{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,5,18]],"date-time":"2026-05-18T15:11:04Z","timestamp":1779117064489,"version":"3.51.4"},"reference-count":44,"publisher":"MDPI AG","issue":"5","license":[{"start":{"date-parts":[[2026,5,15]],"date-time":"2026-05-15T00:00:00Z","timestamp":1778803200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Science and Technology Major Project of Gansu Province","award":["23ZDNA007"],"award-info":[{"award-number":["23ZDNA007"]}]},{"name":"Science and Technology Major Project of Gansu Province","award":["22ZD6NA001"],"award-info":[{"award-number":["22ZD6NA001"]}]},{"name":"National Key Research and Development Program of China","award":["2021YFC2301503"],"award-info":[{"award-number":["2021YFC2301503"]}]},{"name":"Gansu Science and Technology Project","award":["21JR7RA534"],"award-info":[{"award-number":["21JR7RA534"]}]},{"name":"Science and Technology Major Project of Gansu Province"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Vaccines"],"abstract":"<jats:p>Background\/Objectives: Several subunit vaccines for tuberculosis (TB), such as MVA85A and H4:IC31, have not demonstrated ideal protective efficacy in clinical trials, which may be attributed to their limited antigenic profile and lack of effective latency-associated antigens. In this study, we combined bioinformatics with experimental validation to screen for latency-associated antigens that have immune-protective effects. Methods: Highly expressed antigens were identified from models related to latent infections, such as hypoxia and nutritional starvation. Their physicochemical properties and immunogenicity were predicted using online tools such as Expasy-ProParam, IEBD, and VaxiJen. The immunogenicity of these antigens was then evaluated in multiple mycobacterium infection models. Finally, a systematic evaluation of the immune response and protective effects induced by the candidate antigens was performed in a mouse model using intracellular cytokine detection, mycobacterium growth inhibition assays (MGIAs), antibody-dependent cellular phagocytosis (ADCP), and a latent tuberculosis infection (LTBI) mouse model. Results: The antigen Rv2656c is highly expressed in the nutritional starvation model and demonstrates strong immunogenicity in both infected humans and cattle. Moreover, Rv2656c exerted a significant inhibitory effect against Mycobacterium tuberculosis (M. tuberculosis) and Mycobacterium avium (M. avium) infections in MGIA. The humoral immune response elicited by Rv2656c enhanced the phagocytosis and killing of Mycobacteria by macrophages in vitro. Furthermore, in a mouse model of LTBI established using the attenuated M. tuberculosis H37Ra strain, treatment with Rv2656c significantly decreased the bacterial load in the lungs of the mice. Conclusions: Latency-associated Rv2656c may serve as an immune-protective antigen, offering potential for the development of novel multi-stage antigen subunit vaccine against TB.<\/jats:p>","DOI":"10.3390\/vaccines14050442","type":"journal-article","created":{"date-parts":[[2026,5,15]],"date-time":"2026-05-15T10:33:21Z","timestamp":1778841201000},"page":"442","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Rv2656c: A Potential Candidate Antigen Associated with Latent Tuberculosis Infection"],"prefix":"10.3390","volume":"14","author":[{"ORCID":"https:\/\/orcid.org\/0009-0003-2763-5268","authenticated-orcid":false,"given":"Yunjie","family":"Du","sequence":"first","affiliation":[{"name":"State Key Laboratory of Animal Disease Control and Prevention and Lanzhou Center for Tuberculosis Research, Institute of Pathogen Biology, School of Basic Medical Sciences, Lanzhou University, Lanzhou 730000, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Pu","family":"He","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Animal Disease Control and Prevention and Lanzhou Center for Tuberculosis Research, Institute of Pathogen Biology, School of Basic Medical Sciences, Lanzhou University, Lanzhou 730000, China"},{"name":"School of Life