Protective efficacy of a Plasmodium vivax circumsporozoite protein-based vaccine in Aotus nancymaae is associated with antibodies to the repeat region

Abstract

We have previously reported that Vivax Malaria Protein 001 (VMP001), a vaccine candidate based on the circumsporozoite protein of Plasmodium vivax, is immunogenic in mice and rhesus monkeys in the presence of various adjuvants. In the present study, we evaluated the immunogenicity and efficacy of VMP001 formulated with a TLR9 agonist in a water-in-oil emulsion. Following immunization, the vaccine efficacy was assessed by challenging Aotus nancymaae monkeys with P. vivax sporozoites. Monkeys from both the low- and high-dose vaccine groups generated strong humoral immune responses to the vaccine (peak median titers of 291,622), and its subunits (peak median titers to the N-term, central repeat and C-term regions of 22,188; 66,120 and 179,947, respectively). 66.7% of vaccinated monkeys demonstrated sterile protection following challenge. Protection was associated with antibodies directed against the central repeat region. The protected monkeys had a median anti-repeat titer of 97,841 compared to 14,822 in the non-protected monkeys. This is the first report demonstrating P. vivax CSP vaccine-induced protection of Aotus monkeys challenged with P. vivax sporozoites.

Figure 1. Aotus nancymaae immunized with VMP001 generated antigen-specific antibodies that were boosted following each immunization.

Groups of 8 monkeys were immunized three times with either 15 µg (panel A) or 50 µg (panel B) of VMP001 formulated with 200 µg CpG 10104 and Montanide ISA 720. Sera, drawn at two week intervals, were tested for presence of antibodies to VMP001. Antibody titers waned over time, but were boosted with each immunization. Titers are defined as serum dilution that gives an OD₄₁₄ of 1.0. Each symbol represents an individual monkey. Some time points have less than eight data points due to the unavailibility of serum sample at that time point. Horizontal bars represent the median titer for each group at the given time point.

Figure 2. Onset and magnitude of antibody response to VMP001 is similar in both the low (15 µg) and high (50 µg) dose groups.

In the presence of a strong adjuvant formulation the differences in anti-VMP001 titers between the two immunized groups were not stastically significant (ns = p>0.05; 2-tailed Mann Whitney U test) at any timepoint. Data is represented as Box (25–75 percentile) and Whisker (minimum and maximum) plot with horizontal line at the median value.

Figure 3. Protective efficacy of VMP001 in Aotus monkeys challenged intravenously with P. vivax Brazil VII strain post primary and rechallenge.

Monkeys were immunized with either 15 µg or 50 µg of VMP001. Control monkeys were immunized with 50 µg of P. falciparum LSA-1 (PfLSA-1). All antigens were formulated with Montanide ISA 720 and CpG 10104. Kaplan-Meier survival curves represent percentage of uninfected monkeys over time. Vaccine Efficacy was calculated as a ratio between the vaccinated and control groups (see results). 3A. Six weeks following the third immunization, all monkeys were challenged intravenously with 10,000 P. vivax Brazil VII parasites. 3B. Eight weeks after the primary challenge, monkeys were rechallenged with 15,000 P. vivax Brazil VII parasites.

Figure 4. Infection with P. vivax Brazil VII sporozoites induces antibodies to VMP001.

Control monkeys immunized with PfLSA-1 did not generate antibodies against P. vivax (denoted as “pre challenge” in the figure). Following challenge with P. vivax sporozoites, the control monkeys developed anti-P. vivax CSP antibodies, as assessed by ELISA reactivity to VMP001, that were significantly boosted following rechallenge (2-tailed Mann-Whitney U test). Data is represented as Box (25–75 percentile) and Whisker (minimum and maximum) plot with horizontal line at the median value.

Figure 5. Protected monkeys from both the low- and high dose groups had significantly higher anti-repeat antibodies compared to the non-protected monkeys.

Pre-primary challenge sera from immunized monkeys from the low (15 µg) and high (50 µg) dose groups were pooled from the protected (n = 12) and non-protected (n = 4) groups. Antibody reactivity between the protected and non-protected groups were similar for VMP001, N- and C-terminal regions. However, anti-repeat antibodies were significantly different between the protected and non-protected groups (p = 0.034, 2-tailed Mann Whitney U test). Data is represented as Box (25–75 percentile) and Whisker (minimum and maximum) plot with horizontal line at the median value. Data also represented in a tabular form to demonstrate correlation bewteen protected and non-protected groups.

Figure 6. Loss of protective efficacy following rechallenge could be attributed to a drop in anti-repeat antibody titers.

Kaplan-Meier survival curves were generated by pooling data from the low (15 µg) and high (50 µg) dose groups (from Figure 3), and the combined VMP001 immunized group was compared to the control group. 6A. Following primary challenge, 12 of 16 immunized monkeys and 2 of 8 control monkeys remained uninfected, resulting in a calculated vaccine efficay of 66.7% (p = 0.004). 6B. Following rechallenge, one immunized monkey remained uninfected and several others had a delay compared to the controls (p = .04). 6C. Sera drawn two weeks prior to the first challenge from protected monkeys (n = 12) had significantly higher anti-repeat antibody titers compared to sera drawn pre-rechallenge from the non-protected monkeys (n = 13); p = 0.02 (2-taled Mann Whitney U-test). Titers indicate serum dilution that gives an OD₄₁₄ of 1.0. Each dot represents an individual monkey. Horizontal bar represents the median titer for each group.