A multivalent Plasmodium falciparum circumsporozoite protein-based nanoparticle malaria vaccine elicits a robust and durable antibody response against the junctional epitope and the major repeats

Abstract

Plasmodium falciparum (Pf) malaria continues to cause considerable morbidity and mortality worldwide. The circumsporozoite protein (CSP) is a particularly attractive candidate for designing vaccines that target sporozoites-the first vertebrate stage in a malaria infection. Current PfCSP-based vaccines, however, do not include epitopes that have recently been shown to be the target of potent neutralizing antibodies. We report the design of a SpyCatcher-mi3-nanoparticle-based vaccine presenting multiple copies of a chimeric PfCSP (cPfCSP) antigen that incorporates these important "T1/junctional" epitopes as well as a reduced number of (NANP)_n repeats. cPfCSP-SpyCatcher-mi3 was immunogenic in mice eliciting high and durable IgG antibody levels as well as a balanced antibody response against the T1/junctional region and the (NANP)_n repeats. Notably, the antibody concentration elicited by immunization was significantly greater than the reported protective threshold defined in a murine challenge model. Refocusing the immune response toward functionally relevant subdominant epitopes to induce a more balanced and durable immune response may enable the design of a more effective second generation PfCSP-based vaccine.

Keywords: Plasmodium falciparum; circumsporozoite; junctional epitope; malaria; vaccine.

FIGURE 1

Schematic representation of cPfCSP variant and assembly of cPfCSP variant on SpyCatcher‐mi3. (a) cPfCSP. (b) Schematic illustrating the generation of chimeric Plasmodium falciparum circumsporozoite (cPfCSP)‐SpyCatcher‐mi3 conjugates by the reaction of cPfCSP‐SpyTag variants with SpyCatcher‐mi3 nanoparticles. (cPfCSP: green; SpyTag: red; SpyCatcher: yellow; mi3: cyan).

FIGURE 2

Characterization of chimeric PfCSP and cPfCSP‐SpyCatcher‐mi3. (a) Characterization of SpyCatcher‐mi3, cPfCSP, and cPfCSP‐SpyCatcher‐mi3 by SDS‐PAGE. (b) Characterization of the cPfCSP‐SpyCatcher‐mi3 (solid line) and SpyCatcher‐mi3 (dashed line) by dynamic light scattering (DLS). (c) Characterization of the binding of (i) CIS43 antibody (PDB:6B5M) and (ii) 2A10 antibody (PDB: 5T0Y) to cPfCSP (○), cPfCSP‐SpyCatcher‐mi3 (■), and BSA (control) (▲) by enzyme‐linked immunosorbent assay (ELISA) (mean ± SD, n = 2: one assay with two technical replicates).

FIGURE 3

Characterization of immunogenicity elicited by cPfCSP‐SpyCatcher‐mi3. (a) Schedule for mice vaccination and serum collection. (b) Antibody endpoint titers of sera from mice immunized with cPfCSP‐SpyCatcher‐mi3 (gray), or SpyCatcher‐mi3 (white) (geometric mean with geometric SD, n = 5) (i) after the first immunization (day 20) and (ii) after the second immunization (day 36) against peptides T1, B3 ((NANP)₃), and PfCSP: one assay with sera from 5 mice; n = 5. ****P < 0.0001, determined by unpaired t test for (i) and, unpaired t test (against T1 and B3), and Welch's t test (against PfCSP) for (ii). (c) Anti‐T1(NANP)₃ IgG antibody concentration in mice immunized with cPfCSP‐SpyCatcher‐mi3 was determined by comparison to a standard curve generated with the mAb 2A10; sera from mice immunized with SpyCatcher‐mi3 alone were used as control. The broken line indicates the protective anti‐repeat antibody level reported to confer protection in murine models: one assay with sera from 5 mice; n = 5; **P < 0.01, determined by Mann–Whitney test. (d) Characterization of antibody specificity. 5 ng/mL mAb 2A10 (▲) or a pool of sera collected from mice immunized with SpyCatcher‐mi3‐cPfCSP diluted at 1:60,000 (●) were pre‐incubated with three different concentrations (0.5, 5, and 50 μg/mL) of (i) (NANP)₃ (B3) and (ii) T1 competitor peptides and then allowed to bind to PfCSP. The intensity was normalized relative to that in the absence of competing peptides. (e) Anti‐T1(NANP)₃ IgG antibody concentration in mice 118, 188 and 300 days after first immunization with cPfCSP‐SpyCatcher‐mi3 or SpyCatcher‐mi3 (control) was determined by comparison to a standard curve generated with the mAb 2A10. The broken line indicates the protective anti‐repeat antibody level reported to confer protection in murine models; one assay with sera from 5 mice; n = 5; *P < 0.05, determined by Welch's t test; **P < 0.01, determined by Mann–Whitney test at 118 days, and Welch's t test for 188 and 300 days.