{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,9,18]],"date-time":"2026-09-18T18:04:52Z","timestamp":1789754692371,"version":"4.0.1"},"posted":{"date-parts":[[2026,9,14]]},"group-title":"Immunology","reference-count":37,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,9,14]],"date-time":"2026-09-14T00:00:00Z","timestamp":1789344000000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc-nd\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,9,14]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>\n                  Infections due to\n                  <jats:italic>Cryptococcus<\/jats:italic>\n                  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\n                  <jats:italic>C. neoformans<\/jats:italic>\n                  chitin deacetylase (Cda)2 and Cda1 adjuvanted with Cationic Adjuvant Formulation 01 (CAF01) protect mice against experimental cryptococcosis by a mechanism dependent upon CD4\n                  <jats:sup>+<\/jats:sup>\n                  T cells. Here, using overlapping peptide libraries, we mapped epitopes in Cda2 and Cda1 that stimulated interferon-gamma (IFN\u03b3) 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\n                  <jats:sup>+<\/jats:sup>\n                  T cells were responsible for cytokine release as IFN\u03b3 production was lost if CD4\n                  <jats:sup>+<\/jats:sup>\n                  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\n                  <jats:sup>+<\/jats:sup>\n                  T cell epitopes which induce CD4\n                  <jats:sup>+<\/jats:sup>\n                  T cells in DR4 mice. When administered as a CAF01-adjuvanted vaccine, the hybrid protein protected DR4 mice from an otherwise lethal\n                  <jats:italic>C. neoformans<\/jats:italic>\n                  pulmonary challenge. These results provide a proof of principle regarding the utility of MHC-II\/CD4\n                  <jats:sup>+<\/jats:sup>\n                  T cell epitope mapping in cryptococcal vaccine development.\n                <\/jats:p>\n                <jats:sec>\n                  <jats:title>Importance<\/jats:title>\n                  <jats:p>\n                    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\n                    <jats:italic>Cryptococcus neoformans<\/jats:italic>\n                    protein antigens, chitin deacetylase (Cda)2 and Cda1, protect mice against experimental cryptococcosis by a mechanism dependent upon CD4\n                    <jats:sup>+<\/jats:sup>\n                    T cells. In this study, we determined which 15 amino acid peptides of Cda2 and Cda1 stimulate CD4\n                    <jats:sup>+<\/jats:sup>\n                    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\n                    <jats:sup>+<\/jats:sup>\n                    T cell responses in DR4 mice. As an adjuvanted vaccine, the hybrid Cda2\/1 protein protected DR4 mice from a\n                    <jats:italic>C. neoformans<\/jats:italic>\n                    pulmonary challenge. Thus, CD4\n                    <jats:sup>+<\/jats:sup>\n                    T cell\/MHCII epitope mapping experimentally validated the design of a protective multi-epitope protein for use in cryptococcal vaccines.\n                  <\/jats:p>\n                <\/jats:sec>","DOI":"10.64898\/2026.09.07.749995","type":"posted-content","created":{"date-parts":[[2026,9,14]],"date-time":"2026-09-14T17:15:22Z","timestamp":1789406122000},"source":"Crossref","is-referenced-by-count":0,"title":["A protective hybrid protein vaccine informed by mapping major histocompatibility class II epitopes in\n                  <i>Cryptococcus neoformans<\/i>\n                  chitin deacetylases that stimulate CD4\n                  <sup>+<\/sup>\n                  T cell responses"],"prefix":"10.64898","author":[{"given":"Charles A.","family":"Specht","sequence":"first","affiliation":[{"name":"Department of Medicine, University of Massachusetts Chan Medical School, Worcester, Massachusetts, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lorena V.N.","family":"Oliveira","sequence":"additional","affiliation":[{"name":"Department of Medicine, University of Massachusetts Chan Medical School, Worcester, Massachusetts, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Diana","family":"Carlson","sequence":"additional","affiliation":[{"name":"Department of Medicine, University of Massachusetts Chan Medical School, Worcester, Massachusetts, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Maureen M.","family":"Hester","sequence":"additional","affiliation":[{"name":"Department of Medicine, University of Massachusetts Chan Medical School, Worcester, Massachusetts, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Satya","family":"Sahu","sequence":"additional","affiliation":[{"name":"Department of Medicine, University of Massachusetts Chan Medical School, Worcester, Massachusetts, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Gabriel Kristian","family":"Pedersen","sequence":"additional","affiliation":[{"name":"Center for Vaccine Research, Statens Serum Institut, Copenhagen, Denmark"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3799-3064","authenticated-orcid":false,"given":"Stuart M.","family":"Levitz","sequence":"additional","affiliation":[{"name":"Department of Medicine, University of Massachusetts Chan Medical School, Worcester, Massachusetts, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2026091810202627000_2026.09.07.749995v1.1","doi-asserted-by":"publisher","DOI":"10.1016\/s1473-3099(22)00499-6"},{"key":"2026091810202627000_2026.09.07.749995v1.2","doi-asserted-by":"publisher","DOI":"10.1016\/S1473-3099(23)00692-8"},{"key":"2026091810202627000_2026.09.07.749995v1.3","doi-asserted-by":"crossref","first-page":"33","DOI":"10.1038\/s41541-021-00294-8","article-title":"Vaccines for human fungal diseases: close but still a long way to go","volume":"6","year":"2021","journal-title":"NPJ Vaccines"},{"key":"2026091810202627000_2026.09.07.749995v1.4","doi-asserted-by":"publisher","DOI":"10.1371\/journal.ppat.1011115"},{"key":"2026091810202627000_2026.09.07.749995v1.5","doi-asserted-by":"crossref","first-page":"840","DOI":"10.3390\/jof10120840","article-title":"Will the Real Immunogens Please Stand Up: Exploiting the Immunogenic Potential of Cryptococcal Cell Antigens in Fungal Vaccine Development","volume":"10","year":"2024","journal-title":"Journal of Fungi"},{"key":"2026091810202627000_2026.09.07.749995v1.6","doi-asserted-by":"crossref","first-page":"106521","DOI":"10.1016\/j.jinf.2025.106521","article-title":"Peripheral blood CD4+ and CD8+ T cell responses to Cryptococcus candidate vaccine antigens in human subjects with and without cryptococcosis","volume":"91","year":"2025","journal-title":"Journal of Infection"},{"key":"2026091810202627000_2026.09.07.749995v1.7","doi-asserted-by":"crossref","unstructured":"Carlson D , Wang R , Hastings Z , Oliveira LVN , Hester MM , Rodriguez N , Pedersen GK , Tenor JL , Perfect JR , Specht CA , Levitz SM . 2025. Vaccine-Mediated Protection of Mice Against African and Asian Clinical Strains of Cryptococcus neoformans. 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Targeting infection-specific peptides in immunopeptidomics studies for vaccine target discovery. J Exp Med 222.","DOI":"10.1084\/jem.20250444"},{"key":"2026091810202627000_2026.09.07.749995v1.15","doi-asserted-by":"crossref","first-page":"319","DOI":"10.1002\/eji.201545930","article-title":"High-resolution analysis of the murine MHC class II immunopeptidome","volume":"46","year":"2016","journal-title":"European Journal of Immunology"},{"key":"2026091810202627000_2026.09.07.749995v1.16","doi-asserted-by":"publisher","DOI":"10.1016\/j.immuni.2023.03.009"},{"key":"2026091810202627000_2026.09.07.749995v1.17","doi-asserted-by":"crossref","unstructured":"Homan EJ , Bremel RD . 2023. Determinants of tumor immune evasion: the role of T cell exposed motif frequency and mutant amino acid exposure. 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