---
title: "HPV mRNA vaccine candidate — article content not available for clinical details"
id: "frontiers-in-immunology-17-a-novel-hpv-mrna-vaccine-candidate-immunogenicity-and-definition-of-immune"
canonical_url: "https://medichelpline.com/clinical-feed/frontiers-in-immunology-17-a-novel-hpv-mrna-vaccine-candidate-immunogenicity-and-definition-of-immune"
content_type: "clinical_feed_article"
specialty: "Infectious Disease"
source_name: "Frontiers in Immunology"
source_url: "https://www.frontiersin.org/articles/10.3389/fimmu.2026.1925901"
published_at: "2026-10-21T00:00:00.000Z"
evidence_level: "Journal Feed"
license: "CC-BY-NC-4.0 / Informational Use"
---
# HPV mRNA vaccine candidate — article content not available for clinical details
## Provenance & Clinical Metadata
- **Canonical URL:** https://medichelpline.com/clinical-feed/frontiers-in-immunology-17-a-novel-hpv-mrna-vaccine-candidate-immunogenicity-and-definition-of-immune
- **Specialty:** [Infectious Disease](https://medichelpline.com/clinical-feed/infectious-disease.md)
- **Primary Source:** Frontiers in Immunology
- **Source URL:** [Original Journal Publication](https://www.frontiersin.org/articles/10.3389/fimmu.2026.1925901)
- **Published At:** 2026-10-21T00:00:00.000Z
- **Evidence Rating:** Journal Feed
## Executive GIST (TL;DR)
- The source article title and citation metadata were accessible, identifying a study of an **HPV** **mRNA vaccine** and its immunogenicity plus immune correlates, but the published page content with results and methods was not present in the provided source. - No cohort characteristics, dosing schedules, or administration routes were reported in the provided source material; investigators’ methods were not available to summarize. - The source did not provide immunogenicity data — antibody titers, T-cell responses, or timepoints — so no quantitative efficacy or immune-correlate statements can be extracted. - Safety and tolerability outcomes were absent from the supplied content; the provided source did not report adverse event incidence, severity grading, or related monitoring procedures. - Trial design details (randomization, control arms, blinding, endpoints) were not accessible in the source; the study phase and sample size also were not presented. - Regulatory context, next steps, or prespecified statistical analysis plans were not found in the supplied material; the article page appears truncated or inaccessible in the source snapshot. - The clinical feed and editorial summary below therefore focus on what should be reported for an **mRNA vaccine** clinical study and document the missing items explicitly, avoiding any unreported assumptions.
## Clinical Analysis & Structured Key Points
Frontiers | A novel HPV mRNA vaccine candidate: immunogenicity and definition of immune correlates of protection ORIGINAL RESEARCH article Front. Immunol. , 21 October 2026 Sec. Vaccines and Molecular Therapeutics Volume 17 - 2026 | https://doi.org/10.3389/fimmu.2026.1925901 Published in Frontiers in Immunology Vaccines and Molecular Therapeutics 7 impact factor 11.3 citescore Editor & Reviewers Edited by S R Srinivasa Reddy Bonam Reviewed by M R Moutih Rafei A D Anna Duarte Outline Figures and Tables Figure 1 View in article Figure 2 View in article Figure 3 View in article Figure 4 View in article Figure 5 View in article Figure 6 View in article ORIGINAL RESEARCH article Front. Immunol. , 21 October 2026 Sec. Vaccines and Molecular Therapeutics Volume 17 - 2026 | https://doi.org/10.3389/fimmu.2026.1925901 A novel HPV mRNA vaccine candidate: immunogenicity and definition of immune correlates of protection H H Hyogyeong Hwang 1 T A Timothy An 1 E S Eun-Hye Seo 2 P P Pil-Gu Park 3 K H Kee-Jong Hong 1,2,4 * 1. Department of Bio-Medical Science, GAIST, Gachon University, Incheon, Republic of Korea 2. Department of Microbiology, College of Medicine, Gachon University, Incheon, Republic of Korea 3. Department of Life Science, College of BioNano Technology, Gachon University, Seongnam, Republic of Korea 4. Lee Gil Ya Cancer and Diabetes Institute, Gachon University, Incheon, Republic of Korea See more Article metrics View details Abstract Human papillomavirus (HPV), the major cause of cervical cancer, continues to be associated with an increasing number of diagnosed cases despite the availability of licensed vaccines such as Cervarix™ and GARDASIL ® . In addition, cervical cancer