Computational design, expression, and characterization of a Plasmodium falciparum multi-epitope, multi-stage vaccine candidate (PfCTMAG)

Abstract

Malaria, a tropical disease, claims the lives of thousands of people annually and the development of resistance to insecticides and antimalarial drugs poses a great challenge to current prevention and control strategies. Current malaria vaccines are limited in efficacy, duration of protection, and safety, due to the high antigenic diversity and complex life cycle of the Plasmodium parasite. This study sought to design and assess a more effective multi-stage, multi-epitope vaccine candidate for the control of malaria. A multi-epitope malaria vaccine candidate was designed in silico using multiple antigens from both the pre-erythrocytic and erythrocytic stages, expressed in bacteria, and its sero-reactivity to antibodies in plasma from malaria-positive (cases) and negative individuals (controls) was assessed using enzyme-linked immunosorbent assay (ELISA). Immunization experiments were equally conducted with BALB/c mice. In-silico analysis revealed that the designed antigen, PfCTMAG (Plasmodium falciparum Circumsporozoite, Thrombospondin-related adhesion protein, Merozoite surface protein 2, Apical asparagine (Asn)-rich protein and Glutamate-Rich Protein), effectively bound to Toll-like receptor 4 (TLR-4) and triggered a strong immune response. In sero-reactivity studies, malaria-positives (cases) had higher anti-PfCTMAG IgG ( p = 0.024) and IgM ( p < 0.001) levels compared to malaria negatives (controls). The mice immunized with PfCTMAG did not show adverse reactions and had higher levels of IgG antibodies (p = 0.002) compared to controls, thereby validating the safety and immunogenicity of PfCTMAG as a promising vaccine candidate.

Keywords: Immunogenicity; In silico; Multi-epitope; Sero-reactivity; Vaccine candidate.

Fig. 1

Primary structure of PfCTMAG (A) Schematic view (B) Linear sequence.

Fig. 2

Disordered regions predicted by IUpred3 showing a majority of residues located above the threshold score.

Fig. 3

Secondary structure predicted by PSIPRED mostly composed of grey residues which indicate coils.

Fig. 4

Immune profile of PfCTMAG (A)Antigens and antibodies (B) Cytokines and Interleukins.

Fig. 5

SDS-PAGE Gels: Gel obtained by GenScript after expression and purification (A). Gel obtained from quality check after receiving the candidate from GenScript (B). The full non-adjusted gel images are available as supplementary material ‘Gel Fig. 5A’ and ‘Gel Fig. 5B’ respectively.

Fig. 6

Mean Anti-PfCTMAG IgG and IgM (A) Anti-PfCTMAG IgG and IgM seropositivity of cases and controls (B).

Fig. 7

Mean IgG levels of Prime vs Control (A), Booster vs Control (B), and Mean IgM of Prime vs Control (C), Booster vs Control (D) Groups Across Days.

Fig. 8

Seropositivity of PfCTMAG IgG and IgM across mice treatment groups on days 0, 14, and 28.

figs1

Original SDS-PAGE gel obtained by GenScript after expression and purification.

figs2

Original SDS-PAGE gel obtained from quality check after receiving the candidate from GenScript.