A protective hybrid protein vaccine informed by mapping major histocompatibility class II epitopes in Cryptococcus neoformans chitin deacetylases that stimulate CD4+ T-cell responses.

Abstract

Infections due to Cryptococcus species are estimated to cause over 100,000 deaths a year. No cryptococcal vaccines are approved for human use. We have shown that vaccine formulations consisting of C. neoformans chitin deacetylase 2 (Cda2) and Cda1 adjuvanted with Cationic Adjuvant Formulation 01 (CAF01) protect mice against experimental cryptococcosis by a mechanism dependent upon CD4⁺ T cells. Here, using overlapping peptide libraries, we mapped epitopes in Cda2 and Cda1 that stimulated interferon-gamma (IFNγ) release from splenocytes of C57BL/6 and DR4 mice immunized with these proteins. DR4 mice lack mouse major histocompatibility class II (MHC-II) proteins and express chimeric MHC-II proteins with the specificity of the human HLA-DR allele, HLA-DRB1*04:01. Experimental results were then compared with MHC-II binding predictions using the Immune Epitope Database NetMHCIIpan 4.1 BA MHC-II epitope prediction tool. Unique epitopes in Cda2 and Cda1 were discovered for each mouse strain, some of which were not predicted to bind well to MHC-II. CD4⁺ T cells were responsible for cytokine release as IFNγ production was lost if CD4⁺ T cells were depleted from the splenocyte population. Finally, we tested a hybrid recombinant protein that consisted of portions of Cda2 and Cda1 containing the MHC-II/CD4⁺ T-cell epitopes that induce CD4⁺ T cells in DR4 mice. When administered as a CAF01-adjuvanted vaccine, the hybrid protein protected DR4 mice from an otherwise lethal C. neoformans pulmonary challenge. These results provide a proof of principle regarding the utility of MHC-II/CD4⁺ T-cell epitope mapping in cryptococcal vaccine development.

Importance: The development of vaccines to prevent cryptococcosis, a fungal disease which causes over 100,000 deaths annually, is a public health priority. We have demonstrated that adjuvanted vaccines containing two Cryptococcus neoformans protein antigens, chitin deacetylase (Cda)2 and Cda1, protect mice against experimental cryptococcosis by a mechanism dependent upon CD4⁺ T cells. In this study, we determined which 15-amino-acid peptides of Cda2 and Cda1 stimulate CD4⁺ T cells in C57BL/6 and DR4 mice; the latter mouse strain has human major histocompatibility class II (MHC-II) specificity. We then synthesized a small hybrid protein containing those portions of Cda2 and Cda1 that strongly stimulated CD4⁺ T-cell responses in DR4 mice. As an adjuvanted vaccine, the hybrid Cda2/1 protein protected DR4 mice from a C. neoformans pulmonary challenge. Thus, CD4⁺ T-cell/MHC-II epitope mapping experimentally validated the design of a protective multi-epitope protein for use in cryptococcal vaccines.