incidence remains especially high in low- and middle-income countries, while vaccine coverage remains low due to high costs and instability in supply. We developed a novel HPV vaccine candidate using mRNA platform and to propose immune correlates of protection (CoP) for efficacy evaluation. A protection threshold was established based on GARDASIL ® 9, and the corresponding antibody titers were determined. These antibody titers were used as Reference for the immunogenicity analysis of the generated HPV16 mRNA. As a result, the vaccine was shown to induce a sufficiently significant level of protective efficacy. Furthermore, based on the immunogenicity data obtained from GARDASIL ® 9 and HPV16 mRNA, the minimum antibody titers required for protection were determined, thereby defining CoP for HPV vaccine. Additionally, HPV16 mRNA induced cellular immune responses associated with CD4- and CD8-specific cytokine secretion, suggesting potential protective mechanism in which cellular immunity complements humoral immunity. This study proposes an objective evaluation framework for comparing and analyzing HPV vaccine efficacy across different vaccine platforms. 1 Introduction Human Papillomavirus (HPV) is a non-enveloped virus with over 100 subtypes ( 1 ). It is primarily transmitted through sexual contact and can cause benign or malignant lesions ( 2 ). Persistent infection with HPV is the leading cause of cervical cancer in women, accounting for over 90% of cases ( 1 , 3 , 4 ). The major capsid protein L1 mediates HPV attachment to and entry into host cells and serves as the primary target of prophylactic vaccines ( 5 , 6 ). Commercially available HPV vaccines, such as Cervarix™ and GARDASIL ® , are recombinant protein vaccines using L1 virus-like particles (VLPs) ( 3 , 7 ). However, their high cost restricts vaccine uptake in low- and middle-income countries as well as in Korea, despite the rising incidence of cervical cancer ( 8 – 11 ). Therefore, the development of new platform-based HPV vaccines capable of overcoming the limitations of existing vaccines is required. mRNA vaccines began to attract significant attention during COVID-19 pandemic as their efficacy and efficiency were demonstrated ( 12 ). This platform can be rapidly designed and manufactured at large scale, which may enable them to be cost-effective ( 13 ). In addition, mRNA vaccines also have the advantage of enhancing vaccine efficacy through the intrinsic immunostimulatory effects of nucleic acids, without the need for additional adjuvants ( 14 , 15 ). HPV VLPs exhibit type-specific surface structural characteristics, and accordingly, the optimal conditions required to efficiently induce self-assembly may vary among HPV types ( 16 , 17 ). Therefore, individual process development and optimization may be required for each HPV type. These characteristics can lead to increased manufacturing costs as well as prolonged development and production timelines. In contrast, once mRNA vaccine platform has been established, new vaccines can be readily designed by simply replacing the genetic sequence encoding the target HPV type, thereby offering temporal and economic advantages over VLP-based vaccines in terms of development and manufacturing. Based on the advantages of mRNA vaccines, we established an mRNA-based platform as a novel HPV vaccine candidate to overcome the limitations of existing HPV vaccines. Currently, the efficacy of HPV vaccines is primarily evaluated based on the incidence of cervical precancerous lesions, including cervical intraepithelial neoplasia grades 2 and 3 (CIN2/3) and adenocarcinoma in situ (AIS) ( 18 ). However, progression from HPV infection to cervical cancer typically requires several years, and more than 90% of HPV infections resolve spontaneously within two years ( 18 – 20 ). Consequently, efficacy assessment based on disease incidence necessitates long-term follow-up, leading to prolonged study durations as well as increased costs and logistical complexity. Moreover, because HPV infection also causes genital diseases in men, disease-based endpoints focused on cervical lesions may not fully capture the overall protective efficacy of HPV vaccines ( 21 ). Accordingly, using GARDASIL ® 9 as Reference, we defined the minimum antibody titer required to inhibit viral infection and established antibody quantification methods based on ELISA and neutralization assay. In HPV