Conserved genetic and antigenic profiles of Pf12 and Pf41 in asymptomatic Plasmodium falciparum carriers in Tanzania

Plasmodium falciparum remains a major cause of mortality in malaria-endemic regions, and strain-specific immunity limits vaccine efficacy. The 6-cysteine proteins Pf12 and Pf41 are proposed blood-stage vaccine candidates, yet their diversity and antigenicity in asymptomatic carriers are poorly defined. Genetic variation and natural selection pressure were analyzed in 172 P. falciparum isolates from asymptomatic individuals in Tanzania. Genomic DNA extracted from dried blood spots was amplified by PCR and subsequently sequenced to evaluate genetic diversity. IgG responses to recombinant Pf12 and Pf41 were quantified using a protein microarray. Statistical analyses included Spearman's correlation to assess antibody associations and unpaired t-tests to compare IgG levels between parasite-positive and parasite-negative individuals. Both genes exhibited low nucleotide diversity (pf12 π = 0.00042 ± 0.00007; pf41 π = 0.00061 ± 0.00004) and negative Tajima's D values suggest that both genes are under selective constraints, consistent with purifying or directional selection. Neutrality test indicated an excess of rare variants consistent with recent population expansion. Haplotype network showed limited regional differentiation, supporting strong conservation. Pf12 and Pf41 elicited high IgG seropositivity (97.7% and 98.3%), with strongly correlated antibody levels (ρ = 0.721, p < 0.001). A predominant Pf41 substitution, S232R (47.8%), and rare Asn/His variants (0.6%) were detected. Overall, both proteins display limited polymorphism and robust IgG recognition, features consistent with exposure-driven immunity. These findings highlight conserved merozoite antigens that warrant further investigations for their biological relevance and vaccine potential, particularly to determine their functional role in protective immunity.