Humoral immune response to mixed PfAMA1 alleles; multivalent PfAMA1 vaccines induce broad specificity

Abstract

Apical Membrane Antigen 1 (AMA1), a merozoite protein essential for red cell invasion, is a candidate malaria vaccine component. Immune responses to AMA1 can protect in experimental animal models and antibodies isolated from AMA1-vaccinated or malaria-exposed humans can inhibit parasite multiplication in vitro. The parasite is haploid in the vertebrate host and the genome contains a single copy of AMA1, yet on a population basis a number of AMA1 molecular surface residues are polymorphic, a property thought to be primarily as a result of selective immune pressure. After immunisation with AMA1, antibodies more effectively inhibit strains carrying homologous AMA1 genes, suggesting that polymorphism may compromise vaccine efficacy. Here, we analyse induction of broad strain inhibitory antibodies with a multi-allele Plasmodium falciparum AMA1 (PfAMA1) vaccine, and determine the relative importance of cross-reactive and strain-specific IgG fractions by competition ELISA and in vitro parasite growth inhibition assays. Immunisation of rabbits with a PfAMA1 allele mixture yielded an increased proportion of antibodies to epitopes common to all vaccine alleles, compared to single allele immunisation. Competition ELISA with the anti-PfAMA1 antibody fraction that is cross-reactive between FVO and 3D7 AMA1 alleles showed that over 80% of these common antibodies were shared with other PfAMA1 alleles. Furthermore, growth inhibition assays revealed that for any PfAMA1 allele (FVO or 3D7), the cross-reactive fraction alone, on basis of weight, had the same functional capacity on homologous parasites as the total affinity-purified IgGs (cross-reactive+strain-specific). By contrast, the strain-specific IgG fraction of either PfAMA1 allele showed slightly less inhibition of red cell invasion by homologous strains. Thus multi-allele immunisation relatively increases the levels of antibodies to common allele epitopes. This explains the broadened cross inhibition of diverse malaria parasites, and suggests multi-allele approaches warrant further clinical investigation.

Figure 1. Competition ELISA with protein A-purified antibodies and antibody pools made from single allele immunisations.

A) Assay on 3D7 AMA1-coated plates with anti-3D7 AMA1, anti-Combi (mixed allele immunisation) and anti-FVO/HB3/3D7 antibody pool. B) Assay on FVO AMA1-coated plates with anti- FVO AMA1, anti-Combi and anti-FVO/HB3/3D7 antibody pool. C) Assay on HB3 AMA1-coated plates with anti- HB3 AMA1, anti-Combi and anti-FVO/HB3/3D7 antibody pool. IgG pools were made from antibodies raised in single allele immunisations with FVO, HB3 and 3D7 AMA1. All assays were performed with FVO, HB3, 3D7 and CAMP AMA1 proteins as competitor antigens. All IgGs were used at 2 times the pre-determined antibody titre. Plots are representative of at least 2 assay repeats using IgGs from one rabbit per group since the depletion patterns were similar for both rabbits in each immunisation group.

Figure 2. Growth inhibition levels exhibited by protein A-purified IgGs from single/mixed PfAMA1 immunisations and IgG pools.

All IgG fractions were tested in a single growth cycle assay with FCR3, HB3, NF54 and CAMP strains of P. falciparum. For all strains, assays were performed with 0.3±0.1% parasitaemia and a final haematocrit of 2%. IgG samples were tested at 4 dilutions (3-fold titration from 6 mg/ml). IgG pools were made from antibodies raised in single allele immunisations with FVO, HB3 and 3D7 AMA1. The data presented is representative of at least two assay repeats using IgGs from one of the two rabbits per group.

Figure 3. Competition ELISA with PfAMA1-specific IgG fractions.

Anti-3D7 AMA1 IgGs were affinity-purified from total (Protein A) IgG of one of the 3D7 AMA1-immunised rabbits. A portion of this IgG fraction was afterwards fractionated into 3D7 AMA1 strain-specific IgG (flow through) and 3D7/FVO cross-reactive IgG (eluate) by passage over an FVO AMA1 affinity matrix. Similar specific fractions were made from total IgGs from one of the FVO AMA1-immunised rabbits, first over an FVO AMA1 matrix, and then over a 3D7 AMA1 matrix. All IgG fractions were used for competition assays at 2 times the pre-determined antibody titre. AMA1 antigens from the 3D7, HB3, FVO and CAMP parasite strains were used as competitor antigens in all assays. Assays were done using plates coated with 3D7 AMA1 (A) and FVO AMA1 (B), and plots are representative of data from at least 2 repeat assays.

Figure 4. Representative data showing levels of parasite growth inhibition exhibited by affinity-purified AMA1-specific IgG fractions.

All fractions were tested for functional activity on FCR3, NF54 and CAMP strains of P. falciparum. For all strains, assays were done with 0.3±0.1% parasitaemia and a final haematocrit of 2%. IgG samples were tested at 4 dilutions (3-fold titration from 1 mg/ml). Anti-FVO and anti-3D7 are the respective total affinity-purified IgGs, anti-FVO CR and anti-3D7 CR designate the cross-reactive fractions, while anti-FVO spec and anti-3D7 spec designate the respective strain-specific fractions.