Science, Lanzhou University, Lanzhou 730000, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Wenrui","family":"Dang","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Animal Disease Control and Prevention and Lanzhou Center for Tuberculosis Research, Institute of Pathogen Biology, School of Basic Medical Sciences, Lanzhou University, Lanzhou 730000, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Ting","family":"Zhou","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Animal Disease Control and Prevention and Lanzhou Center for Tuberculosis Research, Institute of Pathogen Biology, School of Basic Medical Sciences, Lanzhou University, Lanzhou 730000, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yinjuan","family":"Song","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Animal Disease Control and Prevention, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730000, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Xiaoping","family":"Li","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Animal Disease Control and Prevention, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730000, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yuhao","family":"Zhao","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Fei","family":"Li","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Animal Disease Control and Prevention and Lanzhou Center for Tuberculosis Research, Institute of Pathogen Biology, School of Basic Medical Sciences, Lanzhou University, Lanzhou 730000, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Aizhen","family":"Guo","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8422-4594","authenticated-orcid":false,"given":"Bingdong","family":"Zhu","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Animal Disease Control and Prevention and Lanzhou Center for Tuberculosis Research, Institute of Pathogen Biology, School of Basic Medical Sciences, Lanzhou University, Lanzhou 730000, China"},{"name":"College of Veterinary Medicine, Lanzhou University, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730000, China"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2026,5,15]]},"reference":[{"key":"ref_1","unstructured":"WHO (2025). Global Tuberculosis Report 2025, World Health Organization."},{"key":"ref_2","doi-asserted-by":"crossref","unstructured":"Zhuang, L., Ye, Z., Li, L., Yang, L., and Gong, W. (2023). Next-Generation TB Vaccines: Progress, Challenges, and Prospects. Vaccines, 11.","DOI":"10.3390\/vaccines11081304"},{"key":"ref_3","doi-asserted-by":"crossref","unstructured":"Sia, J.K., and Rengarajan, J. (2019). Immunology of Mycobacterium tuberculosis Infections. Microbiol. Spectr., 7.","DOI":"10.1128\/microbiolspec.GPP3-0022-2018"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"810","DOI":"10.1016\/S2213-2600(19)30274-7","article-title":"Insights and challenges in tuberculosis vaccine development","volume":"7","author":"McShane","year":"2019","journal-title":"Lancet Respir. Med."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"591","DOI":"10.1038\/s41577-018-0028-0","article-title":"Antibodies and tuberculosis: Finally coming of age?","volume":"18","author":"Li","year":"2018","journal-title":"Nat. Rev. Immunol."},{"key":"ref_6","doi-asserted-by":"crossref","unstructured":"Liang, Y., Li, X., Yang, Y., Xiao, L., Liang, Y., Mi, J., Xue, Y., Gong, W., Wang, L., and Wang, J. (2023). Preventive effects of Mycobacterium tuberculosis DNA vaccines on the mouse model with latent tuberculosis infection. Front. Immunol., 14.","DOI":"10.3389\/fimmu.2023.1110843"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"2505","DOI":"10.1128\/IAI.00072-16","article-title":"Transcriptional Profiling of Mycobacterium tuberculosis Exposed to In Vitro Lysosomal Stress","volume":"84","author":"Lin","year":"2016","journal-title":"Infect. Immun."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"17665","DOI":"10.1038\/s41598-017-17751-x","article-title":"The transcriptome of Mycobacterium tuberculosis in a lipid-rich dormancy model through RNAseq analysis","volume":"7","author":"Tilleman","year":"2017","journal-title":"Sci. Rep."