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

Abstract

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 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.