Individuals living in areas where malaria is endemic are repeatedly exposed to many different malaria parasite antigens. Studies on naturally acquired antibody-mediated immunity to clinical malaria have largely focused on the presence of responses to individual antigens and their associations with decreased morbidity. We hypothesized that the breadth (number of important targets to which antibodies were made) and magnitude (antibody level measured in a random serum sample) of the antibody response were important predictors of protection from clinical malaria. We analyzed naturally acquired antibodies to five leading Plasmodium falciparum merozoite-stage vaccine candidate antigens, and schizont extract, in Kenyan children monitored for uncomplicated malaria for 6 months (n ‫؍‬ 119). Serum antibody levels to apical membrane antigen 1 (AMA1) and merozoite surface protein antigens (MSP-1 block 2, MSP-2, and MSP-3) were inversely related to the probability of developing malaria, but levels to MSP-1 19 and erythrocyte binding antigen (EBA-175) were not. The risk of malaria was also inversely associated with increasing breadth of antibody specificities, with none of the children who simultaneously had high antibody levels to five or more antigens experiencing a clinical episode (17/119; 15%; P ‫؍‬ 0.0006). Particular combinations of antibodies (AMA1, MSP-2, and MSP-3) were more strongly predictive of protection than others. The results were validated in a larger, separate case-control study whose end point was malaria severe enough to warrant hospital admission (n ‫؍‬ 387). These findings suggest that under natural exposure, immunity to malaria may result from high titers antibodies to multiple antigenic targets and support the idea of testing combination blood-stage vaccines optimized to induce similar antibody profiles.

are named inventors on patent applications relating to RH5 and/or other malaria vaccines and immunization regimens. L. Siani and S. Di Marco are employees of ReiThera (formerly Okairos), which is currently developing vectored vaccines for a number of diseases. J. Vekemans was an employee of GSK, which has acquired the ChAd63 vector from Okairos. R. Ashfield is a director of Ducentis and holds shares in the company, which is developing a therapy for autoimmune disease. A.M. Minassian has an immediate family member who is an inventor on patents relating to RH5 and/or other malaria vaccines and immunization regimens and who is a cofounder of, shareholder in, and consultant for SpyBiotech. S. Biswas is a cofounder and CEO of, and shareholder in, SpyBiotech and is a contributor in a patent application relating to multimerisation technology. J. Jin is a cofounder of and shareholder in SpyBiotech.

No vaccine has yet proven effective against the blood-stages of Plasmodium falciparum, which cause the symptoms and severe manifestations of malaria. We recently found that PfRH5, a P. falciparum-specific protein expressed in merozoites, is efficiently targeted by broadly-neutralizing, vaccine-induced antibodies. Here we show that antibodies against PfRH5 efficiently inhibit the in vitro growth of short-term-adapted parasite isolates from Cambodia, and that the EC50 values of antigen-specific antibodies against PfRH5 are lower than those against PfAMA1. Since antibody responses elicited by multiple antigens are speculated to improve the efficacy of blood-stage vaccines, we conducted detailed assessments of parasite growth inhibition by antibodies against PfRH5 in combination with antibodies against seven other merozoite antigens. We found that antibodies against PfRH5 act synergistically with antibodies against certain other merozoite antigens, most notably with antibodies against other erythrocyte-binding antigens such as PfRH4, to inhibit the growth of a homologous P. falciparum clone. A combination of antibodies against PfRH4 and basigin, the erythrocyte receptor for PfRH5, also potently inhibited parasite growth. This methodology provides the first quantitative evidence that polyclonal vaccine-induced antibodies can act synergistically against P. falciparum antigens and should help to guide the rational development of future multi-antigen vaccines.

HighlightsWe hypothesized that a threshold antibody concentration is required for protection.Thresholds were derived using statistical methods in a high transmission setting.Thresholds were applied to a low transmission setting.Most children had antibodies below the threshold in the low transmission setting.

Background Plasmodium falciparum malaria remains a major cause of illness and death in sub-Saharan Africa. Young children bear the brunt of the disease and though older children and adults suffer relatively fewer clinical attacks, they remain susceptible to asymptomatic P. falciparum infection. A better understanding of the host factors associated with immunity to clinical malaria and the ability to sustain asymptomatic P. falciparum infection will aid the development of improved strategies for disease prevention.Methods and FindingsHere we investigate whether full differential blood counts can predict susceptibility to clinical malaria among Kenyan children sampled at five annual cross-sectional surveys. We find that the ratio of monocytes to lymphocytes, measured in peripheral blood at the time of survey, directly correlates with risk of clinical malaria during follow-up. This association is evident among children with asymptomatic P. falciparum infection at the time the cell counts are measured (Hazard ratio (HR)  =  2.7 (95% CI 1.42, 5.01, P  =  0.002) but not in those without detectable parasitaemia (HR  =  1.0 (95% CI 0.74, 1.42, P  =  0.9).ConclusionsWe propose that the monocyte to lymphocyte ratio, which is easily derived from routine full differential blood counts, reflects an individual's capacity to mount an effective immune response to P. falciparum infection.

