• Allele-specific efficacy of two malaria subunit vaccines following immunization with the polymorphic antigen apical membrane antigen-1 (AMA1)

      Ouattara, Amed; Plowe, Christopher V. (2012)
      Background: Antigenic diversity of malaria parasites poses a major obstacle to the development of an effective malaria vaccine. Population genetics analyses suggest that polymorphic regions of the vaccine antigen apical membrane antigen 1 (AMA1) are targets for protective immune responses. Objectives: The study exploited a unique opportunity to assess strain-specific efficacy in clinical trials of two AMA1-based vaccines, with the ultimate goal of developing an AMA1 malaria vaccine that protects against genetically diverse parasites. Methods: We used data and samples collected during two randomized, double-blind, controlled phase 2 trials (Bancoumana AMA1-C1 and Bandiagara FMP2.1/AS02A vaccine trials) of malaria vaccine safety and efficacy to assess allele-specific in AMA1 vaccinees compared to control groups. In each study, we sequenced the Plasmodium falciparum ama1 gene from samples collected at baseline, during clinical episodes, at scheduled intervals during post-vaccination follow-up period, and whenever blood smears were collected for malaria diagnosis. Using a homology definition based on clusters c1, c1L, c2, c3 and domains 1, 2 and 3, we assessed the time to first malaria episode with an AMA1 sequence identical to that of the vaccine strains 3D7 or FVO type. In addition, we measured the hazard of having a clinical episode with the vaccine strain in the two treatment groups. Finally, genetic diversity parameters were assessed and compared between treatment arms. Results: The bivalent malaria vaccine showed no overall efficacy. AMA1 vaccine group and controls groups were found to have genetic parameters comparable at baseline and during the follow-up period. No difference was found between the 2 treatment arms regarding the time to first clinical episode with a 3D7 or FVO AMA1 c1L haplotype. In Bandiagara, the monovalent vaccine has a 20% overall efficacy against malaria first clinical episodes. The time to first clinical episode with a 3D7 c1L allele was longer in the AMA1 vaccine group compared to the rabies vaccine, Likelihood Ratio p-value respectively equal to 0.025 (ITT) and 0.018 (PP). The vaccine efficacy against c1L allele was 64% (ITT) and 72% (PP). Conclusion: It may be possible to design a polyvalent or chimeric AMA1 vaccine that provides broader cross-protection.
    • Development and validation of a novel tool to assess naturally acquired and vaccine induced antibody diversity to Plasmodium falciparum apical membrane antigen 1 in a pediatric and adult cohort in Bandiagara, Mali: Implications for vaccine design

      Bailey, Jason Andrew; Plowe, Christopher V. (2015)
      Immunity to clinical malaria disease is acquired after years of exposure to malaria pathogens, but sterile immunity is not achieved. Malaria parasite surface antigens have used antigenic diversity in an arms race to subvert the host immune system. Nearly all vaccines targeting individual malaria antigens have proven ineffective at preventing infection or disease. Plasmodium falciparum apical membrane antigen 1 is a parasite surface antigen that is crucial for erythrocyte invasion, and a leading vaccine target. Antibodies against AMA1 have been shown in vitro, as well as in murine, non-human primate, and human models to prevent homologous parasite infection after challenge. AMA1 is an antigenically diverse molecule, with a seemingly limitless number of unique variants surveyed in the field. We populated a protein microarray with 263 unique whole-ectodomain variants of AMA1 proteins isolated from parasite genomic DNA from malaria-infected blood samples collected during a phase 2 malaria vaccine trial conducted in Bandiagara, Mali. We screened Malian children and adults at seasonal time points to measure the diversity of their immune response. Age, parasitemia, and seasonality were significant predictors of the seroreactivity to PfAMA1 variants. Children and adults vaccinated with monovalent, AMA1 subunit vaccine FMP2.1/AS02A saw a dramatic increase in seroreactivity to all AMA1 variants on the array, regardless of genetic similarity to the vaccine strain 3D7 compared to rabies vaccinated control cohorts. Seroreactivity to PfAMA1 variants is extremely collinear, and genetic variation of the strain isolated at the time of a single acute infection did not correlate with antibody seroreactivity. In a post-hoc analysis, we saw a positive correlation with preseason seroreactivity to AMA1 and odds of symptomatic versus asymptomatic infection using multivariable logistic regression. The research demonstrates the need to have an understanding of both parasite antigenic diversity in the field and a functional epitope map prior to the development of a vaccine based on AMA1.
    • Genomic Epidemiology of the Malaria Parasite Plasmodium falciparum: Implications for Whole-Organism Malaria Vaccine Development

