Naturally acquired antibodies to polymorphic and conserved epitopes of Plasmodium falciparum merozoite surface protein 3

Many studies on the role of merozoite surface protein 3 (MSP3) in immunity against malaria have focused on a conserved section of MSP3. New evidence suggests that polymorphic sequences within MSP3 are under immune selection. We report a detailed analysis of naturally-acquired antibodies to allele-specific and conserved parts of MSP3 in a Kenyan cohort. Indirect and competition ELISA to heterologous recombinant MSP3 proteins were used for antibody assays, and parasites were genotyped for msp3 alleles. Antibody reactivity to allele-specific and conserved epitopes of MSP3 was heterogenous between individuals. Overall, the prevalence of allele-specific antibody reactivity was significantly higher (3D7-specific 54%, K1-specific 41%) than that to a recombinant protein representing a conserved portion of C-terminal MSP3 (24%, P < 0·01). The most abundant IgG subclass was IgG3, followed by IgG1. Allele-specific reactivity to the K1-type of MSP3 was associated with a lower risk of clinical malaria episodes during a 6-month follow-up in individuals who were parasitized at the start of the malaria transmission season (Relative risk 0·41 with 95% confidence interval 0·20–0·81, P = 0·011). The potential importance of allele-specific immunity to MSP3 should be considered in addition to immunity to conserved epitopes, in the development of an MSP3 malaria vaccine

Genetic Diversity of Polymorphic Vaccine Candidate Antigens (Apical Membrane Antigen-1, Merozoite Surface Protein-3, and Erythrocyte Binding Antigen-175) in Plasmodium falciparum Isolates from Western and Central Africa

The malaria vaccine candidate antigens erythrocyte binding antigen 175 (EBA-175), merozoite surface protein 3 (MSP-3), and apical membrane antigen (AMA-1) from Plasmodium falciparum isolates from countries in central and west Africa were assessed for allelic diversity. Samples were collected on filter paper from 600 P. falciparum-infected symptomatic patients in Cameroon, Republic of Congo, Burkina Faso, Ghana, and Senegal and screened for class-specific amplification fragments. Genetic diversity, assessed by mean heterozygosity, was comparable among countries. We detected a clinical increase in eba 175 F-allele frequency from west to east across the study region. No statistical difference in msp-3 allele distribution between countries was observed. The ama-1 3D7 alleles were present at a lower frequency in central Africa than in West Africa. We also detected little to no genetic differentiation among sampling locations. This finding indicates that, at least at the level of resolution offered by restriction fragment length polymorphism analysis, these antigens showed remarkable genetic homogeneity throughout the region sampled, perhaps caused by balancing selection to maintain a diverse array of antigen haplotyes

Antibody responses to <i>P. falciparum</i> blood stage antigens and incidence of clinical malaria in children living in endemic area in Burkina Faso

Abstract Background High parasite-specific antibody levels are generally associated with low susceptibility to Plasmodium falciparum malaria. This has been supported by several studies in which clinical malaria cases of P. falciparum malaria were reported to be associated with low antibody avidities. This study was conducted to evaluate the role of age, malaria transmission intensity and incidence of clinical malaria in the induction of protective humoral immune response against P. falciparum malaria in children living in Burkina Faso. Methods We combined levels of IgG and IgG subclasses responses to P. falciparum antigens: Merozoite Surface Protein 3 (MSP3), Merozoite Surface Protein 2a (MSP2a), Merozoite Surface Protein 2b (MSP2b), Glutamate Rich Protein R0 (GLURP R0) and Glutamate Rich Protein R2 (GLURP R2) in plasma samples from 325 children under five (05) years with age, malaria transmission season and malaria incidence. Results We notice higher prevalence of P. falciparum infection in low transmission season compared to high malaria transmission season. While, parasite density was lower in low transmission than high transmission season. IgG against all antigens investigated increased with age. High levels of IgG and IgG subclasses to all tested antigens except for GLURP R2 were associated with the intensity of malaria transmission. IgG to MSP3, MSP2b, GLURP R2 and GLURP R0 were associated with low incidence of malaria. All IgG subclasses were associated with low incidence of P. falciparum malaria, but these associations were stronger for cytophilic IgGs. Conclusions On the basis of the data presented in this study, we conclude that the induction of humoral immune response to tested malaria antigens is related to age, transmission season level and incidence of clinical malaria

