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

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.目前尚无针对重症疟疾的有效疫苗。恶性疟原虫（Plasmodium falciparum）感染红细胞表面表达的STEVOR抗原与重症疟疾发病密切相关，且可被中和抗体靶向识别，但其具体表位仍未被阐明。本研究通过计算免疫学手段，在与重症疟疾及临床免疫相关的4种STEVOR蛋白的半保守结构域中，筛选得到了高免疫原性的重叠B细胞与T细胞表位，将其命名为多表位（长度为7~27个氨基酸）。结构分析证实，该类表位在138株临床分离株（采自多哥与巴西）及342株全球毒株的同源序列中均保持高度保守。经设计的融合多表位重组蛋白在恶性疟原虫感染者的血清中表现出较高的IgG抗体结合活性。该融合多表位无致敏性与毒性；通过流式细胞术与免疫学实验进行表型分析后发现，其可分别诱导BALB/c小鼠产生CD4+和CD8+ T细胞增殖以及IgG抗体。基于结构引导设计的多表位融合抗原（multiepitope fusion antigen, MEFA）疫苗构建体实现了97.15%的全球人群HLA联合覆盖率，并在计算机模拟中成功诱导了细胞免疫与体液免疫。重组MEFA可在大肠杆菌（Escherichia coli）中稳定表达，且在非重症疟疾患者血清中识别的抗STEVOR IgG抗体水平显著高于重症疟疾患者，这一结果凸显了其潜在免疫原性及与轻症疾病的关联。综上，STEVOR来源的MEFA构建体有望成为一款极具前景的重症疟疾疫苗候选株，其兼具全球HLA覆盖范围广、安全性高及广谱免疫原性等优势，并与轻症临床结局相关。