Cryptococcosis is an opportunistic invasive fungal infection caused by fungi of the genus Cryptococcus, mainly C. neoformans and C. gattii. In most persons, host defenses are sufficient to kill or contain the fungus in a latent state following inhalation of aerosolized cells from the environment, however, pneumonia and life-threatening disseminated infection, especially to the central nervous system can occur in immunocompromised hosts. Despite currently available treatment, the morbidity and mortality of cryptococcosis remain high with over 100,000 deaths annually. C. neoformans was placed into the critical priority group in the WHO fungal priority pathogens list released in 2022. Thus, preventative measures, such as the development of efficacious vaccines are urgently needed. However, no licensed cryptococcal vaccines are available.

One of the most important research directions in our lab focus on identification of immunoreactive fungal proteins that could serve as components of C. neoformans vaccines. In previous studies, we identified two promising vaccine antigens, Cda1 and Cda2, which are members of the chitin deacetylase (Cda) family. Cda1 and Cda2 are attractive candidate vaccine antigens because they have no significant homology to human proteins,  are strongly immunogenic, and catalyze the deacetylation of chitin to the virulence determinant chitosan. Accordingly, we worked with the laboratory of our collaborator, Dr. Gary R. Ostroff to load these two antigens into the glucan particle (GP)-based delivery system.  GPs are purified from Saccharomyces with a hollow, porous structure that enables high antigen loading. It is recognized by the pattern recognition receptor Dectin-1 expressed on antigen-presenting cells. Encouragingly, GP-based subunit vaccines, including GP-Cda1 and GP-Cda2 (alone and in combination), afforded a significant survival advantage following pulmonary challenge of mice with a highly virulent C. neoformans strain.

Here we addressed  the mechanisms responsible for vaccine-induced immunity. During natural cryptococcal infection, CD4⁺ T cells and, to a much lesser extent, antibody are critical for protection. However, the arms of the immune system responsible for vaccine-induced immunity could be quite different. Thus, further understanding the mechanism behind it is critical  for vaccine development and to help predict which populations are likely to benefit.

To probe the immunological correlates of GP-vaccine-mediated protection, we examined mice with congenital and acquired deficiencies in specific aspects of immune function. First, we explored the role of B cells in two different B cell deficiency mouse stains: µMT mice on C57BL/6 background and Jh^-/- mice on the BALB/c background. Protection against cryptococcosis mediated by the GP-Cda1 and GP-Cda2 vaccines was retained in both strains, indicating B cells are not essential in these settings. Our data also demonstrate that CD8⁺ T cells are dispensable for GP-Cda1 and GP-Cda2 vaccine-mediated protection. Vaccinated β2m^-/- mice, deficient in CD8⁺ T cells, survived as well as the vaccinated wild-type mice following lethal challenge with C. neoformans.

While B cells and CD8⁺ T cells were not required, two lines of evidence demonstrate the non-redundant contribution that CD4⁺ T cells make to GP-vaccine mediated protection. First, vaccination with GP-Cda1 and GP-Cda2 failed to protect CD4⁺ T cell-deficient MHCII^-/- mice against cryptococcal challenge. Second, vaccine-mediated protection was lost when mice were depleted of CD4⁺ T cells using a monoclonal antibody targeting CD4. Notably, protection was abrogated regardless of whether the CD4⁺ T cells were depleted at the time of vaccination or during infection. These findings raise the question of whether the immunocompromised populations most at risk for cryptococcosis could still benefit from the vaccine. Taking into consideration that most immunocompromised individuals are only partially deficient in CD4⁺ T cell number or function, we established a partial CD4 deletion animal model using a range of doses of the anti-CD4 mAb, GK1.5. The protection elicited by the GP-Cda1/Cda2 combination vaccine was inversely correlated with GK1.5 dosage. Importantly, mice with very low levels of blood CD4⁺ T cells at the time of challenge were protected. While the translational significance of these findings remains speculative, these data suggest it may be possible to vaccinate persons living with HIV while their CD4⁺ T cells counts are relatively high, either at early diagnosis or after antiretroviral treatment.

The durability and pattern of compartmentalization of vaccine-induced immunity are dependent, at least in part, on the vaccine antigens, delivery system, and route of administration. The vast majority of vaccines in clinical use elicit protective responses that are antibody-dependent.  This is true for vaccines that use traditional adjuvants such as alum, newer adjuvants such as AS01_B, and RNA vaccines. In comparison, we have previously demonstrated GP-based vaccines could elicit strong antigen-specific Th1- and Th17-biased CD4⁺ T cell responses in mice and rats. Consistent with these observations, following GP-Cda1/Cda2 vaccination and infection, mouse lungs had a robust influx of activated CD4⁺ T cells that produced IFNγ (Th1), IL-17 (Th17), and TNFα following ex vivo antigen stimulation. Moreover, significantly enhanced IFNγ concentrations in ex vivo culture supernatants were detected by ELISA in the PBMCs, spleens, and lungs of mice that were both vaccinated and challenged. These results suggest that GP-Cda1/Cda2 vaccine induced immunity are systemic and feature enhanced Th1 and Th17 responses. Furthermore, responses were durable as vaccinated mice had robust T cell recall responses in the lungs at 70 days post-infection.

Finally, we explored GP-Cda1 and GP-Cda2 vaccine-induced protection in a panel of mice with genetic deficiencies in selected cytokines and a cytokine receptor implicated in host defenses against cryptococcosis. Protection was abrogated in mice congenitally deficient in interferon (IFN) γ, IFNγ receptor, interleukin (IL)-1β, IL-6, or IL-23. Thus, specific pro-inflammatory cytokines are required for GP-vaccine mediated protection.

In summary, our preclinical studies illuminate the arms of the immune system which are required for GP-Cda1 and GP-Cda2 vaccines to protect mice from cryptococcosis, and provide encouragement that GP-vaccines could be employed in some immunodeficient hosts. One of the future challenges and directions will be how to maximize protection in hosts with CD4⁺ T cell impairment. One strategy which we are considering is to stimulate other arms of the immune system, such as exploring whether vaccines containing cryptococcal protein antigens exposed on the capsular surface will stimulate protective opsonophagocytic antibody responses.