{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,20]],"date-time":"2026-08-20T09:24:33Z","timestamp":1787217873348,"version":"build-2736575974"},"reference-count":40,"publisher":"MDPI AG","issue":"8","license":[{"start":{"date-parts":[[2026,8,19]],"date-time":"2026-08-19T00:00:00Z","timestamp":1787097600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Pinion Immunotherapeutics"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Vaccines"],"abstract":"<jats:p>Background\/Objective: Persistent infection with human papillomavirus type 16 (HPV-16) is the principal cause of cervical high-grade squamous intraepithelial lesions (cHSIL) and cervical cancer, yet the established treatments remain limited to ablative or excisional procedures that carry reproductive risk and do not eliminate the underlying infection. We report the preclinical development of ARV-2001, a messenger RNA (mRNA)\u2013lipid nanoparticle (LNP) immunotherapeutic encoding mutated, non-oncogenic HPV-16 E6 and E7 fused to a SARS-CoV-2 spike S2 subdomain enriched in human CD4 helper epitopes, formulated in a novel cholesterol-derived ionizable lipid (ARV-T1). Methods: Interactions of ARV-2001-expressed antigens with p53 and retinoblastoma (Rb) were evaluated in human cervical carcinoma cell line C33A, in lentiviral constructs in primary human keratinocytes, and in soft-agar colony-formation assays. ARV-2001 was administrated by intramuscular (IM) or intradermal (ID) injection in naive mice or in the TC-1 tumor models. Tumor size and survival were monitored over time and tumor-infiltrated lymphocytes were characterized by flow cytometry. Intracellular cytokine staining and Elispot were used to evaluate immunogenicity. Results: In vitro, the mutated E6\/E7\u2013S2 antigen lost the ability to degrade p53, to deregulate the retinoblastoma (Rb) pathway, and to support anchorage-independent growth, suggesting abrogation of oncogenic activity. The S2 domain and imiquimod administration each augmented antitumor activity and intratumoral CD8+ T-cell infiltration while reducing myeloid-derived suppressor cells in the syngeneic HPV-16 E6\/E7 TC-1 tumor models. In addition, ID administration of ARV-2001 into TC-1 tumor-bearing mice was superior to IM administration in terms of both tumor growth inhibition and survival. ID vaccination with ARV-2001 in mice consistently elicited a more potent E6\/E7-specific T-cell response than the same dose given IM. Dose-escalation studies showed a dose-dependent T cell response against E6\/E7 in ID-injected mice. Conclusions: This study supports future human evaluation of intradermally administrated ARV-2001 for treatment of HPV-16+ cHSIL in clinical trials.<\/jats:p>","DOI":"10.3390\/vaccines14080714","type":"journal-article","created":{"date-parts":[[2026,8,19]],"date-time":"2026-08-19T13:14:01Z","timestamp":1787145241000},"page":"714","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Preclinical Development of ARV-2001, an Intradermally Administered mRNA\u2013Lipid Nanoparticle Immunotherapeutic for the Treatment of HPV-16-Positive Cervical High-Grade Squamous Intraepithelial Lesions"],"prefix":"10.3390","volume":"14","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-8961-859X","authenticated-orcid":false,"given":"Zhengxiang","family":"He","sequence":"first","affiliation":[{"name":"Pinion Immunotherapeutics, North Bethesda, MD 20852, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Huabin","family":"Zhu","sequence":"additional","affiliation":[{"name":"Pinion Immunotherapeutics, North Bethesda, MD 20852, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ju Hyeong","family":"Jeon","sequence":"additional","affiliation":[{"name":"Pinion Immunotherapeutics, North Bethesda, MD 20852, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5687-6154","authenticated-orcid":false,"given":"Jianzhu","family":"Chen","sequence":"additional","affiliation":[{"name":"Department of Biology and Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Gregory M.","family":"Glenn","sequence":"additional","affiliation":[{"name":"Pinion Immunotherapeutics, North Bethesda, MD 