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"218","DOI":"10.1016\/j.tube.2004.02.003","article-title":"Mycobacterium tuberculosis gene expression during adaptation to stationary phase and low-oxygen dormancy","volume":"84","author":"Voskuil","year":"2004","journal-title":"Tuberculosis"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"717","DOI":"10.1046\/j.1365-2958.2002.02779.x","article-title":"Evaluation of a nutrient starvation model of Mycobacterium tuberculosis persistence by gene and protein expression profiling","volume":"43","author":"Betts","year":"2002","journal-title":"Mol. Microbiol."},{"key":"ref_11","doi-asserted-by":"crossref","unstructured":"Keren, I., Minami, S., Rubin, E., and Lewis, K. (2011). Characterization and Transcriptome Analysis of Mycobacterium tuberculosis Persisters. mBio, 2.","DOI":"10.1128\/mBio.00100-11"},{"key":"ref_12","doi-asserted-by":"crossref","unstructured":"Gong, W., and Wu, X. (2021). Differential Diagnosis of Latent Tuberculosis Infection and Active Tuberculosis: A Key to a Successful Tuberculosis Control Strategy. Front. Microbiol., 12.","DOI":"10.3389\/fmicb.2021.745592"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"303","DOI":"10.1172\/JCI46252","article-title":"The multistage vaccine H56 boosts the effects of BCG to protect cynomolgus macaques against active tuberculosis and reactivation of latent Mycobacterium tuberculosis infection","volume":"122","author":"Lin","year":"2012","journal-title":"J. Clin. Investig."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"373","DOI":"10.1016\/S2213-2600(20)30319-2","article-title":"Safety and immunogenicity of the adjunct therapeutic vaccine ID93 + GLA-SE in adults who have completed treatment for tuberculosis: A randomised, double-blind, placebo-controlled, phase 2a trial","volume":"9","author":"Day","year":"2021","journal-title":"Lancet Respir. Med."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"1138","DOI":"10.1038\/s41467-023-36789-2","article-title":"Safety and immunogenicity of a thermostable ID93 + GLA-SE tuberculosis vaccine candidate in healthy adults","volume":"14","author":"Sagawa","year":"2023","journal-title":"Nat. Commun."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"2300463","DOI":"10.1080\/22221751.2023.2300463","article-title":"A multistage Sendai virus vaccine incorporating latency-associated antigens induces protection against acute and latent tuberculosis","volume":"13","author":"Hu","year":"2024","journal-title":"Emerg. Microbes Infect."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"2429","DOI":"10.1056\/NEJMoa1909953","article-title":"Final Analysis of a Trial of M72\/AS01(E) Vaccine to Prevent Tuberculosis","volume":"381","author":"Tait","year":"2019","journal-title":"N. Engl. J. Med."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"1021","DOI":"10.1016\/S0140-6736(13)60177-4","article-title":"Safety and efficacy of MVA85A, a new tuberculosis vaccine, in infants previously vaccinated with BCG: A randomised, placebo-controlled phase 2b trial","volume":"381","author":"Tameris","year":"2013","journal-title":"Lancet"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"1969","DOI":"10.1056\/NEJMc1811046","article-title":"H4:IC31 Vaccine or BCG Revaccination for Tuberculosis","volume":"379","author":"Netea","year":"2018","journal-title":"N. Engl. J. Med."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"138","DOI":"10.1056\/NEJMoa1714021","article-title":"Prevention of M. tuberculosis Infection with H4:IC31 Vaccine or BCG Revaccination","volume":"379","author":"Nemes","year":"2018","journal-title":"N. Engl. J. Med."