Evolutionarily Conserved Plasmodium falciparum HSP70-3 Enhances the Immunogenicity of the Established Malaria Vaccine Antigen Circumsporozoite Protein through Cross-Presentation

Background: Clinically approved P. falciparum circumsporozoite protein (PfCSP)-based pre-erythrocytic stage malaria vaccines suffer from sub-optimal efficacy and rapidly waning memory. By decoding these limitations, we fast-tracked vaccine optimization by correcting CSPs fragile immunogenicity using a parasite-derived chaperone. Unlike other orthologues of P. falciparum heat shock protein 70 (PfHSP70), substrate-binding domain (SBD) of PfHSP70-3 is an evolutionarily conserved internal mitochondrial matrix chaperone. We demonstrate that PfHSP70-3-SBD hydrophobic region complexes with PfCSP and targets host CD91 on antigen-presenting cells to trigger cross-presentation, resulting in enhanced immune responses to PfCSP. Methods: In silico analyses predicted interactions among PfHSP70-3-SBD, PfCSP, and CD91 receptor. Recombinant PfHSP70-3-SBD and PfCSP proteins were purified and assessed for in vitro interaction using ELISA and far-western dot blot assays. BALB/c mice were immunized with alum-formulated PfCSP, with or without PfHSP70-3-SBD. PfCSP-specific IgG and IFN-{gamma} expression were evaluated by ELISA and RT-qPCR, respectively. Functional activity was assessed using transgenic Plasmodium berghei expressing PfCSP (Pb/Pf CSP), by sporozoite neutralization assay. Findings: PfHSP70-3-SBD demonstrated a specific interaction with PfCSP. The complex primarily engages CD91 receptor cluster IV, providing a potential mechanistic basis for antigen cross-presentation. Incorporation of PfHSP70-3-SBD enhanced PfCSP-specific total IgG responses and increased IFN-gamma expression in BALB/c mice, indicating improved immunogenicity upon co-formulation. Notably, immune sera neutralized Pb/Pf CSP sporozoites, delaying blood-stage patency until day 12 in 40% of C57BL/6 mice, with no detectable parasitemia in surviving mice for up to 2 months, indicating sustained efficacy against the sporozoite stage. Interpretation: Molecular chaperones, including HSPs, are currently under intense investigation for their immune-potentiating activity, leading to next-generation vaccine development. By utilizing chaperonic domains derived directly from the pathogenic agent itself to serve an essential adjuvant function, this approach reduces reliance on heterologous adjuvants while enhancing antigen-specific immunity. Funding: This work was supported by funding from Indian Council of Medical Research (IIRPSG-2024-01-03940). Keywords: Substrate-binding domain, PfHSP70-3, Adjuvant, Cross-presentation, Pre-erythrocytic stage, Plasmodium falciparum