ANALYSIS OF IMMUNE RESPONSES TO CANDIDATE VACCINE ANTIGENS IN NON-ALBICANS CANDIDA MEDIATED SYSTEMIC CANDIDIASIS

Abstract

Systemic candidiasis is fourth most common blood-stream infection in ICU patients worldwide. Every year, systemic candidiasis affects approx. 250,000 people world-wide causing ~50,000 deaths. According to CDC, mortality associated with systemic candidiasis is ~40-70% worldwide, even with use of antifungal therapies. Although C. albicans is a predominant species causing systemic candidiasis, in recent decades, infections caused by non-albicans Candida (NAC) species have increased. Among NAC species, C. tropicalis is frequently identified in Asia-Pacific region, is more invasive and persistent, and exhibits increased antifungal resistance. Therefore, there is need for alternative immune-therapies against C. tropicalis infections. Most research to-date focuses on C. albicans infections and few vaccine candidates are identified. The aim of this study was to investigate the protective potential of candidate vaccine antigens (from C. albicans) in murine systemic candidiasis caused by C. tropicalis. Two vaccine candidates: ALS3 (agglutinin like sequence gene) and SAP2 (secreted aspartyl proteinase gene) were selected. Initially, mice model of systemic candidiasis using C. albicans and C. tropicalis was established. Subsequently, ALS3 gene was cloned, expressed and purified as recombinant protein from C. albicans. After dose optimization, ALS3-albicans protein was used for mice immunization followed by challenge with either C. albicans or C. tropicalis species. Using survival analysis and fungal burden assay, it was observed that ALS3-albicans protein did not confer protection in either C. albicans or C. tropicalis mediated systemic candidiasis. Cloning and expression of rALS3 was also performed using C. tropicalis and C. parapsilosis species. However, in absence of protective potential, further research was discontinued using ALS3. Thereafter, SAP2 gene was cloned, expressed and purified as recombinant protein from C. albicans. After dose optimization, recombinant SAP2-albicans protein was used for mice immunization followed by challenge with lethal dose of C. albicans. Immunization using SAP2-albicans protein increased survival and reduced organ fungal burden upon C. albicans infection, compared to sham-immunized controls. However, immunization with SAP2-albicans protein did not confer protection during C. tropicalis infection. Next, rSAP2 gene was cloned and expressed from C. tropicalis and C. parapsilosis fungal species and their protective potential was evaluated in C. tropicalis infection. Similar to SAP2-albicans protein, immunization with SAP2-tropicalis protein was not found protective when mice were infected with C. tropicalis. However, mice immunized with SAP2-parapsilosis exhibited increased survival and significantly reduced organ fungal burdens compared to sham-immunized controls. It was observed that SAP2- vaccination induced significantly high titers of antigen-specific antibodies. Further, sera from SAP2-vaccinated mice exhibited increased C. tropicalis biofilm inhibition ability and enhanced neutrophil-mediated fungal killing. Passive transfer of SAP2-immune serum in naïve mice significantly reduced fungal burdens and improved survival, compared to mice receiving sham immune serum. Histopathologically, kidneys of SAP2-vaccinated mice had increased neutrophil recruitment and reduced fungal dissemination compared to control mice. Increased levels of serum Th1/Th2/Th17 cytokines in SAP2-immunized mice suggests immunomodulatory role of SAP2 vaccine-candidate during C. tropicalis infection. In summary, results suggest that SAP2- vaccination can improve mice survival during C. tropicalis infection by inducing both humoral and cellular immunity, and higher titers of SAP2-specific antibodies are beneficial during systemic candidiasis.