20852, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6817-1875","authenticated-orcid":false,"given":"Renhuan","family":"Xu","sequence":"additional","affiliation":[{"name":"Pinion Immunotherapeutics, North Bethesda, MD 20852, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,8,19]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"16086","DOI":"10.1038\/nrdp.2016.86","article-title":"Carcinogenic human papillomavirus infection","volume":"2","author":"Schiffman","year":"2016","journal-title":"Nat. Rev. Dis. Primers"},{"key":"ref_2","first-page":"209","article-title":"Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries","volume":"71","author":"Sung","year":"2021","journal-title":"CA Cancer J. Clin."},{"key":"ref_3","doi-asserted-by":"crossref","unstructured":"Yousefi, Z., Aria, H., Ghaedrahmati, F., Bakhtiari, T., Azizi, M., Bastan, R., Hosseini, R., and Eskandari, N. (2021). An Update on Human Papilloma Virus Vaccines: History, Types, Protection, and Efficacy. Front. Immunol., 12.","DOI":"10.3389\/fimmu.2021.805695"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"1266","DOI":"10.5858\/arpa.LGT200570","article-title":"The Lower Anogenital Squamous Terminology Standardization Project for HPV-Associated Lesions: Background and consensus recommendations from the College of American Pathologists and the American Society for Colposcopy and Cervical Pathology","volume":"136","author":"Darragh","year":"2012","journal-title":"Arch. Pathol. Lab. Med."},{"key":"ref_5","first-page":"CD001318","article-title":"Surgery for cervical intraepithelial neoplasia","volume":"2013","author":"Paraskevaidis","year":"2013","journal-title":"Cochrane Database Syst. Rev."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"i3633","DOI":"10.1136\/bmj.i3633","article-title":"Adverse obstetric outcomes after local treatment for cervical preinvasive and early invasive disease according to cone depth: Systematic review and meta-analysis","volume":"354","author":"Kyrgiou","year":"2016","journal-title":"BMJ"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"211","DOI":"10.1002\/ijc.26349","article-title":"Long-term risk of recurrent cervical human papillomavirus infection and precancer and cancer following excisional treatment","volume":"131","author":"Kreimer","year":"2012","journal-title":"Int. J. Cancer"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"215","DOI":"10.1128\/CMR.05028-11","article-title":"Epithelial cell responses to infection with human papillomavirus","volume":"25","author":"Stanley","year":"2012","journal-title":"Clin. Microbiol. Rev."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"97","DOI":"10.1016\/j.it.2010.12.006","article-title":"Immunotherapy for persistent viral infections and associated disease","volume":"32","author":"Arens","year":"2011","journal-title":"Trends Immunol."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"1164","DOI":"10.1016\/j.cell.2017.08.001","article-title":"HPV16 E7 Genetic Conservation Is Critical to Carcinogenesis","volume":"170","author":"Mirabello","year":"2017","journal-title":"Cell"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"550","DOI":"10.1038\/nrc2886","article-title":"Human papillomavirus oncoproteins: Pathways to transformation","volume":"10","author":"Moody","year":"2010","journal-title":"Nat. Rev. Cancer"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"1838","DOI":"10.1056\/NEJMoa0810097","article-title":"Vaccination against HPV-16 oncoproteins for vulvar intraepithelial neoplasia","volume":"361","author":"Kenter","year":"2009","journal-title":"N. Engl. J. Med."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"2342","DOI":"10.1158\/1078-0432.CCR-15-2594","article-title":"Vaccination against Oncoproteins of HPV16 for Noninvasive Vulvar\/Vaginal Lesions: Lesion Clearance Is Related to the Strength of the T-Cell Response","volume":"22","author":"Welters","year":"2016","journal-title":"Clin. Cancer Res."