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

UNLABELLED: 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 (Cda)2 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 (IEDB) 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 which 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 (MHCII) 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/MHCII epitope mapping experimentally validated the design of a protective multi-epitope protein for use in cryptococcal vaccines.