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"34","DOI":"10.1038\/s41541-018-0057-5","article-title":"The TLR-4 agonist adjuvant, GLA-SE, improves magnitude and quality of immune responses elicited by the ID93 tuberculosis vaccine: First-in-human trial","volume":"3","author":"Coler","year":"2018","journal-title":"npj Vaccines"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"220","DOI":"10.1164\/rccm.201802-0366OC","article-title":"Dose Optimization of H56:IC31 Vaccine for Tuberculosis-Endemic Populations. A Double-Blind, Placebo-controlled, Dose-Selection Trial","volume":"199","author":"Suliman","year":"2019","journal-title":"Am. J. Respir. Crit. Care Med."},{"key":"ref_23","doi-asserted-by":"crossref","unstructured":"Kapoor, N., Pawar, S., Sirakova, T.D., Deb, C., Warren, W.L., and Kolattukudy, P.E. (2013). Human Granuloma In Vitro Model, for TB Dormancy and Resuscitation. PLoS ONE, 8.","DOI":"10.1371\/journal.pone.0053657"},{"key":"ref_24","doi-asserted-by":"crossref","unstructured":"Kruh, N.A., Troudt, J., Izzo, A., Prenni, J., and Dobos, K.M. (2010). Portrait of a Pathogen: The Mycobacterium tuberculosis Proteome In Vivo. PLoS ONE, 5.","DOI":"10.1371\/journal.pone.0013938"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"445","DOI":"10.1016\/j.tube.2005.10.002","article-title":"ICAT-based comparative proteomic analysis of non-replicating persistent Mycobacterium tuberculosis","volume":"86","author":"Cho","year":"2006","journal-title":"Tuberculosis"},{"key":"ref_26","doi-asserted-by":"crossref","unstructured":"Zhao, Y., Fei, W., Yang, L., Xiang, Z., Chen, X., Chen, Y., Hu, C., Chen, J., and Guo, A. (2024). The Establishment of a Novel \u03b3-Interferon In Vitro Release Assay for the Differentiation of Mycobacterial Bovis-Infected and BCG-Vaccinated Cattle. Vet. Sci., 11.","DOI":"10.3390\/vetsci11050198"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"1370","DOI":"10.1016\/j.vaccine.2016.01.049","article-title":"A novel liposome adjuvant DPC mediates Mycobacterium tuberculosis subunit vaccine well to induce cell-mediated immunity and high protective efficacy in mice","volume":"34","author":"Liu","year":"2016","journal-title":"Vaccine"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"S809","DOI":"10.1093\/ofid\/ofaa439.1817","article-title":"1637. SPR720, A Novel Benzamidazole Gyrase Inhibitor, Demonstrates Potent Efficacy Against Mycobacterium avium ATCC 700898 in a Chronic C3HeBFeJ Mouse Infection Model","volume":"7","author":"Verma","year":"2020","journal-title":"Open Forum Infect. Dis."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"118","DOI":"10.1016\/j.tube.2017.07.008","article-title":"A mycobacterial growth inhibition assay (MGIA) for bovine TB vaccine development","volume":"106","author":"Pepponi","year":"2017","journal-title":"Tuberculosis"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"3836","DOI":"10.1016\/j.vaccine.2023.04.003","article-title":"Immunogenicity and efficacy analyses of EPC002, ECA006, and EPCP009 protein subunit combinations as tuberculosis vaccine candidates","volume":"41","author":"Wang","year":"2023","journal-title":"Vaccine"},{"key":"ref_31","doi-asserted-by":"crossref","unstructured":"Li, H., Wu, M., Shi, Y., and Javid, B. (2016). Over-Expression of the Mycobacterial Trehalose-Phosphate Phosphatase OtsB2 Results in a Defect in Macrophage Phagocytosis Associated with Increased Mycobacterial-Macrophage Adhesion. Front. Microbiol., 7.","DOI":"10.3389\/fmicb.2016.01754"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"425","DOI":"10.1111\/sji.12556","article-title":"Subunit Vaccines Consisting of Antigens from Dormant and Replicating Bacteria Show Promising Therapeutic Effect against Mycobacterium Bovis BCG Latent Infection","volume":"85","author":"Li","year":"2017","journal-title":"Scand. J. Immunol."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"2323","DOI":"10.1128\/IAI.68.4.2323-2327.2000","article-title":"Identification of Secreted Proteins of Mycobacterium tuberculosis by a Bioinformatic Approach","volume":"68","author":"Gomez","year":"2000","journal-title":"Infect. Immun."