infection, as protective antibodies are known to recognize conformational epitopes formed by the capsomere structure composed of L1 pentamers, virus-like particles (VLPs) with intact conformational structures were used as antigens in ELISA ( 22 , 23 ). Neutralizing antibody responses were assessed using luciferase-based pseudovirus neutralization assay (PBNA) to ensure robustness and experimental convenience ( 24 , 25 ). To establish a standardized approach for evaluating the efficacy of HPV vaccine candidates, these assays were standardized to quantitatively assess vaccine-induced binding and neutralizing antibody responses. By relating these antibody responses to protection in vivo HPV PsV challenge model, antibody-based immune correlates of protection (CoP) were established to provide quantitative criteria for evaluation of HPV vaccine efficacy. In this study, we established standardized assays and CoP for the evaluation of HPV vaccine efficacy and applied these to a novel HPV mRNA vaccine candidate to assess its immunogenicity. In addition to antibody responses, based on previous studies demonstrating that mRNA vaccines elicit both humoral and cellular immune responses, T cell–mediated immunity was additionally evaluated ( 12 , 13 ). Notably, mRNA vaccines have been reported to preferentially induce Th1-biased immune responses ( 26 , 27 ). To evaluate these immunological characteristics, antigen-specific IgG subclass analysis was performed. Collectively, this study supports the reliability of the proposed CoP and analytical assays and provide a quantitative framework for the objective assessment of HPV vaccine candidates, including mRNA-based platforms. 2 Materials and methods 2.1 Preparation and characterization of mRNA vaccine HPV16 L1 sequence (accession number: P03101) from p16sheLL plasmid, which is commonly used for PsV production, was used as a template in this study ( 28 ). CUK3–1 plasmid vector was provided by Catholic University (Seoul, Korea). The target coding region was inserted into CUK3–1 plasmid vector. This vector included T7 promoter, 5’- and 3’-untranslated regions (UTRs), and a 100-nucleotide poly-A tail necessary for in vitro transcription (IVT). To improve mRNA stability and translational efficiency, a 20-nucleotides linker element (A50L50LO) was incorporated within the poly-A tail ( 29 ). The plasmids were linearized by restriction enzyme digestion for 2 hours at 37 °C and purified using QIAquick PCR & Gel clean up kit (QIAGEN, Hilden, Germany) prior to IVT. mRNA was synthesized using HiScribe ® T7 High Yield RNA Synthesis Kit (NEB, Ipswich, USA) with CleanCap (TriLink, San Diego, USA) to enhance stability. Synthesized mRNA was purified by lithium chloride (LiCl) precipitation and treated with double-stranded RNA removal kit (CELLSCRIPT, Madison, USA). mRNA concentration was measured using NanoDrop2000 v1.5 (Thermofisher, Waltham, USA). 2.2 Denaturing agarose gel electrophoresis mRNA samples were mixed with RNA loading dye and heated at 70 °C for 10 minutes. RNA (100 ng per lane) and ladder were loaded onto a 1.2% agarose gel containing 2.2 M formaldehyde in 1X MOPS-EDTA-Sodium Acetate (MESA) buffer (Sigma-Aldrich, St. Louis, USA) and electrophoresed at 100 V for approximately 35 minutes. The images were acquired using Gel Doc (Bio-Rad, Hercules, USA) and analyzed with Image Lab v5.2. 2.3 Dot blot 500 ng of RNA was spotted onto positively charged nylon membrane (Sigma-Aldrich, St. Louis, USA) and allowed to air-dry. The membrane was blocked with 5% skim milk in TBST for 1 hour, followed by incubation with J2 anti-dsRNA antibody (Cell Signaling Technology, Danvers, USA) (1:1,000 in 1% skim milk in TBST) for 1 hour. After three washing with TBST, the membrane was incubated with HRP-conjugated anti-mouse IgG secondary antibody (1:3,000 in 1% skim milk in TBST) for 1 hour. Signals were detected using ECL substrate and quantified using Image J v1.54g. 2.4 Cell transfection HEK293TT cells, provided by Yonsei University (Seoul, Korea) were cultured in DMEM with 10% FBS, 1% NEAA, 1% Glutamax, and 1% antibiotic-antimycotic. Cells at 70–80% confluency were transfected with 4 μg of mRNA using Lipofectamine 2000 (Invitrogen, Waltham, USA). 24 hours post-transfection, cells were lysed with RIPA buffer containing protease inhibitors. Protein concentration was quantified via BCA assay. 