Abstract
ABSTRACTDevelopment of a vaccine to protect against cryptococcosis is a priority given the enormous global burden of disease in at-risk individuals. Using glucan particles (GPs) as a delivery system, we previously demonstrated that mice vaccinated with crude Cryptococcus-derived alkaline extracts were protected against lethal challenge with Cryptococcus neoformans and Cryptococcus gattii. The goal of the present study was to identify protective protein antigens that could be used in a subunit vaccine. Using biased and unbiased approaches, six candidate antigens (Cda1, Cda2, Cda3, Fpd1, MP88, and Sod1) were selected, recombinantly expressed in Escherichia coli, purified, and loaded into GPs. Three mouse strains (C57BL/6, BALB/c, and DR4) were then vaccinated with the antigen-laden GPs, following which they received a pulmonary challenge with virulent C. neoformans and C. gattii strains. Four candidate vaccines (GP-Cda1, GP-Cda2, GP-Cda3, and GP-Sod1) afforded a significant survival advantage in at least one mouse model; some vaccine combinations provided added protection over that seen with either antigen alone. Vaccine-mediated protection against C. neoformans did not necessarily predict protection against C. gattii. Vaccinated mice developed pulmonary inflammatory responses that effectively contained the infection; many surviving mice developed sterilizing immunity. Predicted T helper cell epitopes differed between mouse strains and in the degree to which they matched epitopes predicted in humans. Thus, we have discovered cryptococcal proteins that make promising candidate vaccine antigens. Protection varied depending on the mouse strain and cryptococcal species, suggesting that a successful human subunit vaccine will need to contain multiple antigens, including ones that are species specific.
Highlights
IMPORTANCE The encapsulated fungi Cryptococcus neoformans and Cryptococcus gattii are responsible for nearly 200,000 deaths annually, mostly in immunocompromised individuals
Vaccines comprised of GXM and other cell wall carbohydrates conjugated to carrier proteins elicit antibody responses to GXM that protect in experimental models of cryptococcosis [13, 14]
Cda1 and Cda3 were chosen as they are chitin deacetylases (Cda) with protein sequence homology to Cda2 [25] and for the importance of Cda proteins for virulence [26]
Summary
IMPORTANCE The encapsulated fungi Cryptococcus neoformans and Cryptococcus gattii are responsible for nearly 200,000 deaths annually, mostly in immunocompromised individuals. An adaptive antigen-specific, T cell-mediated immune response becomes critical for preventing local spread and dissemination. Antibody responses, those directed against glucuronoxylomannan (GXM) and other polysaccharide components in the capsule, appear to contribute to the adaptive response in at least some individuals [8]. Vaccines comprised of GXM and other cell wall carbohydrates conjugated to carrier proteins elicit antibody responses to GXM that protect in experimental models of cryptococcosis [13, 14]. We and others have focused on the discovery of C. neoformans antigens that stimulate T cell responses and could serve as candidate antigens in cryptococcal vaccines [15,16,17,18]. Because most vaccine adjuvants presently in clinical use stimulate predominantly protective antibody responses [19], we postulate that a successful vaccine will need to incorporate adjuvants and/or delivery systems that elicit strong T cell responses
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