T-cell and serological responses to Erp, an exported Mycobacterium tuberculosis protein, in tuberculosis patients and healthy individuals

<p>Abstract</p> <p>Background</p> <p>The identification of antigens able to differentiate tuberculosis (TB) disease from TB infection would be valuable. Cellular and humoral immune responses to Erp (Exported repetitive protein) – a recently identified <it>M. tuberculosis </it>protein – have not yet been investigated in humans and may contribute to this aim.</p> <p>Methods</p> <p>We analyzed the cellular and humoral immune responses to Erp, ESAT-6, Ag85B and PPD in TB patients, in BCG⁺individuals without infection, BCG⁺individuals with latent TB infection (LTBI) and BCG^-controls. We used lymphoproliferation, ELISpot IFN-γ, cytokine production assays and detection of specific human antibodies against recombinant <it>M. tuberculosis </it>proteins.</p> <p>Results</p> <p>We included 22 TB patients, 9 BCG⁺individuals without TB infection, 7 LTBI and 7 BCG^-controls. Erp-specific T cell counts were higher in LTBI than in the other groups. Erp-specific T cell counts were higher in LTBI subjects than TB patients (median positive frequency of 211 SFC/10⁶PBMC (range 118–2000) for LTBI subjects compared to 80 SFC/10⁶PBMC (range 50–191), p = 0.019); responses to PPD and ESAT-6 antigens did not differ between these groups. IFN-γ secretion after Erp stimulation differed between TB patients and LTBI subjects (p = 0.02). Moreover, LTBI subjects but not TB patients or healthy subjects produced IgG3 against Erp.</p> <p>Conclusion</p> <p>The frequencies of IFN-γ-producing specific T cells, the IFN-γ secretion and the production of IgG3 after Erp stimulation are higher in LTBI subjects than in TB patients, whereas PPD and ESAT-6 are not.</p

Pre-Clinical Assessment of Novel Multivalent MSP3 Malaria Vaccine Constructs

BACKGROUND: MSP3 has been shown to induce protection against malaria in African children. The characterization of a family of Plasmodium falciparum merozoite surface protein 3 (MSP3) antigens sharing a similar structural organization, simultaneously expressed on the merozoite surface and targeted by a cross-reactive network of protective antibodies, is intriguing and offers new perspectives for the development of subunit vaccines against malaria. METHODS: Eight recombinant polyproteins containing carefully selected regions of this family covalently linked in different combinations were all efficiently produced in Escherichia coli. The polyproteins consisted of one monovalent, one bivalent, one trivalent, two tetravalents, one hexavalent construct, and two tetravalents incorporating coiled-coil repeats regions from LSA3 and p27 vaccine candidates. RESULTS: All eight polyproteins induced a strong and homogeneous antibody response in mice of three distinct genotypes, with a dominance of cytophilic IgG subclasses, lasting up to six months after the last immunization. Vaccine-induced antibodies exerted a strong monocyte-mediated in vitro inhibition of P. falciparum growth. Naturally acquired antibodies from individuals living in an endemic area of Senegal recognized the polyproteins with a reactivity mainly constituted of cytophilic IgG subclasses. CONCLUSIONS: Combination of genetically conserved and antigenically related MSP3 proteins provides promising subunit vaccine constructs, with improved features as compared to the first generation construct employed in clinical trials (MSP3-LSP). These multivalent MSP3 vaccine constructs expand the epitope display of MSP3 family proteins, and lead to the efficient induction of a wider range of antibody subclasses, even in genetically different mice. These findings are promising for future immunization of genetically diverse human populations

A defined mechanistic correlate of protection against Plasmodium falciparum malaria in non-human primates.

