P120 Vaccine induced protection by secreted aspartyl proteinase 2 from Candida parapsilosis in Candida tropicalis mediated murine systemic candidiasis: a role of B-cells and antibodies
Authors: Shukla Manisha , Chandley Pankaj , Rohatgi Soma 
Published: September 2022

Abstract

Poster session 1, September 21, 2022, 12:30 PM - 1:30 PM

Objectives
Non-albicans Candida species are increasingly becoming more prevalent globally and emergence of drug resistance is another cause of concern. These challenges merit the need for developing novel immune therapies against Candida infections. We investigated the protective potential of secreted aspartyl proteinase (Sap2), a leading Candidate vaccine antigen identified from research based on C. albicans, in murine systemic candidiasis caused by non-albicans Candida species. Moreover, while the role of cellular immune responses in anti-Candida immunity is well established, less is known about the role of humoral immunity against systemic candidiasis. As such, we also investigated the role of humoral immunity in vaccine-mediated protection during murine systemic candidiasis.

Methods
The Sap2 genes were successfully cloned and expressed as recombinant proteins from C. albicans, C. tropicalis and C. parapsilosis strains prevalent in India. Groups of wild-type BALb/c mice were vaccinated with individual rSap2 protein along with alum as adjuvant, followed by systemic infection with a lethal dose of C. tropicalis. The protective potential of each rSap2 protein was evaluated using survival analysis and estimation of organ fungal burden. Histopathological assessment was performed using H&E and PAS staining. Serum cytokine levels and antigen-specific antibody titers were measured by ELISA. Cellular responses were analyzed in detail using flow cytometry. Functional evaluation of antibody role was performed using in vitro (biofilm inhibition and neutrophil-mediated killing) and in vivo (passive transfer) studies. B-cell epitope analysis was carried out using immunoinformatics approaches.

Results
Mice vaccinated with rSap2 cloned from C. parapsilosis (Sap2-parapsilosis) showed highest increase in survival time (P = .02) and maximum reduction in organ fungal burden (spleen, kidney, lungs, brain) (P &lt;.05); compared with sham immunized controls. Vaccination with rSap2 cloned from C. albicans did not improve survival in non-albicans C. tropicalis infection, despite the protein having ∼60% homology across species. Mice vaccinated with rSap2-parapsilosis also exhibited significantly higher levels of IFN gamma, IL-17, and IL-4 cytokine levels just before infection, which correlated with protection. In addition, rSap2-parapsilosis vaccination induced high titers of Sap2-specific antibodies, and a fraction of antibodies could bind whole fungus (which were predominantly of IgM isotype). Notably, sera from rSap2-parapsilosis vaccinated mice exhibited increased C. tropicalis biofilm inhibition ability and enhanced neutrophil-mediated fungal killing in vitro. Sap2-specificity was further confirmed by depleting antigen-specific antibodies. Passive transfer of Sap2-parapsilosis immune serum significantly reduced fungal burdens in naive mice, as compared to mice receiving sham-immune serum, upon infection. Higher numbers of total CD19 + B-cells, plasma cells, and Candida-binding B-cells in rSap2-parapsilosis vaccinated mice indicate a role of B-cells during early stages of Sap2-mediated immune response. Epitope analysis performed using identified B-cell epitopes provides insights about including important IgM and IgG epitopes, when designing multivalent or multi-epitope anti-Candida vaccine/s.

Conclusion
Mice immunized with recombinant Sap2 cloned from C. parapsilosis exhibited increased survival during C. tropicalis mediated systemic candidiasis. Both Th1/Th17 mediated cellular immunity and humoral immunity contribute toward protection. Our results establish a role of B-cells and antibodies in generation of protective immune responses against Sap2 vaccine antigen during non-albicans Candida mediated murine systemic candidiasis.