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 ( Pf CSP)-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 CSP’s fragile immunogenicity using a parasite-derived chaperone. Unlike other orthologues of P. falciparum heat shock protein 70 ( Pf HSP70), substrate-binding domain (SBD) of Pf HSP70-3 is an evolutionarily conserved internal mitochondrial matrix chaperone. We demonstrate that Pf HSP70-3-SBD hydrophobic region complexes with Pf CSP and targets host CD91 on antigen-presenting cells to trigger cross-presentation, resulting in enhanced immune responses to Pf CSP.

Methods

In silico analyses predicted interactions among Pf HSP70-3-SBD, Pf CSP, and CD91 receptor. Recombinant Pf HSP70-3-SBD and Pf CSP 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 Pf CSP, with or without Pf HSP70-3-SBD. Pf CSP-specific IgG and IFN-γ expression were evaluated by ELISA and RT-qPCR, respectively. Functional activity was assessed using transgenic Plasmodium berghei expressing Pf CSP ( Pb/Pf CSP), by sporozoite neutralization assay.

Findings

Pf HSP70-3-SBD demonstrated a specific interaction with Pf CSP. The complex primarily engages CD91 receptor cluster IV, providing a potential mechanistic basis for antigen cross-presentation. Incorporation of Pf HSP70-3-SBD enhanced Pf CSP-specific total IgG responses and increased IFN-γ 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).

RESEARCH IN CONTEXT

Evidence before this study

Evidence from Pf CSP-based licensed vaccines, RTS,S/AS01 and R21/Matrix-M, underscores the need to improve the magnitude and durability of vaccine-induced protection. Although alum is an established adjuvant used in human vaccines, its limited capacity to elicit robust Th1-type cellular responses motivates the search for complementary strategies to fight against malaria. Findings on P. falciparum heat shock proteins (HSPs) remain mixed. Pf HSP70 has been reported to lack immunomodulatory activity, whereas an HSP70-derived C-terminal fragment enhanced DNA vaccine potency when linked to a malaria antigen. Whether mitochondrial Pf HSP70-3 substrate-binding domain can enhance Pf CSP immunogenicity and function as adjuvant remains insufficiently characterized. Although CD91-mediated uptake and cross-presentation of HSP-bound antigens have been previously reported, the potential involvement of this pathway in Pf CSP- Pf HSP70-3-SBD complex-mediated antigen presentation remains unexplored.

Added value of this study

This study explores Pf HSP70-3-SBD as a novel parasite-derived adjuvant, offering an alternative approach to fast-track the improvement of Pf CSP-based subunit vaccine immunogenicity. By linking antigen-chaperone interactions with the potential involvement of host CD91 receptor, it provides a plausible mechanistic framework for investigating enhancement in Pf CSP antigen cross-presentation. The integration of immunological evaluation with a functional neutralizing activity against transgenic P. berghei sporozoites expressing P. falciparum CSP, extends the findings beyond immunogenicity.

Implications of all the available evidence

Pf HSP70-3-SBD represents a promising candidate for further development as a parasite-derived chaperonic domain with modular adjuvant activity for next-generation malaria subunit vaccines. Incorporating Pf HSP70-3-SBD into Pf CSP-based formulations may offer a complementary approach to existing adjuvant strategies while providing a basis for investigating antigen-specific cross-presentation. This strategy may ultimately facilitate more streamlined vaccine development by integrating antigen delivery with immune potentiation. This approach further represents an initial step towards developing parasite-derived adjuvants, to reduce reliance on heterologous adjuvant sources.