      Moser, Kara; Carneiro da Silva, Joana; Plowe, Christopher V. (2018)
      Whole-organism malaria vaccines have shown great promise in initial clinical trials against controlled human malaria infections (CHMI) with homologous P. falciparum strains. However, efficacy against heterologous CHMI and against natural infection is somewhat lower. While the reason for lower efficacy against non-vaccine strains is unknown, several knowledge gaps, if addressed, would assist in developing highly-efficacious whole-organism malaria vaccines. First, regions of the parasite genome responsible for protection have not been identified. Such identification could be achieved by comparing breakthrough P. falciparum infections in vaccinated individuals with the vaccine strain; however, it is not clear how best to genetically characterize breakthrough infections, in vaccinated individuals. Second, vaccine and challenge strains have not been characterized and compared at the genomic level to circulating strains from parasite populations in malaria endemic regions. Finally, a rigorous investigation of allele frequency fluctuations between temporally isolated parasite populations would help predict if such changes would affect vaccine efficacy. Using next- and third-generation sequencing technologies, new reference assemblies for whole-organism malaria vaccine and CHMI strains were generated, along with reference assemblies for 19 clinical isolates to use for improved read mapping and characterization of clinical P. falciparum isolates. Using a geographic-specific reference assembly improved the ability to characterize clinical isolates through increased read coverage, and so these references may be helpful to characterize breakthrough infections (particularly for studies in Southeast Asian populations). In addition, assemblies for vaccine and CHMI strains reveal thousands of variants between these parasites. CHMI strains also have variants which convey differences in immunological potential, thus confirming their appropriateness as heterologous CHMI strains. Vaccine and CHMI strains were also shown to representative of their respective geographic origins when compared to extant parasite populations from malaria endemic regions. Finally, we show that while there were fluctuations in allele frequencies between West and East African parasite populations over almost a decade, none affected the frequency of vaccine candidate alleles in a way that would meaningfully impact vaccine efficacy. These results will greatly assist in the interpretation of whole-organism malaria vaccine trials, and will pave the way for the design of next-generation whole-organism malaria vaccines.
    • Humoral Immune Responses to a Malaria Vaccine Candidate: Towards a Correlate of Vaccine-Induced Protection

      Gottlieb, Eric Raphael; Berry, Andrea A. (2012)
      Introduction: Identifying immune correlates of protection is a priority for malaria vaccine research. A successful pediatric Phase 2 clinical trial in Mali of FMP2.1/AS02A, a recombinant apical membrane antigen 1 (AMA1)-based vaccine candidate, provided a source of serum samples from subjects who may have developed vaccine-induced, strain-specific protective immunity to clinical malaria illness. We studied IgG subclass and avidity patterns of antibodies to the malaria protein AMA1 in a subset of participants, with the objective of identifying immune responses that may be associated with protection against malaria. We hypothesized that the AMA1 vaccine candidate would induce production of cytophilic antibody subclasses IgG1 and IgG3, as well as overall IgG avidity maturation. Methods: Titers of IgG1, IgG2, IgG3, and IgG4, as well as avidity of antibodies to AMA1, were determined by ELISA for ten AMA1 vaccine recipients and ten control subjects who had been randomized to receive a rabies vaccine in this double-blind trial at days 0, 90, and 150 after the first of three vaccinations. To identify statistically significant differences between the groups, responses in vaccine recipients were evaluated longitudinally and compared with responses in control subjects. Results: IgG1, IgG2, IgG3, and IgG4 were induced more strongly in vaccine recipients than in control subjects. Additionally, vaccine recipients had higher ratios of cytophilic to non-cytophilic antibodies than control subjects. Avidity indices were not significantly different between the two groups at the three time points tested, and there were no significant differences in avidity between time points in either group. Conclusion: Contrary to our hypothesis, both cytophilic and non-cytophilic antibodies were induced by the FMP2.1/AS02A vaccine candidate and immunization did not appear to stimulate avidity maturation. Therefore, IgG1, IgG2, IgG3, and IgG4 titers are candidate variables for a humoral immune correlate of vaccine-induced protection. These results are among the first reports of the subclasses and avidity of antibodies produced in response to a malaria vaccine candidate with allele-specific protective efficacy.
    • Variation in the Circumsporozoite Protein of Plasmodium falciparum: Implications for Vaccine Development

      Gandhi, Kavita; Plowe, Christopher V. (2013)
      Background: A leading malaria vaccine candidate, RTS,S/AS01, is based on immunogenic regions of Plasmodium falciparum circumsporozoite protein (CSP) from the 3D7 variant, and has shown modest efficacy against clinical disease in African children. It is unclear, however, what aspect(s) of the immune response elicited by this vaccine are protective. Better understanding of how diversity in the immunogenic regions of CSP (T-cell and B-cell epitopes) may relate to clinical immunity is needed to evaluate and improve the efficacy of vaccines based on CSP. Objectives: The goals of this study were to measure diversity in immunogenic regions of CSP in a natural population of parasites and identify associations between variation in amino acid sequences in CSP and the risk of infection and clinical disease caused by P. falciparum in African children. Methods: One hundred children were selected from those who had participated in a prospective cohort study designed to measure incidence of malaria infection in Bandiagara, Mali. DNA was extracted from 769 parasite-positive blood samples corresponding to both acute clinical malaria episodes and asymptomatic infections detected in monthly surveys and B- and T-cell epitope-encoding portions of the cs gene were sequenced. Non-synonymous SNP data were generated via 454, a next generation sequencing technology, for the T-cell epitopes and repeat length data was generated for the B-cell epitopes of the cs gene. Cox proportional hazards models were used to determine the effect of sequence variation in consecutive infections occurring within individuals on the time to new infection and new clinical malaria episode. Conclusions: Extensive diversity was found in the T-cell epitopes, but no associations were found between sequence variation in either the T-cell epitopes or the repeat region, and hazard of infection or clinical malaria, suggesting that naturally acquired immunity to CSP may not be allele-specific.