binding affinity, thus lacking fine lymphocyte specificity. To address this question, we examined malaria Ag stimulation of human cord and adult blood mononuclear cells in samples from residents of a malaria endemic area in Kenya. We constructed overlapping 18-mer peptides derived from sequences contained in dimorphic alleles of the C-terminal 33-kDa fragment of Plasmodium falciparum merozoite protein 1. This study identified a dominant T cell epitope for one MSP1(33) allele (MAD20) and two T cell epitopes for the second allele (K1); these epitopes were nonoverlapping and allele specific. In a given donor, peptide-specific proliferation and IFN-gamma secretion were highly concordant. However, IL-10 and IL-13 secretion were not correlated. Importantly, the fine specificity of lymphocyte proliferation and cytokine secretion in cord and adult blood mononuclear cells was similar. Cord blood cells obtained from malaria-infected pregnant women were 4-fold more likely to acquire a peptide-specific immune response. We conclude that the fetal malaria response functions in a fully adaptive manner and that this response may serve to help protect the infant from severe malaria during infancy.

Malaria vaccine-related benefits of a single protein comprising Plasmodium falciparum apical membrane antigen 1 domains I and II fused to a modified form of the 19-kilodalton C-terminal fragment of merozoite surface protein 1.

We show that the smallest module of Plasmodium falciparum AMA1 (PfAMA1) that can be expressed in the yeast Pichia pastoris while retaining the capacity to induce high levels of parasite-inhibitory antibodies comprises domains I and II. Based on this, two fusion proteins, differing in the order of the modules, were developed. Each comprised one module of PfAMA1 (FVO strain, amino acids [aa] 97 to 442) (module A) and one module of PfMSP1(19) (Wellcome strain, aa 1526 to 1621) (module Mm) in which a cystine had been removed to improve immune responses. Both fusion proteins retained the antigenicity of each component and yielded over 30 mg/liter purified protein under fed-batch fermentation. Rabbits immunized with purified fusion proteins MmA and AMm had up to eightfold-higher immune responses to MSP1(19) than those of rabbits immunized with module Mm alone or Mm mixed with module A. In terms of parasite growth inhibition, fusion did not diminish the induction of inhibitory antibodies compared with immunization with module A alone or module A mixed with module Mm, and fusion outperformed antibodies induced by immunization with module M or Mm alone. When tested against parasites expressing AMA1 heterologous to the immunogen, antibodies to the fusion proteins inhibited parasite growth to a greater extent than did antibodies either to the individual antigens or to the mixture. These results suggest that compared with the individual modules delivered separately or as a mixture, fusion proteins containing these two modules offer the potential for significant vaccine-related advantages in terms of ease of production, immunogenicity, and functionality.

The effects of varying exposure to malaria transmission on development of antimalarial antibody responses in preschool children. XVI. Asembo Bay Cohort Project.

In areas of intense malaria transmission, malaria morbidity and mortality is highest in children 3-18 months old. Interventions that reduce malaria exposure early in life reduce morbidity but may also delay development of clinical immunity. We assessed the relationship between intensity of malaria exposure and development of antibody responses. Thirty-nine children were monitored monthly, from birth to > or =2.5 years old (1238 observations), and were divided into 3 exposure categories, on the basis of parasitemic episodes or entomological data. Children with low exposure during the first 2 years of life had higher subsequent levels of antibody to merozoite surface protein-1(19-kDa) (a marker of blood-stage responses) by months 24-35 (P<.05). This inverse relationship decreased as children aged. There was no consistent relationship between exposure early in life and subsequent levels of antibody to circumsporozoite protein (a marker of sporozoite-stage responses). These data suggest that, in areas of intense malaria transmission, during the first 3 years of life, interventions that either reduce the number of asexual parasitemic episodes or lower entomological exposure do not delay the development of antibody responses to blood-stage malarial antigens.

Does infection with Human Immunodeficiency Virus affect the antibody responses to Plasmodium falciparum antigenic determinants in asymptomatic pregnant women?

OBJECTIVES: HIV-seropositive pregnant women are more susceptible to malaria than HIV-seronegative women. We assessed whether HIV infection alters maternal and cord plasma malarial antibody responses and the mother-to-infant transfer of malaria antibodies. METHODS: We determined plasma levels of maternal and cord antibodies [Immunoglobulin (IgG)] to recombinant malarial proteins [merozoite surface protein 1 (MSP-1(19kD)), the erythrocyte binding antigen (EBA-175)], the synthetic peptides [MSP-2, MSP-3, rhoptry associated protein 1 (RAP-1), and the pre-erythrocytic stage, circumsporozoite protein (NANP)(5)] antigenic determinants of Plasmodium falciparum; and tetanus toxoid (TT) by ELISA among samples of 99 HIV-seropositive mothers, 69 of their infants, 102 HIV-seronegative mothers and 62 of their infants. RESULTS: The prevalence of maternal antibodies to the malarial antigenic determinants ranged from 18% on MSP3 to 91% on EBA-175; in cord plasma it ranged from 13% to 91%, respectively. More than 97% of maternal and cord samples had antibodies to TT. In multivariate analysis, HIV infection was only associated with reduced antibodies to (NANP)(5) in maternal (P=0.001) and cord plasma (P=0.001); and reduced mother-to-infant antibody transfer to (NANP)(5) (P=0.012). This effect of HIV was independent of maternal age, gravidity and placental malaria. No consistent HIV-associated differences were observed for other antigenic determinants. CONCLUSION: An effect of HIV infection was only observed on one malarial antigenic determinant, suggesting that the increased susceptibility to malaria among HIV-infected pregnant women may not be explained on the basis of their reduced antibody response to malaria antigens.