Immunogenicity of a Plasmodium vivax vaccine based on the duffy binding protein formulated using adjuvants compatible for use in humans

Abstract

The invasion of reticulocytes by Plasmodium vivax merozoites is dependent on the interaction of the Plasmodium vivax Duffy Binding Protein (PvDBP) with the Duffy antigen receptor for chemokines (DARC). The N-terminal cysteine-rich region II of PvDBP (PvDBPII), which binds DARC, is a leading P. vivax malaria vaccine candidate. Here, we have evaluated the immunogenicity of recombinant PvDBPII formulated with the adjuvants Matrix-M and GLA-SE in mice. Analysis of the antibody responses revealed comparable ELISA recognition titres as well as similar recognition of native PvDBP in P. vivax schizonts by immunofluorescence assay. Moreover, antibodies elicited by the two adjuvant formulations had similar functional properties such as avidity, isotype profile and inhibition of PvDBPII-DARC binding. Furthermore, the anti-PvDBPII antibodies were able to block the interaction of DARC with the homologous PvDBPII SalI allele as well as the heterologous PvDBPII PvW1 allele from a Thai clinical isolate that is used for controlled human malaria infections (CHMI). The cross-reactivity of these antibodies with PvW1 suggest that immunization with the PvDBPII SalI strain should neutralize reticulocyte invasion by the challenge P. vivax strain PvW1.

Figure 1

Polymorphisms and binding residues in the PvDBPII SalI and PvW1. (A) Binding residues that remain conserved (blue) and polymorphisms (red) between PvDBPII SalI and PvW1 are shown. (B) PvDBPII SalI structure (green) including the binding residues (blue) and the PvW1 polymorphisms (red) are shown. The leucine insertion between positions 429 and 430 in the SalI sequence is indicated with a dashed arrow. The amino and carboxyl termini of PvDBPII are indicated with N and C, respectively. Structures were obtained from the Protein Data Bank (PDB) structure 4NUV and modified using PyMOL software version 1.2.

Figure 2

Binding of the recombinant PvDBPII variants SalI and PvW1 to DARC. (A) Binding to Duffy positive (Fy+) or Duffy negative (Fy−) erythrocytes of the recombinant PvDBPII variants SalI and PvW1 were detected by Western blotting. Erythrocyte Binding assay with no recombinant protein was used as a negative control (N). (B,C) Binding to nDARC(Y)-Fc and nDARC(F)-Fc of PvDBPII SalI and PvW1 at different concentrations (B) and at 25 ng/ml (C). Means and SD are shown. p values **p < 0.01, ***p < 0.001, pairwise comparisons with Bonferroni’s multiple comparison tests. (D) Binding kinetics of the PvDBPII SalI and PvW1 to nDARC(Y)-Fc by BLI. Different concentrations of PvDBPII domains ranging from 1 to 120 nM were tested to bind nDARC(Y)-Fc. The affinity constant, K_D, was calculated by fitting the association/dissociation profiles with a 1:2 binding model and performing a Steady-state analysis. Two independent experiments were performed, and the K_D was averaged and reported for each PvDBPII protein ± SD.

Figure 3

Anti-PvDBPII antibodies elicited in mice can recognize both SalI and PvW1. (A) Scheme of mice immunizations with PvDBPII. Five mice received PvDBPII SalI formulated with Matrix-M or GLA-SE at days 0, 28 and 56. Mice sera was collected at days − 1, 27, 55 and 70. Images were generated in online portal Biorender (B) Antibody kinetics measured by ELISA over the course of the immunizations. Median OD₄₅₀ at dilution 1:50,000 of each mice group including range is shown. (C) Antibody titres reported as effective concentrations (EC₅₀) that can recognize PvDBPII SalI and PvW1 in both mice groups. Median and range are shown.

Figure 4

Reactivity of mouse sera with P. vivax schizonts from infected P. vivax malaria patients. Representative images of P. vivax schizonts incubated with sera from mice immunized with PvDBPII formulated with GLA-SE and mice immunized with PvDBPII formulated with Matrix-M showing apical staining of merozoites (green) in mature P. vivax schizonts compared to a pool of mice sera at day − 1 (Naive). This clinical isolate contains 3 mutations in the PvDBP RII sequence compared to Sal1 reference sequence: R263S, D339G and R345H.

Figure 5

Functional characterization of anti-PvDBPII antibodies. (A) Avidity of PvDBPII-specific antibodies at day 70 elicited in GLA-SE and Matrix-M adjuvant groups to PvDBPII SalI and PvW1. (B) Binding inhibitory titres at day 70 that block interaction between DARC and PvDBPII SalI and PvW1. (C) Isotypes of antibodies raised against PvDBPII for each adjuvant group. (A–C) Median and range are shown.

Figure 6

Correlations of functional properties between PvDBPII SalI and PvW1. The PvW1-specific antibody titres, binding inhibitory titres and avidity are associated with the PvDBPII SalI responses. Spearman’s rank correlation coefficient and p values are shown.