Malaria vaccine design and prioritization has been hindered by the lack of a mechanistic correlate of protection. We previously demonstrated a strong association between protection and merozoite-neutralizing antibody responses following vaccination of non-human primates against Plasmodium falciparum reticulocyte binding protein homolog 5 (PfRH5). Here, we test the mechanism of protection. Using mutant human IgG1 Fc regions engineered not to engage complement or FcR-dependent effector mechanisms, we produce merozoite-neutralizing and non-neutralizing anti-PfRH5 chimeric monoclonal antibodies (mAbs) and perform a passive transfer-P. falciparum challenge study in Aotus nancymaae monkeys. At the highest dose tested, 6/6 animals given the neutralizing PfRH5-binding mAb c2AC7 survive the challenge without treatment, compared to 0/6 animals given non-neutralizing PfRH5-binding mAb c4BA7 and 0/6 animals given an isotype control mAb. Our results address the controversy regarding whether merozoite-neutralizing antibody can cause protection against P. falciparum blood-stage infections, and highlight the quantitative challenge of achieving such protection

Diagnostic comparison of malaria infection in peripheral blood, placental blood and placental biopsies in Cameroonian parturient women

<p>Abstract</p> <p>Background</p> <p>In sub-Saharan Africa, <it>Plasmodium falciparum </it>malaria in pregnancy presents an enormous diagnostic challenge. The epidemiological and clinical relevance of the different types of malaria diagnosis as well as risk factors associated with malaria infection at delivery were investigated.</p> <p>Method</p> <p>In a cross-sectional survey, 306 women reporting for delivery in the Mutenegene maternity clinic, Fako division, South West province, Cameroon were screened for <it>P. falciparum </it>in peripheral blood, placental blood and placental tissue sections by microscopy. Information relating to the use of intermittent preventive treatment in pregnancy with sulphadoxine/pyrimethamine, history of fever attack, infant birth weights and maternal anaemia were recorded.</p> <p>Results</p> <p>Among these women, <it>P. falciparum </it>infection was detected in 5.6%, 25.5% and 60.5% of the cases in peripheral blood, placental blood and placental histological sections respectively. Placental histology was more sensitive (97.4%) than placental blood film (41.5%) and peripheral blood (8.0%) microscopy. In multivariate analysis, age (≤ 20 years old) (OR = 4.61, 95% CI = 1.47 – 14.70), history of fever attack (OR = 2.98, 95% CI = 1.58 – 5.73) were significant risk factors associated with microscopically detected parasitaemia. The use of ≥ 2 SP doses (OR = 0.18, 95% CI = 0.06 – 0.52) was associated with a significant reduction in the prevalence of microscopic parasitaemia at delivery. Age (>20 years) (OR = 0.34, 95% CI = 0.15 – 0.75) was the only significant risk factor associated with parasitaemia diagnosed by histology only in univariate analysis. Microscopic parasitaemia (OR = 2.74, 95% CI = 1.33–5.62) was a significant risk factor for maternal anaemia at delivery, but neither infection detected by histology only, nor past infection were associated with increased risk of anaemia.</p> <p>Conclusion</p> <p>Placenta histological examination was the most sensitive indicator of malaria infection at delivery. Microscopically detected parasitaemia was associated with increased risk of maternal anaemia at delivery, but not low-grade parasitaemia detected by placental histology only.</p

Understanding Human-Plasmodium falciparum Immune Interactions Uncovers the Immunological Role of Worms

BACKGROUND: Former studies have pointed to a monocyte-dependent effect of antibodies in protection against malaria and thereby to cytophilic antibodies IgG1 and IgG3, which trigger monocyte receptors. Field investigations have further documented that a switch from non-cytophilic to cytophilic classes of antimalarial antibodies was associated with protection. The hypothesis that the non-cytophilic isotype imbalance could be related to concomittant helminthic infections was supported by several interventions and case-control studies. METHODS AND FINDINGS: We investigated here the hypothesis that the delayed acquisition of immunity to malaria could be related to a worm-induced Th2 drive on antimalarial immune responses. IgG1 to IgG4 responses against 6 different parasite-derived antigens were analyzed in sera from 203 Senegalese children, half carrying intestinal worms, presenting 421 clinical malaria attacks over 51 months. Results show a significant correlation between the occurrence of malaria attacks, worm carriage (particularly that of hookworms) and a decrease in cytophilic IgG1 and IgG3 responses and an increase in non-cytophilic IgG4 response to the merozoite stage protein 3 (MSP3) vaccine candidate. CONCLUSION: The results confirm the association with protection of anti-MSP3 cytophilic responses, confirm in one additional setting that worms increase malaria morbidity and show a Th2 worm-driven pattern of anti-malarial immune responses. They document why large anthelminthic mass treatments may be worth being assessed as malaria control policies

