Use of Immunodampening To Overcome Diversity in the Malarial Vaccine Candidate Apical Membrane Antigen 1

Abstract

c Apical membrane antigen 1 (AMA1) is a leading malarial vaccine candidate; however, its polymorphic nature may limit its success in the field. This study aimed to circumvent AMA1 diversity by dampening the antibody response to the highly polymorphic loop Id, previously identified as a major target of strain-specific, invasion-inhibitory antibodies. To achieve this, five polymorphic residues within this loop were mutated to alanine, glycine, or serine in AMA1 of the 3D7 and FVO Plasmodium falciparum strains. Initially, the corresponding antigens were displayed on the surface of bacteriophage, where the alanine and serine but not glycine mutants folded correctly. The alanine and serine AMA1 mutants were expressed in Escherichia coli, refolded in vitro, and used to immunize rabbits. Serological analyses indicated that immunization with a single mutated form of 3D7 AMA1 was sufficient to increase the cross-reactive antibody response. Targeting the corresponding residues in an FVO backbone did not achieve this outcome. The inclusion of at least one engineered form of AMA1 in a biallelic formulation resulted in an antibody response with broader reactivity against different AMA1 alleles than combining the wild-type forms of 3D7 and FVO AMA1 alleles. For one combination, this extended to an enhanced relative growth inhibition of a heterologous parasite line, although this was at the cost of reduced overall inhibitory activity. These results suggest that targeted mutagenesis of AMA1 is a promising strategy for overcoming antigenic diversity in AMA1 and reducing the number of variants required to induce an antibody response that protects against a broad range of Plasmodium falciparum AMA1 genotypes. However, optimization of the immunization regime and mutation strategy will be required for this potential to be realized.T he apicomplexan parasite Plasmodium falciparum, the causative agent of the most severe form of human malaria, is responsible for Ͼ0.5 million deaths annually. There are currently no licensed vaccines for malaria; however, the development of clinical immunity in naturally exposed individuals suggests that a vaccine that reduces the morbidity and mortality associated with malaria is likely to be achievable (1). RTS,S/ASO1, a vaccine targeting the preerythrocytic stages of P. falciparum, is showing partial efficacy in a large multicenter phase III trial in Africa (2), but ultimately, an improved vaccine with higher efficacy and longer duration of protection will be required (3). This may be achieved through new vaccine approaches or by incorporating one or more asexual blood-stage antigens into a vaccine containing RTS,S.Of the asexual blood-stage antigens assessed as potential vaccine components, apical membrane antigen 1 (AMA1) has been considered particularly promising, and a large body of preclinical data supported the decision by several groups to take AMA1 vaccines into clinical development (recently reviewed in reference 4). Additionally, AMA1 is an important target of acquired immunity; antibodies t...

Recent advances in recombinant protein-based malaria vaccines

“…Indeed, it remains unknown, for now, what proportion of vaccine-induced PfAMA1 antibodies in humans can provide functional anti-parasitic activity. Recent efforts have largely aimed at enhancing strain-transcendent, neutralizing antibodies by incorporating multiple PfAMA1 alleles or mutagenesis to redirect the immune response toward conserved regions [39] , [40] , [41] , [42] , [43] , [44] , and one ‘diversity covering’ (DiCo) approach has just entered Phase Ia testing in Europe (Clinicaltrials.gov NCT02014727 ). These approaches increase the repertoire of antibodies against multiple PfAMA1 alleles but do not, however, appear to enhance inhibition of homologous parasites.…”

Far from the Madding Crowd: the Molecular Basis for Immunological Escape ofPlasmodium falciparum

“…T h e s e polymorphic residues surround a hydrophobic pocket suggested as being critical for AMA-1 function. Domain I has the major polymorphic sites, grouped into 3 clusters (C1, C2 and C3); regarding C1-L (loop containing residues 196, 197, 199, 200, 201, 204, 206 and 207), 1F9 mAb binds in loop Id and most human antibodies react against it (Bai et al, 2005;Dutta et al, 2007;Takala et al, 2009;Ouattara et al, 2013;Harris et al, 2014). However, once the epitope has been recognised it inhibits in vitro invasion in a strain-specific manner, as in the case of 1F9 mAb inhibiting in vitro growth of 3D7 and D10 parasite strains, but not HB3 or W2mef where a single aa substitution at the most highly polymorphic site (residue 197) in AMA-1 abolishes 1F9 mAb binding (Coley et al, 2006), thereby limiting the effectiveness of these antigens as vaccine components.…”

Progress and prospects for blood-stage malaria vaccines

“…The results of a non-human primate study were promising [ 110 ], and a phase I trial is underway (ClinicalTrial.gov Identifier NCT02014727). One more unique approach is to mutate major polymorphic residues to alanine, glycine, or serine [ 111 ]. While the chimeric AMA1 induced more cross-reactive antibodies judged by GIA, the levels of inhibition were lower compared to the antibodies raised with non-chimeric AMA1 for the same strains of parasites.…”