Severe malaria (SM) is a life-threatening complication of infection with Plasmodium falciparum. Epidemiological observations have long indicated that immunity against SM is acquired relatively rapidly, but prospective studies to investigate its immunological basis are logistically challenging and have rarely been undertaken. We investigated the merozoite targets and antibody-mediated mechanisms associated with protection against SM in Kenyan children aged 0 to 2 years. We designed a unique prospective matched case-control study of well-characterized SM clinical phenotypes nested within a longitudinal birth cohort of children (n = 5,949) monitored over the first 2 years of life. We quantified immunological parameters in sera collected before the SM event in cases and their individually matched controls to evaluate the prospective odds of developing SM in the first 2 years of life. Anti-AMA1 antibodies were associated with a significant reduction in the odds of developing SM (odds ratio [OR] = 0.37; 95% confidence interval [CI] = 0.15 to 0.90; P = 0.029) after adjustment for responses to all other merozoite antigens tested, while those against MSP-2, MSP-3, Plasmodium falciparum Rh2 [PfRh2], MSP-119, and the infected red blood cell surface antigens were not. The combined ability of total IgG to inhibit parasite growth and mediate the release of reactive oxygen species from neutrophils was associated with a marked reduction in the odds of developing SM (OR = 0.07; 95% CI = 0.006 to 0.82; P = 0.03). Assays of these two functional mechanisms were poorly correlated (Spearman rank correlation coefficient [rs] = 0.12; P = 0.07). Our data provide epidemiological evidence that multiple antibody-dependent mechanisms contribute to protective immunity via distinct targets whose identification could accelerate the development of vaccines to protect against SM.

Although Plasmodium falciparum apical membrane antigen 1 (AMA1) is a leading malaria vaccine candidate, extensive allelic diversity may compromise its vaccine potential. We have previously shown that naturally acquired antibodies to AMA1 were associated with protection from clinical malaria in this Kenyan population. To assess the impact of allelic diversity on naturally acquired immunity, we first sequenced the ectodomainencoding region of P. falciparum ama1 from subjects with asymptomatic, mild, and severe malaria and measured allele frequency distributions. We then measured antibodies to three allelic AMA1 proteins (AMA1_3D7, AMA1_FVO, and AMA1_HB3) and used competition enzyme-linked immunosorbent assays (ELISAs) to analyze allele-specific antibodies. Seventy-eight unique haplotypes were identified from 129 alleles sampled. No clustering of allelic haplotypes with disease severity or year of sampling was observed. Differences in nucleotide frequencies in clinical (severe plus mild malaria) versus asymptomatic infections were observed at 16 polymorphic positions. Allele frequency distributions were indicative of balancing selection, with the strongest signature being identified in domain III (Tajima's D ‫؍‬ 2.51; P < 0.05). Antibody reactivities to each of the three allelic AMA1 proteins were highly correlated (P < 0.001 for all pairwise comparisons). Although antibodies to conserved epitopes were abundant, 48% of selected children with anti-AMA1 IgG (n ‫؍‬ 106) had detectable reactivity to allele-specific epitopes as determined by a competition ELISA. Antibodies to both conserved and allele-specific epitopes in AMA1 may contribute to clinical protection.

IFN-γ and T cells are both required for the development of experimental cerebral malaria during P. berghei ANKA infection. Surprisingly, however, the role of IFN-γ in shaping the effector CD4+ and CD8+ T cell response during this infection has not been examined in detail. To address this, we have compared the effector T cell responses in wild-type and IFN- γ−/− mice during P. berghei ANKA infection. The expansion of splenic CD4+ and CD8+ T cells during P. berghei ANKA infection was unaffected by the absence of IFN-γ but the contraction phase of the T cell response was significantly attenuated. Splenic T cell activation and effector function were essentially normal in IFN- γ−/− mice, however, the migration to, and accumulation of, effector CD4+ and CD8+ T cells in the lung, liver and brain was altered in IFN- γ−/− mice. Interestingly, activation and accumulation of T cells in various non-lymphoid organs was differently affected by lack of IFN-γ, suggesting that IFN- γ influences T cell effector function to varying levels in different anatomical locations. Importantly, control of splenic T cell numbers during P. berghei ANKA infection depended upon active IFN- γ-dependent environmental signals – leading to T cell apoptosis - rather than upon intrinsic alterations in T cell programming. This is the first study to fully investigate the role of IFN- γ in modulating T cell function during P. berghei ANKA infection and reveals that IFN-γ is required for efficient contraction of the pool of activated T cells.