{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T01:37:08Z","timestamp":1760060228645,"version":"build-2065373602"},"reference-count":34,"publisher":"MDPI AG","issue":"8","license":[{"start":{"date-parts":[[2025,8,15]],"date-time":"2025-08-15T00:00:00Z","timestamp":1755216000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Biomolecules"],"abstract":"<jats:p>Glycosylation plays a pivotal role in regulating the functions and immunogenicity of antigens. Targeting the receptor-binding domain (RBD) of the spike protein (S protein) of SARS-CoV-2, we examined the impact of different glycoforms on RBD antigen immunogenicity and the underlying mechanisms. IgG-specific antibody titers and pseudovirus neutralization were compared in mice immunized with RBD antigens bearing different glycoforms, which were prepared using glycoengineering-capable Pichia pastoris and mammalian cell expression systems with distinct glycosylation pathways. The glycosylation impacted the surface charges of the RBD antigen, and influenced its adsorption onto alum. This may further lead to variations in the antigen\u2019s immunogenicity. The high-mannose variant of the RBD antigen (H-MAN\/RBD) expressed in wild-type Pichia pastoris induced significantly higher IgG-specific antibody titers and pseudovirus neutralization activity compared with the complex RBD variant (Complex\/RBD) expressed in mammalian cells (293F) or glycoengineering-capable Pichia pastoris. The rate of H-MAN\/RBD adsorption onto aluminum hydroxide (alum) adjuvant was significantly higher than that of Complex\/RBD. It was assumed that H-MAN\/RBD might carry more negative charges because of its phosphomannose-modified surfaces, leading to a higher rate of adsorption onto the positively charged alum and enhancing the immune response. To assess the impact of phosphomannose modification on antigen immunogenicity, a yeast strain was engineered to prepare a low-mannose RBD antigen (L-MAN\/RBD); additionally, a yeast strain was constructed to generate a low-phosphomannose-modified RBD antigen (L-MAN-P\/RBD). In conclusion, phosphomannose modification substantially enhanced the immunogenicity of RBD by altering the surface charges of the RBD antigen and facilitating its adsorption onto alum. These findings offer novel insights and strategies for vaccine design and immunotherapeutic approaches.<\/jats:p>","DOI":"10.3390\/biom15081172","type":"journal-article","created":{"date-parts":[[2025,8,15]],"date-time":"2025-08-15T14:24:28Z","timestamp":1755267868000},"page":"1172","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Glycosylated SARS-CoV-2 RBD Antigens Expressed in Glycoengineered Yeast Induce Strong Immune Responses Through High Antigen\u2013Alum Adsorption"],"prefix":"10.3390","volume":"15","author":[{"given":"Ai","family":"Li","sequence":"first","affiliation":[{"name":"Department of Microorganism Engineering, Beijing Institute of Biotechnology, Beijing 100071, China"},{"name":"Institute of Physical Science and Information Technology, Anhui University, Hefei 230601, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Tiantian","family":"Wang","sequence":"additional","affiliation":[{"name":"Department of Microorganism Engineering, Beijing Institute of Biotechnology, Beijing 100071, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Bin","family":"Zhang","sequence":"additional","affiliation":[{"name":"Department of Microorganism Engineering, Beijing Institute of Biotechnology, Beijing 100071, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Xuchen","family":"Hou","sequence":"additional","affiliation":[{"name":"Department of Microorganism Engineering, Beijing Institute of Biotechnology, Beijing 100071, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Peng","family":"Sun","sequence":"additional","affiliation":[{"name":"Department of Microorganism Engineering, Beijing Institute of Biotechnology, Beijing 100071, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Hao","family":"Wang","sequence":"additional","affiliation":[{"name":"Department of Microorganism Engineering, Beijing Institute of Biotechnology, Beijing 100071, China"},{"name":"Department of Biopharmaceutical Science, Shanghai Ocean University, Shanghai 201306, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Huifang","family":"Xu","sequence":"additional","affiliation":[{"name":"Department of Microorganism Engineering, Beijing Institute of Biotechnology, Beijing 100071, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Min","family":"Tan","sequence":"additional","affiliation":[{"name":"Department of Microorganism Engineering, Beijing Institute of Biotechnology, Beijing 100071, China"},{"name":"Medical College, Hubei Enshi College, Enshi 445000, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Xin","family":"Gong","sequence":"additional","affiliation":[{"name":"Department of Microorganism Engineering, Beijing Institute of Biotechnology, Beijing 100071, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jun","family":"Wu","sequence":"additional","affiliation":[{"name":"Department of Microorganism Engineering, Beijing Institute of Biotechnology, Beijing 100071, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6655-1866","authenticated-orcid":false,"given":"Bo","family":"Liu","sequence":"additional","affiliation":[{"name":"Department of Microorganism Engineering, Beijing Institute of Biotechnology, Beijing 100071, China"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2025,8,15]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"265","DOI":"10.1038\/s41586-020-2008-3","article-title":"A new coronavirus associated with human respiratory disease in China","volume":"579","author":"Wu","year":"2020","journal-title":"Nature"},{"key":"ref_2","doi-asserted-by":"crossref","unstructured":"Coronaviridae Study Group of the International Committee on Taxonomy of Viruses (2020). 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