Effect of treating Schistosoma haematobium infection on Plasmodium falciparum-specific antibody responses

<p>Abstract</p> <p>Background</p> <p>The overlapping geographical and socio-economic distribution of malaria and helminth infection has led to several studies investigating the immunological and pathological interactions of these parasites. This study focuses on the effect of treating schistosome infections on natural human immune responses directed against plasmodia merozoite surface proteins MSP-1 (DPKMWR, MSP1₁₉), and MSP-2 (CH150 and Dd2) which are potential vaccine candidates as well as crude malaria (schizont) and schistosome (whole worm homogenate) proteins.</p> <p>Methods</p> <p>IgG1 and IgG3 antibody responses directed against <it>Schistosoma haematobium </it>crude adult worm antigen (WWH) and <it>Plasmodium falciparum </it>antigens (merozoite surface proteins 1/2 and schizont extract), were measured by enzyme linked immunosorbent assay (ELISA) in 117 Zimbabweans (6–18 years old) exposed to <it>S. haematobium </it>and <it>P. falciparum </it>infection. These responses were measured before and after anti-helminth treatment with praziquantel to determine the effects of treatment on anti-plasmodial/schistosome responses.</p> <p>Results</p> <p>There were no significant associations between antibody responses (IgG1/IgG3) directed against <it>P. falciparum </it>and schistosomes before treatment. Six weeks after schistosome treatment there were significant changes in levels of IgG1 directed against schistosome crude antigens, plasmodia crude antigens, MSP-1₁₉, MSP-2 (Dd2), and in IgG3 directed against MSP-1₁₉. However, only changes in anti-schistosome IgG1 were attributable to the anti-helminth treatment.</p> <p>Conclusion</p> <p>There was no association between anti-<it>P. falciparum </it>and <it>S. haematobium antibody </it>responses in this population and <it>a</it>nti-helminth treatment affected only anti-schistosome responses and not responses against plasmodia crude antigens or MSP-1 and -2 vaccine candidates.</p

A Conserved Multi-Gene Family Induces Cross-Reactive Antibodies Effective in Defense against Plasmodium falciparum

BACKGROUND: Two related merozoite surface proteins, MSP3 and MSP6, have previously been identified as targets of antibody-dependent cellular inhibition (ADCI), a protective mechanism against Plasmodium falciparum malaria. Both MSP3 and MSP6 share a common characteristic small N-terminal signature amino-acid stretch (NLRNA/G), a feature similar to MSP3-like orthologs identified in other human and primate malaria parasites. METHODS/RESULTS: This signature amino-acid sequence led to the identification of eight ORFs contiguously located on P. falciparum chromosome 10. Our subsequent investigations on their expression, localization, sequence conservation, epitope sharing, immunogenicity and the functional role of antibodies in defense are reported here. Six members of P. falciparum MSP3-multigene family share similar sequence organization within their C-terminal regions, are simultaneously expressed as merozoite surface proteins and are highly conserved among parasite isolates. Each of these proteins is a target of naturally occurring antibodies effective at parasite killing in ADCI assays. Moreover, both naturally occurring antibodies and those generated by immunization display cross-reactivity with other members of the family and exhibit varied binding avidities. CONCLUSIONS/SIGNIFICANCE: The unusual characteristics of the MSP3 multi-gene family lead us to hypothesize that the simultaneous expression of targets eliciting cross-reactive antibody responses capable of controlling parasite densities could represent an immune process selected through evolution to maintain homeostasis between P. falciparum and human hosts; a process that allows the continuous transmission of the parasite without killing the host. Our observations also have practical consequences for vaccine development by suggesting MSP3 vaccine efficacy might be improved when combined with the various C-terminus regions of the MSP3 family members to generate a wider range of antibodies acting and to increase vaccine immunogenicity in varied human genetic backgrounds