Generation of humoral immune responses to multi-allele PfAMA1 vaccines; effect of adjuvant and number of component alleles on the breadth of response

Abstract

There is increasing interest in multi-allele vaccines to overcome strain-specificity against polymorphic vaccine targets such as Apical Membrane Antigen 1 (AMA1). These have been shown to induce broad inhibitory antibodies in vitro and formed the basis for the design of three Diversity-Covering (DiCo) proteins with similar immunological effects. The antibodies produced are to epitopes that are shared between vaccine alleles and theoretically, increasing the number of component AMA1 alleles is expected to broaden the antibody response. A plateau effect could however impose a limit on the number of alleles needed to achieve the broadest specificity. Moreover, production cost and the vaccine formulation process would limit the number of component alleles. In this paper, we compare rabbit antibody responses elicited with multi-allele vaccines incorporating seven (three DiCos and four natural AMA1 alleles) and three (DiCo mix) antigens for gains in broadened specificity. We also investigate the effect of three adjuvant platforms on antigen specificity and antibody functionality. Our data confirms a broadened response after immunisation with DiCo mix in all three adjuvants. Higher antibody titres were elicited with either CoVaccine HT™ or Montanide ISA 51, resulting in similar in vitro inhibition (65-82%) of five out of six culture-adapted P. falciparum strains. The antigen binding specificities of elicited antibodies were also similar and independent of the adjuvant used or the number of vaccine component alleles. Thus neither the four extra antigens nor adjuvant had any observable benefits with respect to specificity broadening, although adjuvant choice influenced the absolute antibody levels and thus the extent of parasite inhibition. Our data confirms the feasibility and potential of multi-allele PfAMA1 formulations, and highlights the need for adjuvants with improved antibody potentiation properties for AMA1-based vaccines.

Figure 1. Levels of anti-AMA1 antibody elicited with the four multi-allele vaccine formulations in rabbits.

Gp 1 rabbits were immunised with seven AMA1 antigens (DiCo mix and AMA1 from FVO, HB3, 3D7 and CAMP parasite strains) in CoVaccine HT™, and the other groups were immunised with DiCo mix in CoVaccine HT™ (Gp 2), Montanide IMS (Gp 3) and Montanide ISA 51 (Gp 4) respectively. Antibody titres were determined by a standardized ELISA with DiCo 1, DiCo 2, DiCo 3, FVO, HB3, 3D7 and CAMP AMA1-coated plates. Data is presented on a Log2 scale as dotplots with a boxplot superpose indicating the lower and upper quartiles as well as the median per immunisation group. Within the same immunisation group, plotting symbols represent the antibody titre of individual rabbits on all coating/capture antigens.

Figure 2. Alignment of the protein sequences (aa25-545) of PfAMA1 antigens used in this study.

DiCo proteins were used to immunise rabbits and as capture antigens in ELISA. Natural allele AMA1 proteins were used to immunize rabbits, and as capture and competitor antigens in (competition) ELISAs. All proteins were produced in Pichia pastoris and are devoid of N-glycosylation sites. These have been replaced with amino acid residues (indicated in red) that occur in AMA1 sequences from other malarial species (N162Q, T288V, S373D, N422D, S423K, N499Q). Residue 162 is unique as it is also a polymorphic residue. Additionally, all sequences contain a point mutation at position 376 (K to R, indicated in orange). This was necessary to prevent protein cleavage by P. pastoris proteases.

Figure 3. Growth inhibition of Plasmodium parasites by antibody pools from the 3-antigen and 7-antigen immunisation groups.

The 7-antigen immunisation group (Gp 1) had only a single sample (antibodies purified from a pool of 98 rabbit sera), hence a direct comparison of the functional activities of antibodies from the 3 and 7-antigen immunisation groups could not be made. The growth inhibitory activity of IgGs purified from a pool of all rabbits immunised with DiCo mix (3-antigen, Gp 2) in the same adjuvant (CoVaccine HT™) was therefore compared with that of the single Gp 1 sample against the FCR3, HB3 and NF54 parasite strains. Plots represent the mean % inhibition ± SEM for replicate measurements for each sample. Blue filled circles (•) represent the Gp 1 pooled sample and the red filled squares (▪) represent the Gp 2 pooled sample.

Figure 4. Growth inhibition of Plasmodium parasites by antibodies elicited with the three DiCo mix vaccines.

Antibodies from all immunisation groups were tested on each of six culture-adapted strains (7G8, CAMP, FCR3, HB3, L32 and NF54) of P. falciparum. Plots represent the mean % inhibition ± SEM for all antibody samples within an immunisation group. Blue filled circles (•) represent Gp 2 (DiCo mix in CoVaccine HT™, n = 8), green open diamonds (◊) represent Gp 3 (DiCo mix in Montanide IMS, n = 8) and red filled squares (▪) represent Gp 4 (DiCo mix in Montanide ISA 51, n = 5).

Figure 5. Alignment of DiCo protein sequences (aa25-545) with those of AMA1 present in GIA malaria parasites.

AMA1 protein sequences of malaria parasites used for in vitro growth inhibition assays were accessed from the GenBank database. The DiCo sequences contain point mutations at the cleavage (K376R, indicated in orange) and potential N-glycosylation (N162Q, T288V, S373D, N422D, S423K, N499Q) sites (indicated in red), and differ from parasite AMA1 sequences at these sites. Amino acids at 51 polymorphic sites (within aa25-525) also differ between sequences.

Figure 6. Relationship between ELISA antibody titre and in vitro parasite growth inhibition.

Association of antibody levels with in vitro antibody functionality for three of the four immunisation groups (Gps 2, 3 and 4) is shown for parasite strains whose AMA1 allelic antigens were available for antibody measurement. In order to obtain an optimal estimate of the association, growth inhibition data at all four antibody concentrations tested (6.0, 3.0, 1.5 and 0.75 mg/ml) for each sample were included. Plots are based on a four-parameter logistic function, and each symbol/colour represents individual rabbits in the same immunisation group.