Cell-mediated immunity elicited by the blood stage malaria vaccine apical membrane antigen 1 in Malian adults: Results of a Phase I randomized trial

Abstract

The development of a safe and effective malaria vaccine is impeded by the complexity of the Plasmodium life cycle. A vaccine that elicits both cell-mediated and humoral immune responses might be needed for protection against this multistage parasitic infection. Apical membrane antigen 1 (AMA-1) plays a key role in erythrocytic invasion but is also expressed in sporozoites and in late stage liver schizonts, where it may provide a target of protective cell-mediated immunity (CMI). A Phase 1 trial of a vaccine consisting of recombinant AMA-1 protein and the Adjuvant system AS02A was conducted in 60 Malian adults aged 18–55 years who were randomized to receive either half-dose (25 μg/0.25 ml) or full dose (50 μg/0.5 ml) FMP2.1/AS02A or a control rabies vaccine. Interleukin 5 (IL-5) and interferon-γ (IFN-γ) production as evaluated by ELISpot and lymphocyte proliferation were measured after in vitro AMA-1 stimulation of peripheral blood mononuclear cells (PBMCs) collected on Days 0 and 90. Post-FMP2.1/AS02A immunization mean stimulation indices were significantly elevated as were the number of IL-5 spot forming cells (SFC)/10⁶ PBMC, but no difference was noted in INF-γ production between the AMA-1/AS02A vaccinated group and the rabies group. These results provide evidence that complex immune responses can be induced by this vaccination strategy and add further impetus for the continuing clinical evaluation of this vaccine.

Introduction

Apical membrane antigen 1 (AMA-1) is an asexual blood-stage antigen that has been identified in all Plasmodium species examined and is a leading malaria vaccine candidate [1], [2], [3]. Although its precise biological function remains undefined, AMA-1 appears to play a role in erythrocytic invasion possibly by orienting merozoites and mediating close junctional apical contact [4], [5] or direct erythrocytic binding [6]. The protein is comprised of an ectodomain in which non-overlapping groups of disulfide bonds define four separate domains (pro-domain and domains I, II, and III) [7]. Correct conformational folding is critical for the invocation of protective or inhibitory antibodies, implying that protection is directed at epitopes dependent upon correct disulfide bonding [7], [8], [9].

Antibodies against Plasmodium falciparum AMA-1 (PfAMA-1) inhibit antigen processing and merozoite invasion of erythrocytes [10] as well as P. falciparum growth in vitro [11], [12]. Passive immunization of P. chabaudi-infected mice with AMA-1-specific polyclonal antibodies prevents lethal parasitemia [8]. Protective immunity against malaria has been elicited after immunization of mice and monkeys with recombinant AMA-1 [8], [9], [13], [14], [15]. Seroprevalence studies have demonstrated the presence of antibodies to AMA-1 in malaria endemic areas [16], [17] and in a longitudinal study of subjects parasitemic at entry, antibodies to AMA-1 were significantly associated with fewer episodes of clinical malaria [18].

While antibody-mediated immunity to AMA-1 appears to be critical for immune protection, AMA-1-specific T cell-mediated immunity (CMI) might also play a significant role in protective immunity. AMA-1 is also present on the sporozoite surface and a strong T cell response against AMA-1 may have an inhibitory effect on sporozoite invasion and exoerythrocytic stage development in the liver [19]. Just before the liver phase where CMI plays a key role in host defense, AMA-1 is translocated to the surface of the sporozoite, undergoes proteolytic cleavage and is essential to hepatocyte invasion [20], [21]. T cells specific for AMA-1 may regulate antibody production as demonstrated in immunized BALB/c mice [9]. Evidence of antibody-independent protective CMI can be demonstrated by the finding that AMA-1 immunized mice and immunized B-cell-deficient mice, which are then depleted of CD4⁺ T cells, experience a loss of immunity unrelated to changes in antibody levels. CD4⁺ T cells specific for a conserved AMA-1 cryptic epitope transferred into athymic mice resulted in partial protection from parasite challenge [22]. T cell epitopes that elicit proliferative responses in Kenyan volunteers have been demonstrated in P. falciparum AMA-1 [23]. Strong T cell responses to AMA-1 were also seen in irradiated sporozoite-immunized volunteers protected against experimental malaria challenge [24]. Thus, while antibodies play a crucial role in protection post-AMA-1 immunization, antibody-independent CMI to AMA-1 contributes separately to the overall protective immune response in mice and might also do so in humans.

Immunization strategies that induce both a strong humoral response and CMI may be desirable in development of an AMA-1-based malaria vaccine. A Phase 1 trial of AMA-1/AS02A in malaria-naïve adults conducted at the Walter Reed Army Institute for Research (WRAIR) revealed strong humoral and cellular responses [3]. In the subsequent, presently described trial we measured CMI in semi-immune Malian adults after immunization with three doses of AMA-1 (FMP2.1/AS02A). Enhanced AMA-1-specific CMI was demonstrated in vaccine recipients as compared to control volunteers. These results, together with those demonstrating strong anti-AMA-1 antibody responses [25] provide evidence that multifaceted immune responses can be induced by this vaccination strategy and add further impetus for the continuing clinical evaluation of this vaccine.