Malaria infection by sporozoite challenge induces high functional antibody titres against blood stage antigens after a DNA prime, poxvirus boost vaccination strategy in Rhesus macaques

Abstract

Background: A DNA prime, poxvirus (COPAK) boost vaccination regime with four antigens, i.e. a combination of two Plasmodium knowlesi sporozoite (csp/ssp2) and two blood stage (ama1/msp142) genes, leads to self-limited parasitaemia in 60% of rhesus monkeys and survival from an otherwise lethal infection with P. knowlesi. In the present study, the role of the blood stage antigens in protection was studied in depth, focusing on antibody formation against the blood stage antigens and the functionality thereof.

Methods: Rhesus macaques were immunized with the four-component vaccine and subsequently challenged i.v. with 100 P. knowlesi sporozoites. During immunization and challenge, antibody titres against the two blood stage antigens were determined, as well as the in vitro growth inhibition capacity of those antibodies. Antigen reversal experiments were performed to determine the relative contribution of antibodies against each of the two blood stage antigens to the inhibition.

Results: After vaccination, PkAMA1 and PkMSP1₁₉ antibody titres in vaccinated animals were low, which was reflected in low levels of inhibition by these antibodies as determined by in vitro inhibition assays. Interestingly, after sporozoite challenge antibody titres against blood stage antigens were boosted over 30-fold in both protected and not protected animals. The in vitro inhibition levels increased to high levels (median inhibitions of 59% and 56% at 6 mg/mL total IgG, respectively). As growth inhibition levels were not significantly different between protected and not protected animals, the ability to control infection appeared cannot be explained by GIA levels. Judged by in vitro antigen reversal growth inhibition assays, over 85% of the inhibitory activity of these antibodies was directed against PkAMA1.

Conclusions: This is the first report that demonstrates that a DNA prime/poxvirus boost vaccination regimen induces low levels of malaria parasite growth inhibitory antibodies, which are boosted to high levels upon challenge. No association could, however, be established between the levels of inhibitory capacity in vitro and protection, either after vaccination or after challenge.

Figure 1

ELISA titters against PkMSP1₁₉ and PkAMA1 before and after challenge. IgG antibody titres were measured by ELISA. The superimposed box around the data points indicate the upper and lower quartiles, the line in the middle indicates the median value. A) Antibody titres to PkMSP1₁₉ in the control (mock vaccine receiving) animals and Pk4-vaccinated animals (protected or not protected) before challenge (left panel) and after challenge (right panel). B) Antibody titres to PkAMA1 in the control (mock vaccine receiving) animals and vaccinated animals (protected or not protected) before challenge (left panel) and after challenge (right panel). Geometric shapes represent individual animals in each group, throughout all figures. For one animal in the control group no post challenge data are available, as it died for study-unrelated reasons.

Figure 2

Parasite growth inhibition activity of protected and non-protected monkeys. A) Post vaccination B) post challenge. Mean GIA inhibition levels of total IgG isolated from monkey serum from animals in control (mock) group, CSP/SSP2 group, and Pk4x3/COPAK vaccinated animals (protected or not protected animals are shown). Final IgG concentration added to P. knowlesi parasite culture was 6 mg/mL. Geometric shapes represent individual animals in each group, throughout all figures. For one animal in the control group no post challenge data are available, as it died for study-unrelated reasons.

Figure 3

Reversal of growth inhibitory activity by PkAMA1 and PkMSP1₁₉. Purified IgG from monkey 3086 (protected), post-challenge (black triangle). Mixture of pool purified IgG taken from Pk4-vaccinated monkeys (262, 299, 3086, 3098, AB34, Q120, T152, 228), post-challenge (black square). IgG was pre-incubated with either PkMSP1₁₉ (Panel A) or PkAMA1 (Panel B) in a five-fold serial dilution, prior to mixing with P. knowlesi parasites. Results shown are the mean of two independent assays.