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"1702","DOI":"10.1002\/pmic.200600853","article-title":"Comprehensive analysis of exported proteins from Mycobacterium tuberculosis H37Rv","volume":"7","author":"Berven","year":"2007","journal-title":"Proteomics"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"eabq0132","DOI":"10.1126\/science.abq0132","article-title":"A bacterial phospholipid phosphatase inhibits host pyroptosis by hijacking ubiquitin","volume":"378","author":"Chai","year":"2022","journal-title":"Science"},{"key":"ref_36","doi-asserted-by":"crossref","unstructured":"Hiemstra, A.M., MacDonald, C.E., van Rensburg, I.C., Stanley, K., Maasdorp, E., Mc Anda, S., T\u00f6nsing, S., Shaw, J.A., Tromp, G., and van der Spuy, G.D. (2022). Cascade Immune Mechanisms of Protection against Mycobacterium tuberculosis (IMPAc-TB): Study protocol for the Household Contact Study in the Western Cape, South Africa. BMC Infect. Dis., 22.","DOI":"10.1186\/s12879-022-07349-8"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"4834","DOI":"10.1016\/j.vaccine.2013.07.077","article-title":"An unbiased peptide-wide discovery approach to select Mycobacterium tuberculosis antigens that target CD8+ T cell response during infection","volume":"31","author":"Cayabyab","year":"2013","journal-title":"Vaccine"},{"key":"ref_38","doi-asserted-by":"crossref","unstructured":"Huang, Y., Huang, Y., Fang, Y., Wang, J., Li, Y., Wang, N., Zhang, J., Gao, M., Huang, L., and Yang, F. (2012). Relatively Low Level of Antigen-specific Monocytes Detected in Blood from Untreated Tuberculosis Patients Using CD4+ T-cell Receptor Tetramers. PLoS Pathog., 8.","DOI":"10.1371\/journal.ppat.1003036"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"300","DOI":"10.1093\/infdis\/jiw141","article-title":"Association of Human Antibodies to Arabinomannan With Enhanced Mycobacterial Opsonophagocytosis and Intracellular Growth Reduction","volume":"214","author":"Chen","year":"2016","journal-title":"J. Infect. Dis."},{"key":"ref_40","doi-asserted-by":"crossref","unstructured":"Bhavanam, S., Rayat, G.R., Keelan, M., Kunimoto, D., and Drews, S.J. (2018). Characterization of immune responses of human PBMCs infected with Mycobacterium tuberculosis H37Ra: Impact of donor declared BCG vaccination history on immune responses and M. tuberculosis growth. PLoS ONE, 13.","DOI":"10.1371\/journal.pone.0203822"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"102054","DOI":"10.1016\/j.tube.2021.102054","article-title":"Immunological characterization of chimeras of high specificity antigens from Mycobacterium tuberculosis H37Rv","volume":"127","author":"Fatma","year":"2021","journal-title":"Tuberculosis"},{"key":"ref_42","doi-asserted-by":"crossref","unstructured":"Yang, S.-J., Chen, Y.-Y., Hsu, C.-H., Hsu, C.-W., Chang, C.-Y., Chang, J.-R., and Dou, H.-Y. (2020). Activation of M1 Macrophages in Response to Recombinant TB Vaccines with Enhanced Antimycobacterial Activity. Front. Immunol., 11.","DOI":"10.3389\/fimmu.2020.01298"},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"102479","DOI":"10.1016\/j.tube.2024.102479","article-title":"The use of Mycobacterium tuberculosis H37Ra-infected immunocompetent mice as an in vivo model of persisters","volume":"145","author":"Kumari","year":"2024","journal-title":"Tuberculosis"},{"key":"ref_44","first-page":"e13409","article-title":"Ag85B with c-di-AMP as mucosal adjuvant showed immunotherapeutic effects on persistent Mycobacterium tuberculosis infection in mice","volume":"57","author":"Liang","year":"2024","journal-title":"Braz. J. Med. Biol. Res."}],"container-title":["Vaccines"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2076-393X\/14\/5\/442\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,5,18]],"date-time":"2026-05-18T15:00:03Z","timestamp":1779116403000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2076-393X\/14\/5\/442"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,5,15]]},"references-count":44,"journal-issue":{"issue":"5","published-online":{"date-parts":[[2026,5]]}},"alternative-id":["vaccines14050442"],"URL":"https:\/\/doi.org\/10.3390\/vaccines14050442","relation":{},"ISSN":["2076-393X"],"issn-type":[{"value":"2076-393X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,5,15]]}}}