Micro-geographic variation in antigenic diversity of PfEBA-175 region II in asymptomatic Plasmodium falciparum infections in Tanzania

Jadidan Hada Syahada¹Wang-Jong Lee¹Hojong Jun²JOHNSY MARY SAWARIMUTHU LOUIS¹Fadhila Fitriana¹Fauzi Muh²Feng Lu³Md Atique Ahmed⁴Sunghun Na⁵Wanjoo Chun⁶Won Sun Park⁷BO YOUNG JEON⁸Eun-Teak Han¹Jim Todd^10,9Alphaxard Manjurano¹¹Winifrida Kidima¹²Ernest Mazigo^1,11Sin Jin Lee^5*Jin-Hee Han^1,13,2*

• ¹Department of Medical Environmental Biology and Tropical Medicine, School of Medicine, Kangwon National University, Chuncheon, Republic of Korea, Kangwon National University School of Medicine, Chuncheon-si, Republic of Korea
• ²Department of Epidemiology and Tropical Diseases, Faculty of Public Health, Universitas Diponegoro, Semarang, Indonesia, Semarang, Indonesia
• ³Department of Pathogen Biology and Immunology, School of Medicine, Yangzhou University, Yangzhou, China, Yangzhou, China
• ⁴ICMR - Regional Medical Research Centre Dibrugarh, Dibrugarh, India
• ⁵Department of Obstetrics and Gynecology, Kangwon National University Hospital, Chuncheon, Republic of Korea, Chuncheon, Republic of Korea
• ⁶Department of Pharmacology, School of Medicine, Kangwon National University, Chuncheon, Republic of Korea, Kangwon National University School of Medicine, Chuncheon-si, Republic of Korea
• ⁷Department of Physiology, School of Medicine, Kangwon National University, Chuncheon, Republic of Korea, Kangwon National University School of Medicine, Chuncheon-si, Republic of Korea
• ⁸Department of Biomedical Laboratory Science, College of Software and Digital Healthcare Convergence, Yonsei University, Wonju, Republic of Korea, Wonju, Republic of Korea
• ⁹Department of Population Health, London School of Hygiene and Tropical Medicine, London, UK, London School of Hygiene & Tropical Medicine, London, United Kingdom
• ¹⁰Department of Biostatistics, Catholic University of Health and Allied Sciences (CUHAS), Mwanza, Tanzania, Catholic University of Health and Allied Sciences, Mwanza, Tanzania
• ¹¹Department of Parasitic Diseases, National Institute for Medical Research, Dar es Salaam, Tanzania, Dar es Salaam, Tanzania
• ¹²Department of Zoology, College of Natural and Applied Sciences, University of Dar es Salaam, Dar es Salaam, Tanzania, Dar es Salaam, Tanzania
• ¹³Institute of Medical Sciences, Kangwon National University, Chuncheon, Republic of Korea, Chuncheon, Republic of Korea

Background: Malaria remains a major public health burden, and the development of effective blood-stage vaccines is complicated by extensive antigenic variation in Plasmodium falciparum antigens. PfEBA-175, a leading vaccine candidate, mediates erythrocyte invasion by binding to glycophorin A via its region II (RII), which is known to be highly polymorphic. Methods: We examined the genetic diversity and antigenicity of PfEBA-175 region II (RII) in 172 asymptomatic P. falciparum isolates collected from Geita and Kigoma, Tanzania. Sequence diversity was assessed through nucleotide diversity (π) and analysis of selection pressure using dN-dS and Fu's Fs tests. A recombinant PfEBA-175 RII protein was expressed to evaluate antibody response in plasma samples. Results: Sequence analysis revealed high nucleotide diversity (π = 0.00359 ± 0.00012), with evidence of adaptive evolution driven by immune pressure (dN-dS = 3.15, p < 0.001), and sign of recent population expansion based on Fu's Fs value (-30.614). A recombinant RII protein exhibited high antigenicity, with an average seropositivity of 84.8%, although rates varied across villages, ranging from 95.4% in Rwantaba to 50.0% in Bunyambo. The antibody response was positively correlated with age (ρ = 0.333, p < 0.001), but not with parasitemia or gender. Several important amino acids substitutions, including K478N, K481I, and L482V, were located within B-cell epitopes targeted by the invasion-inhibitory monoclonal antibody R217, and N577K, found at the dimer interface, had little effect on naturally acquired immune responses. However, several charged amino acid substitutions including D168H, T198K, K275I, K448N, and D619H influenced natural acquired antibody recognition. Conclusion: Despite substantial polymorphism, the glycan-binding residues of PfEBA-175 RII remain conserved, and its high antigenicity across diverse geographic and demographic contexts supports its continued evaluation as a blood-stage vaccine candidate. These findings highlight the importance of accounting for naturally occurring antigenic variation in malaria vaccine development.