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"2078","DOI":"10.1016\/S0140-6736(15)00239-1","article-title":"Safety, efficacy, and immunogenicity of VGX-3100, a therapeutic synthetic DNA vaccine targeting human papillomavirus 16 and 18 E6 and E7 proteins for cervical intraepithelial neoplasia 2\/3: A randomised, double-blind, placebo-controlled phase 2b trial","volume":"386","author":"Trimble","year":"2015","journal-title":"Lancet"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"e069616","DOI":"10.1136\/bmjopen-2022-069616","article-title":"Efficacy and safety of therapeutic HPV vaccines to treat CIN 2\/CIN 3 lesions: A systematic review and meta-analysis of phase II\/III clinical trials","volume":"13","author":"Zhang","year":"2023","journal-title":"BMJ Open"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"261","DOI":"10.1038\/nrd.2017.243","article-title":"mRNA vaccines\u2014A new era in vaccinology","volume":"17","author":"Pardi","year":"2018","journal-title":"Nat. Rev. Drug Discov."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"S6","DOI":"10.1067\/mjd.2000.107808","article-title":"Immunomodulatory and pharmacologic properties of imiquimod","volume":"43","author":"Sauder","year":"2000","journal-title":"J. Am. Acad. Dermatol."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"1441","DOI":"10.1084\/jem.20070021","article-title":"Tumoricidal activity of TLR7\/8-activated inflammatory dendritic cells","volume":"204","author":"Stary","year":"2007","journal-title":"J. Exp. Med."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"463","DOI":"10.1038\/nature11522","article-title":"A vaccine strategy that protects against genital herpes by establishing local memory T cells","volume":"491","author":"Shin","year":"2012","journal-title":"Nature"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1038\/s41541-023-00785-w","article-title":"Immunogenicity and reactogenicity of intradermal mRNA-1273 SARS-CoV-2 vaccination: A non-inferiority, randomized-controlled trial","volume":"9","author":"Prins","year":"2024","journal-title":"npj Vaccines"},{"key":"ref_21","doi-asserted-by":"crossref","unstructured":"Rosenbaum, P., Tchitchek, N., Joly, C., Rodriguez Pozo, A., Stimmer, L., Langlois, S., Hocini, H., Gosse, L., Pejoski, D., and Cosma, A. (2021). Vaccine Inoculation Route Modulates Early Immunity and Consequently Antigen-Specific Immune Response. Front. Immunol., 12.","DOI":"10.3389\/fimmu.2021.645210"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"9389","DOI":"10.2147\/IJN.S527822","article-title":"Lipid Nanoparticles Formulated with a Novel Cholesterol-Tailed Ionizable Lipid Markedly Increase mRNA Delivery Both in vitro and in vivo","volume":"20","author":"Jeon","year":"2025","journal-title":"Int. J. Nanomed."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"e39","DOI":"10.1002\/cpz1.39","article-title":"Cap 1 Messenger RNA Synthesis with Co-transcriptional CleanCap\u00ae Analog by In Vitro Transcription","volume":"1","author":"Henderson","year":"2021","journal-title":"Curr. Protoc."},{"key":"ref_24","doi-asserted-by":"crossref","unstructured":"Qin, J., Jeon, J.H., Xu, J., Langston, L.K., Marasini, R., Mou, S., Montoya, B., Melo-Silva, C.R., Jeon, H.J., and Zhu, T. (2023). Design and preclinical evaluation of a universal SARS-CoV-2 mRNA vaccine. Front. Immunol., 14.","DOI":"10.3389\/fimmu.2023.1126392"},{"key":"ref_25","doi-asserted-by":"crossref","unstructured":"Chen, L., He, Z., Qin, L., Li, Q., Shi, X., Zhao, S., Chen, L., Zhong, N., and Chen, X. (2011). Antitumor effect of malaria parasite infection in a murine Lewis lung cancer model through induction of innate and adaptive immunity. PLoS ONE, 6.","DOI":"10.1371\/journal.pone.0024407"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"62","DOI":"10.1097\/CJI.0000000000000156","article-title":"DNA Vaccine Encoding HPV16 Oncogenes E6 and E7 Induces Potent Cell-mediated and Humoral Immunity Which Protects in Tumor Challenge and Drives E7-expressing Skin Graft Rejection","volume":"40","author":"Chandra","year":"2017","journal-title":"J. Immunother."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"2650","DOI":"10.1128\/jvi.63.6.2650-2656.1989","article-title":"A point mutational analysis of human papillomavirus type 16 E7 protein","volume":"63","author":"Edmonds","year":"1989","journal-title":"J. Virol."