Immunological correlates of protection mediated by a whole organism Cryptococcus neoformans vaccine deficient in chitosan

Abstract

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 C. neoformans to create a chitosan-deficient, avirulent strain, designated cda1Δ2Δ3Δ 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 IFNγ, TNFα, or 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 cda1Δ2Δ3Δ 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 cda1Δ2Δ3Δ . When used as a vaccine, cda1Δ2Δ3Δ 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 for future CD4 ⁺ T cell dysfunction.