A protective hybrid protein vaccine informed by mapping major histocompatibility class II epitopes in Cryptococcus neoformans chitin deacetylases that stimulate CD4 ⁺ T-cell responses
- 1Department of Medicine, University of Massachusetts Chan Medical School
- 2Center for Vaccine Research, Statens Serum Institut
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.
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BibTeX
@article{specht2026protective,
title = {{A protective hybrid protein vaccine informed by mapping major histocompatibility class II epitopes in Cryptococcus neoformans chitin deacetylases that stimulate CD4 ⁺ T-cell responses}},
author = {Charles A. Specht and Lorena V. N. Oliveira and Diana Carlson and Maureen M. Hester and Satya Sahu and Gabriel Kristian Pedersen and Stuart M. Levitz},
journal = {mBio},
year = {2026},
month = oct,
doi = {10.1128/mbio.02176-26},
url = {https://journals.asm.org/doi/10.1128/mbio.02176-26},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - A protective hybrid protein vaccine informed by mapping major histocompatibility class II epitopes in Cryptococcus neoformans chitin deacetylases that stimulate CD4 ⁺ T-cell responses
AU - Charles A. Specht
AU - Lorena V. N. Oliveira
AU - Diana Carlson
AU - Maureen M. Hester
AU - Satya Sahu
AU - Gabriel Kristian Pedersen
AU - Stuart M. Levitz
PY - 2026
DA - 2026/10/14
JO - mBio
DO - 10.1128/mbio.02176-26
UR - https://journals.asm.org/doi/10.1128/mbio.02176-26
AB - 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.
SN - 2150-7511
ER -