Despite the substantial global burden of human fungal infections, there are no approved fungal vaccines to protect at risk individuals. Here, we review the progress that has been made and the challenges that lie ahead in the quest towards efficacious fungal vaccines. In mouse studies, protection has been achieved with vaccines directed against fungal pathogens, including species of Candida, Cryptococcus, and Aspergillus, that most commonly cause life-threatening human disease. Encouraging results have been obtained with vaccines composed of live-attenuated and killed fungi, crude extracts, recombinant subunit formulations, and nucleic acid vaccines. Novel adjuvants that instruct the immune system to mount the types of protective responses needed to fight mycotic infections are under development. Candidate vaccines include those that target common antigens expressed on multiple genera of fungi thereby protecting against a broad range of mycoses. Encouragingly, three vaccines have reached human clinical trials. Still, formidable obstacles must be overcome before we will have fungal vaccines licensed for human use.

Vaccination with glucan particles (GP) containing the Cryptococcus neoformans chitin deacetylases Cda1 and Cda2 protect mice against experimental cryptococcosis. Here, immunological correlates of vaccine-mediated protection were explored. Studies comparing knockout and wild-type mice demonstrated CD4⁺ T cells are crucial, while B cells and CD8⁺ T cells are dispensable. Protection was abolished following CD4⁺ T cell depletion during either vaccination or infection but was retained if CD4⁺ T cells were only partially depleted. Vaccination elicited systemic and durable antigen-specific immune responses in peripheral blood mononuclear cells (PBMCs), spleens, and lungs. Following vaccination and fungal challenge, robust T-helper (Th) 1 and Th17 responses were observed in the lungs. Protection was abrogated in mice congenitally deficient in interferon (IFN) γ, IFNγ receptor, interleukin (IL)-1β, IL-6, or IL-23. Thus, CD4⁺ T cells and specific proinflammatory cytokines are required for GP-vaccine-mediated protection. Importantly, retention of protection in the setting of partial CD4⁺ T depletion suggests a pathway for vaccinating at-risk immunocompromised individuals.

The global burden of infections due to the pathogenic fungus Cryptococcus is substantial in persons with low CD4 ⁺ T-cell counts. Previously, we deleted three chitin deacetylase genes from Cryptococcus neoformans to create a chitosan-deficient, avirulent strain, designated as cda1∆2∆3∆ , which, when used as a vaccine, protected mice from challenge with virulent C. neoformans strain KN99. Here, we explored the immunological basis for protection. Vaccine-mediated protection was maintained in mice lacking B cells or CD8 ⁺ T cells. In contrast, protection was lost in mice lacking α/β T cells or CD4 ⁺ T cells. Moreover, CD4 ⁺ T cells from vaccinated mice conferred protection upon adoptive transfer to naive mice. Importantly, while monoclonal antibody-mediated depletion of CD4 ⁺ T cells just prior to vaccination resulted in complete loss of protection, significant protection was retained in mice depleted of CD4 ⁺ T cells after vaccination but prior to challenge. Vaccine-mediated protection was lost in mice genetically deficient in interferon-γ (IFNγ), tumor necrosis factor alpha (TNFα), or interleukin (IL)-23p19. A robust influx of leukocytes and IFNγ- and TNFα-expressing CD4 ⁺ T cells was seen in the lungs of vaccinated and challenged mice. Finally, a higher level of IFNγ production by lung cells stimulated ex vivo correlated with lower fungal burden in the lungs. Thus, while B cells and CD8 ⁺ T cells are dispensable, IFNγ and CD4 ⁺ T cells have overlapping roles in generating protective immunity prior to cda1∆2∆3∆ vaccination. However, once vaccinated, protection becomes less dependent on CD4 ⁺ T cells, suggesting a strategy for vaccinating HIV ⁺ persons prior to loss of CD4 ⁺ T cells. IMPORTANCE: The fungus Cryptococcus neoformans is responsible for >100,000 deaths annually, mostly in persons with impaired CD4 ⁺ T-cell function such as AIDS. There are no approved human vaccines. We previously created a genetically engineered avirulent strain of C. neoformans , designated as cda1∆2∆3∆ . When used as a vaccine, cda1∆2∆3∆ protects mice against a subsequent challenge with a virulent C. neoformans strain. Here, we defined components of the immune system responsible for vaccine-mediated protection. We found that while B cells and CD8 ⁺ T cells were dispensible, protection was lost in mice genetically deficient in CD4 ⁺ T cells and the cytokines IFNγ, TNFα, or IL-23. A robust influx of cytokine-producing CD4 ⁺ T cells was seen in the lungs of vaccinated mice following infection. Importantly, protection was retained in mice depleted of CD4 ⁺ T cells following vaccination, suggesting a strategy to protect persons who are at risk of future CD4 ⁺ T-cell dysfunction.

The fungal infection, cryptococcosis, is responsible for >100,000 deaths annually. No licensed vaccines are available. We explored the efficacy and immune responses of subunit cryptococcal vaccines adjuvanted with Cationic Adjuvant Formulation 01 (CAF01). CAF01 promotes humoral and T helper (Th) 1 and Th17 immune responses and has been safely used in human vaccine trials. Four subcutaneous vaccines, each containing single recombinant Cryptococcus neoformans protein antigens, partially protected mice from experimental cryptococcosis. Protection increased, up to 100%, in mice that received bivalent and quadrivalent vaccine formulations. Vaccinated mice that received a pulmonary challenge with C. neoformans had an influx of leukocytes into the lung including robust numbers of polyfunctional CD4⁺ T cells which produced interferon gamma (IFNγ), tumor necrosis factor alpha (TNFα), and interleukin (IL)-17 upon ex vivo antigenic stimulation. Cytokine-producing lung CD8⁺ T cells were also found, albeit in lesser numbers. A significant, durable IFNγ response was observed in the lungs, spleen, and blood. Moreover, IFNγ secretion following ex vivo stimulation directly correlated with fungal control in the lungs. Thus, we have developed multivalent cryptococcal vaccines which protect mice from experimental cryptococcosis using an adjuvant which has been safely tested in humans. These preclinical studies suggest a path towards human cryptococcal vaccine trials.

Immunological studies of persons with cryptococcal disease without known immunocompromise could inform vaccine development and identify novel requirements for antifungal immunity. We tested recall responses to heat-killed (HK) Cryptococcus and recombinant cryptococcal candidate vaccine antigens in peripheral blood T cells from subjects with a history of cryptococcosis and no recognized predisposing condition at the time of study enrollment, alongside healthy controls. In response to stimulation with HK Cryptococcus, CD4⁺ and CD8⁺ T cells from the cryptococcosis subjects had greater activation compared to healthy controls. Responses to recombinant antigens, while less robust, were also more frequent in patient samples compared to healthy controls. Fungal strains stimulated much higher interferon gamma (IFNγ) secretion compared with interleukin (IL)-17A and IL-4. A mouse model of vaccination and cryptococcal infection was established to determine the degree to which antigen-specific IFNγ responses in the periphery reflect responses in the tissue. Peripherally, the mouse model mimicked the human studies with the additional finding that responses were greatest in mice following both vaccination and infection. Moreover, responses in splenocytes and lung cells exceeded those in PBMCs suggesting PBMC responses underestimate those at sites of infection. In summary, in otherwise immunocompetent patients with cryptococcosis, antigen-stimulated CD4⁺ and CD8⁺ T cell activation, as measured by cytokine, was intact and biased towards a T-helper 1 (Th1) response. The mouse data support vaccination as a strategy to boost immune responses to prevent clinical Cryptococcus infection.