Mixed allele malaria vaccines: host protection and within-host selection

Abstract

Malaria parasites are frequently polymorphic at the antigenic targets of many candidate vaccines, presumably as a consequence of selection pressure from protective immune responses. Conventional wisdom is therefore that vaccines directed against a single variant could select for non-target variants, rendering the vaccine useless. Many people have argued that a solution is to develop vaccines containing the products of more than one variant of the target. However, we are unaware of any evidence that multi-allele vaccines better protect hosts against parasites or morbidity. Moreover, selection of antigen-variants is not the only evolution that could occur in response to vaccination. Increased virulence could also be favored if more aggressive strains are less well controlled by vaccine-induced immunity. Virulence and antigenic identity have been confounded in all studies so far, and so we do not know formally from any animal or human studies whether vaccine failure has been due to evasion of protective responses by variants at target epitopes, or whether vaccines are just less good at protecting against more aggressive strains. Using the rodent malaria model Plasmodium chabaudi and recombinant apical membrane antigen-1 (AMA-1), we tested whether a bi-allelic vaccine afforded greater protection from parasite infection and morbidity than did vaccination with the component alleles alone. We also tested the effect of mono- and bi-allelic vaccination on within-host selection of mixed P. chabaudi infections, and whether parasite virulence mediates pathogen titres in immunized hosts. We found that vaccination with the bi-allelic AMA-1 formulation did not afford the host greater protection from parasite infection or morbidity than did mono-allelic AMA-1 immunization. Mono-allelic immunization increased the frequency of heterologous clones in mixed clone infections. There was no evidence that any type of immunization regime favored virulence. A single AMA-1 variant is a component of candidate malaria vaccines current in human trials; our results suggest that adding extra AMA-1 alleles to these vaccines would not confer clinical benefits, but that that mono-allelic vaccines could alter AMA-1 allele frequencies in natural populations.

Fig. 1

IGg2b antibody levels from the serum of mice in the pilot experiment. Mice were either sham-immunized or immunised with one of the two atingens (DS AMA-1, DK AMA-1). Each of the treatments used to immunize mice and the AMA-1 test antigen used to coat ELISA plates are shown on the x-axis. Dots represent the antibody titre against a particular immunizing antigen for a single mouse. Horizontal lines indicate mean antibody levels. Antibody levels in antigen immunized groups of mice were higher than in sham-immunized controls (p < 0.001) and, among the immunized mice, the levels induced between the antigen immunized groups differed (immunizing treatment × ELISA antigen: p = 0.003) with higher titres against the homologous antigen. Neither of the immunising antigens induced higher titres (p > 0.05).

Fig. 2

IgG2b antibody levels from the serum of mice in the main experiment. Mice were either sham-immunized, or immunized with one of the antigen immunization treatments (DS AMA-1, DK AMA-1 or the bi-allelic formulation). Each of the treatments used to immunize mice and the AMA-1 test antigen used to coat ELISA plates are shown on the x-axis. Dots represent the antibody titre for individual mice against a particular antigen. Mice that were sham-immunized or immunized with the bi-allelic formulation were assayed for antibody responses against both DS and DK AMA-1 antigens. Horizontal lines indicate mean antibody levels. Antibody levels in antigen immunized groups of mice were higher than in sham-immunized controls (p < 0.001), and among the antigen immunized mice, antibody titres did not differ (p = 0.085). The antibody titres in animals immunized with both antigens were not dominated by responses to either one (p = 0.44).

Fig. 3

Effect of Plasmodium chabaudi infection (clone DK alone, DS alone or DS + DK) and immunization (sham-immunized control, DK AMA-1, DS AMA-1, or bi-allelic form) on the kinetics of minimum red blood cell density (left panels) and minimum weight (right panels). In A–F lines represent the change in RBC density (left panels) and weight (right panels) over time. Each line represents the mean of up to 6 mice (±1 S.E.M.) that were infected with DK alone (A and D), DS alone (B and E) or a mixed clone (C and F) infection during immunization with either a sham-inoculation control (solid thick black line), DK AMA-1 (open triangle), DS AMA-1 (open squares), or the bi-allelic mixture (dotted black line). In G–H bars represent the minimum red blood cell density (left panel) and minimum weight (right panel) reached during infection with clone DK alone (grey bars), DS alone (black bars) or a mixture of both clones (black and white bars) under each of the immunization treatments. Each bar represents the least squares mean of up to 6 mice (±1 S.E.M.).

Fig. 4

Kinetics of P. chabaudi infections (clones DK alone, DS alone or both together) following immunization (DK AMA-1, DS AMA-1, or bi-allelic formulation or sham-immunized control). In A–C, lines represent the change in parasite density over time. Each line represents the mean of up to 6 mice (±1 S.E.M.) that were infected with DK alone (A), DS alone (B) or a mixed clone (C) infection during immunization with either a sham-inoculation control (solid thick black line), DK AMA-1 (open triangle), DS AMA-1 (open squares), or the bi-allelic mixture (dotted black line). In (D), bars represent peak parasite densities reached during infection with clone DK alone (grey bars), DS alone (black bars) or a mixture of both clones (black and white diagonal) under each of the immunization treatments. Each bar represents the least squares mean of up to 6 mice (±1 S.E.M.).

Fig. 5

Proportion of clone DS in mixed DS and DK infections following immunization with DK AMA-1, DS AMA-1, the bi-allelic formulation, or in sham-immunized controls. (A) Lines represents the proportion of clone DS through time in control (solid black line), DK AMA-1 (open triangles), DS AMA-1 (open diamonds) or bi-allelic (dotted black line) immunized mice. Each line represents the mean of up to 6 mice (±1 S.E.M.). (B) Bar graphs represent the proportion of total parasites in a mixed infection that were DS under each of the immunization treatments. Each bar represents the least squares mean of up to 6 mice with 95% confidence intervals. The black horizontal dotted line represents the proportion DS present in the inoculum.