Conserved multiepitopes in Plasmodium falciparum STEVORs enable rational design of a fusion antigen vaccine construct with broad immunogenicity

Abstract

There is no vaccine for severe malaria. STEVOR antigens on the surface of Plasmodium falciparum-infected red blood cells are implicated in severe malaria and are targeted by neutralizing antibodies, but their epitopes remain unknown. Using computational immunology, we identified highly immunogenic overlapping B- and T-cell epitopes (referred to as multiepitopes, 7-27 amino acids) in the semiconserved domain of four STEVORs linked with severe malaria and clinical immunity. Structural analyses confirmed the conservation in homologous sequences across 138 clinical isolates (Togo and Brazil) and 342 global strains. Designed fused multiepitopes showed high IgG antibody reactivity in the sera of P. falciparum-infected individuals. The fused multiepitopes had no allergenicity/toxicity, and phenotyping via flow cytometry and immunological assays revealed the induction of CD4+ and CD8+ T-cell proliferation and IgG antibodies in BALB/c mice, respectively. On this basis, structure-guided design of a multiepitope fusion antigen (MEFA) vaccine construct achieved 97.15% global combined HLA coverage and elicited both cellular and humoral immunity in silico. Recombinant MEFA was stably expressed in Escherichia coli and recognized significantly more anti-STEVOR IgG antibodies in the sera of nonsevere malaria cases than in those of severe cases, underscoring its potential immunogenicity and association with milder disease. The STEVOR MEFA construct emerges as a promising severe malaria vaccine candidate, combining global HLA coverage, safety, and broad immunogenicity linked to milder clinical outcomes.

Keywords: Plasmodium falciparum; STEVOR; immunogenicity; multiepitope; severe malaria; vaccine construct.

Figure 1.

Mapping of domains, physicochemical characteristics, and conservation analyses of epitopes within the SC domains of STEVOR antigens. (A) Selected multiepitopes in PF3D7_1040200 (ST1). (B) Selected multiepitopes in PF3D7_0617600 (ST2). (C) Selected multiepitopes in PF3D7_0115400 (ST3). (D) Selected multiepitopes in PF3D7_0300400 (ST3). The horizontal axis of the bar graph represents the position number of the amino acid. (E) Weblogo and entropy sequences showing the amino acid sequences of the selected multiepitopes.

Figure 2.

Differential recognition of multiepitope-specific anti-STEVOR IgG antibodies in malaria-exposed individuals. (A) Total IgG antibody recognition levels in the SMG versus the NMG to the ST1-Grp. (B) Total IgG antibody recognition levels in the SMG versus the NMG to the ST2-Grp. (C) Total IgG antibody recognition levels in the SMG versus the NMG to the ST3-Grp. (D) Total IgG antibody recognition levels in the SMG versus the NMG to the ST4-Grp. (E) Differential total IgG antibody recognition levels between ST1-Grp, ST2-Grp, ST3-Grp, ST4-Grp, and MSP1_{_p83} groups. (F) Differential total IgG antibody recognition levels in the SMG versus the NMG between the ST1-Grp, ST2-Grp, ST3-Grp, and ST4-Grp groups. Seroreactivity intensity indicates the antibody levels. SMG: severe malaria group; NMG: nonsevere malaria group. NMG: Nonsevere malaria group. NCG: (uninfected) normal control group. (G) Seroprevalence of total IgG antibody recognition to STVEVOR fused multiepitopes in SMG vs NMG. The numbers on the blocks represent the number of serum samples that were seroreactive to a particular fused multiepitope. SMG: severe malaria group; NMG: nonsevere malaria group. ***: P < 0.001.

Figure 3.

Levels of total IgG antibodies, IFN-γ, and IL-2 in mice immunized with the fused epitopes. (A) Schedule of mouse immunization and blood collection. (B) Changes in mouse body weight. Each point represents the mean weight of a mouse before and after blood collection. (C) Trends in the levels of IgG antibodies in the immunized following boost injections. (D) Levels of total IgG antibody in the immunized mice on day 28. (E) Levels of total IgG antibody in the immunized mice on day 42. (F) Levels of total IgG antibody in the immunized mice on day 56. (G) Trends in the levels of circulating IFN-γ following boost injections. (H) Trends in the levels of circulating IL-2 following boost injections.

Figure 4.

Levels of CD8+ T- and CD4+ T-cell proliferation in the spleens of immunized mice. (A-G) Proportion of both CD4+ T and CD8+ T cells in mice immunized with ST1-Grp, ST2-Grp, ST3-Grp, ST4-Grp, PBS control, adjuvant control, MSP1_{_p83} control groups. P3 represents the proportion of CD8+ T cells. P4 represents the proportion of CD4+ T cells. (H) Proportion of CD8+ T cells in mice immunized with ST1-Grp, ST2-Grp, ST3-Grp, ST4-Grp, PBS/adjuvant control, and MSP1_{_p83} positive control groups. (I) Proportion of CD4+ T cells in immunized mice with ST1-Grp, ST2-Grp, ST3-Grp, ST4-Grp, PBS/adjuvant control, and MSP1_{_p83} positive control groups.

Figure 5.

Multiepitope fusion antigen (MEFA) vaccine construct and population coverage of T-cell epitopes. (A) MEFA construct consisting of adjuvant, CD8 + and CD4+ T-cell epitopes, B-cell epitopes and linkers. (B) Two-dimensional structural model of the MEFA construct. (C) Three-dimensional structural model of the MEFA construct. (D) Ramachandran plot of the MEFA construct. Residues in most favoured regions [A, B, L]. Residues in additional allowed regions [a, b, l, p]. Residues in generously allowed regions [∼a, ∼b, ∼l, ∼p]. (E) Number of HLA combinations recognized by different percentages of individuals globally. (F) Population coverage of the individual classes and combined classes worldwide.

Figure 6.

Differential recognition of MEFA-specific anti-STEVOR IgG antibodies in malaria-exposed individuals. Recombinant MEFA (A) and MSP1_-42 (B) proteins expressed in E. coli and purified via Ni²⁺ affinity chromatography. SDS-PAGE analysis was performed to assess protein purity (left). Lane M1: molecular weight marker (kDa); Lane BSA: bovine serum albumin control. Western blotting with anti-His antibody was used to confirm protein identity (right). Lane M2: Western blot marker. Arrows indicate the expected positions of MEFA and MSP1_-42 proteins. (C) Differential total IgG antibody recognition levels in the SMG versus the NMG between the MEFA, and MSP1_-42. (D) Differential total IgG antibody recognition levels in the SMG versus the NMG to the MEFA. (E) Total IgG antibody recognition levels in the SMG versus the NMG to the MSP1_-42. (F) Seroprevalence of IgG antibody recognition in SMG vs NMG to MEFA and MSP1_-42.