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"145709","DOI":"10.1016\/j.gene.2021.145709","article-title":"Degradation of p53 by HPV16-E6 variants isolated from cervical cancer specimens of Moroccan women","volume":"791","author":"Hadami","year":"2021","journal-title":"Gene"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"118","DOI":"10.1016\/j.virol.2009.10.018","article-title":"Destabilization of Rb by human papillomavirus E7 is cell cycle dependent: E2-25K is involved in the proteolysis","volume":"396","author":"Oh","year":"2010","journal-title":"Virology"},{"key":"ref_30","first-page":"21","article-title":"Treatment of established tumors with a novel vaccine that enhances major histocompatibility class II presentation of tumor antigen","volume":"56","author":"Lin","year":"1996","journal-title":"Cancer Res."},{"key":"ref_31","doi-asserted-by":"crossref","unstructured":"Pateev, I., Seregina, K., Ivanov, R., and Reshetnikov, V. (2023). Biodistribution of RNA Vaccines and of Their Products: Evidence from Human and Animal Studies. Biomedicines, 12.","DOI":"10.3390\/biomedicines12010059"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"1105","DOI":"10.1016\/j.ymthe.2025.01.008","article-title":"Visualizing lipid nanoparticle trafficking for mRNA vaccine delivery in non-human primates","volume":"33","author":"Buckley","year":"2025","journal-title":"Mol. Ther."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"103479","DOI":"10.1016\/j.isci.2021.103479","article-title":"The mRNA-LNP platform\u2019s lipid nanoparticle component used in preclinical vaccine studies is highly inflammatory","volume":"24","author":"Ndeupen","year":"2021","journal-title":"iScience"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"101402","DOI":"10.1016\/j.omtm.2024.101402","article-title":"Reducing off-target expression of mRNA therapeutics and vaccines in the liver with microRNA binding sites","volume":"33","author":"Parrett","year":"2025","journal-title":"Mol. Ther. Methods Clin. Dev."},{"key":"ref_35","doi-asserted-by":"crossref","unstructured":"Hettinga, J., and Carlisle, R. (2020). Vaccination into the Dermal Compartment: Techniques, Challenges, and Prospects. Vaccines, 8.","DOI":"10.3390\/vaccines8030534"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"e52","DOI":"10.3802\/jgo.2019.30.e52","article-title":"Human papillomavirus genotype-specific risk in cervical carcinogenesis","volume":"30","author":"So","year":"2019","journal-title":"J. Gynecol. Oncol."},{"key":"ref_37","first-page":"CD008054","article-title":"Human papillomavirus testing versus repeat cytology for triage of minor cytological cervical lesions","volume":"2013","author":"Arbyn","year":"2013","journal-title":"Cochrane Database Syst. Rev."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"1231","DOI":"10.1038\/modpathol.2010.109","article-title":"Local immune response in the microenvironment of CIN2-3 with and without spontaneous regression","volume":"23","author":"Ovestad","year":"2010","journal-title":"Mod. Pathol."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"4358","DOI":"10.1128\/jvi.68.7.4358-4368.1994","article-title":"Progressive squamous epithelial neoplasia in K14-human papillomavirus type 16 transgenic mice","volume":"68","author":"Arbeit","year":"1994","journal-title":"J. Virol."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"27","DOI":"10.4103\/0976-0105.177703","article-title":"A simple practice guide for dose conversion between animals and human","volume":"7","author":"Nair","year":"2016","journal-title":"J. Basic Clin. Pharm."}],"container-title":["Vaccines"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2076-393X\/14\/8\/714\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,8,20]],"date-time":"2026-08-20T08:38:34Z","timestamp":1787215114000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2076-393X\/14\/8\/714"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,8,19]]},"references-count":40,"journal-issue":{"issue":"8","published-online":{"date-parts":[[2026,8]]}},"alternative-id":["vaccines14080714"],"URL":"https:\/\/doi.org\/10.3390\/vaccines14080714","relation":{},"ISSN":["2076-393X"],"issn-type":[{"value":"2076-393X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,8,19]]}}}