2.5 Western blot Proteins (10 μg) were mixed with loading buffer and denatured at 95 °C for 5 minutes. Denatured samples were separated by 10% SDS-PAGE and transferred to nitrocellulose membranes using iBlot2 (Invitrogen, Waltham, USA). Membranes were blocked with 5% skim milk in TBST for 1 hour at room temperature. HPV pan-specific primary antibodies (Invitrogen, Waltham, USA) were diluted 1:1,000 in 5% BSA, 1% sodium azide in TBST and incubated overnight at 4 °C. HRP-conjugated secondary antibodies were diluted 1:10,000 in 5% skim milk and incubated 1 hour at room temperature. Protein bands were visualized with ECL substrate by Amersham™ ImageQuant™ 800 (Cytiva, Marlborough, USA). 2.6 Lipid nanoparticle encapsulation of mRNA vaccine LNP encapsulation was performed at Seoul National University Hospital (Seoul, Korea). Lipid composition included SM-102 (ionizable lipids) (MedChemExpress, Monmouth Junction USA), DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), cholesterol and DMG-PEG2000 (1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000) at a molar ratio of 50:10:38.5:1.5. mRNA was mixed with lipids in 50 mM sodium citrate buffer (pH 4.0), and LNPs were generated using enCELL-Master V2 (ENPARTICLE, Busan, Korea) under a 3:1 aqueous-to-organic flow ratio at a total flow rate of 10 mL/min. The resulting LNPs were subsequently concentrated and transferred into PBS using Amicon Ultra centrifugal filter units (Merck, Darmstadt, Germany). For quality control, the particle size, polydispersity index (PDI), zeta potential, and encapsulation efficiency (EE) of LNP-formulated HPV16 L1 mRNA were characterized. Particle size, PDI, and zeta potential were measured by dynamic light scattering (DLS) using Zetasizer Nano ZS (Malvern Panalytical, Malvern, UK). EE was determined using ribogreen assay. 2.7 Immunization and sample collection BALB/c female mice (6 weeks old) received intramuscular injections of either GARDASIL ® 9 (MSD, New Jersey, USA) or LNP–encapsulated mRNA encoding HPV16 L1. Immunizations were administered at a dose of 1, 5, 10 µg on days 0, 14, and 28, while control animals were injected with PBS. Blood samples were obtained every two weeks via retro-orbital collection under inhalational anesthesia using 1–5% isoflurane delivered with oxygen. Two weeks following the final dose, animals were exposed to HPV pseudovirus (PsV) and splenocytes were collected. All animal procedures were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of the Center of Animal Care and Use (CACU), Lee Gil Ya Cancer and Diabetes Institute (Incheon, Korea) (approval number: LCDI-2024-0080). 2.8 HPV16 PsV challenge in mice HPV PsV infection procedure was adapted from a previously described protocol ( 30 ). This model has been previously validated as a quantitative platform for assessing in vivo protective efficacy of HPV vaccine candidates ( 31 – 33 ). PsVs were provided by Yonsei University (Seoul, Korea). Mice received 100 μL of 30 mg/mL Depo-Provera (Sigma-Aldrich, St. Louis, USA) subcutaneously. PsVs were mixed 1:1 (v/v) with 3% carboxymethylcellulose (CMC). Three days later, mice were anesthetized with Isoflurane, and 20 μL of PsVs-3% CMC mixture was administered intravaginally using positive-displacement pipette. A pap-brush was inserted into the vagina and rotated firmly 10–20 times until light reddish blood appeared to induce epithelial abrasion, after which an additional 20 μL of the mixture was administered intravaginally. Three days post-infection, 20 μL of 7.8 mg/mL firefly luciferin (Revvity, Waltham, USA) was administered intravaginally, and infection was monitored using in vivo imaging system (IVIS) (Spectral Instruments Imaging, Tucson, USA). Images were analyzed using AURA v4.0.8. 2.9 Virus-like particle ELISA VLPs were provided by Yonsei University (Seoul, Korea). ELISA plates (Greiner Bio-One, Kremsmünster, Austria) were coated with 100 μL/well of HPV16 VLP diluted in PBS at a concentration of 25 ng/well and incubated overnight at 4 °C. After three washes with PBS, wells were blocked with 150 μL/well of 1% BSA/PBS and incubated at 37 °C for 2 hours. Following three additional PBS washes, serum samples diluted in 1% BSA/PBS were added at 100 μL/well and incubated overnight at 4 °C. Mouse sera were diluted 1:100, and standard serum (10/140, NIBSC, Potters Bar, UK) was diluted 1:200 and serially diluted 2-fold for 8 points. After four washes with PBST, HRP-conjugated secondary antibodies diluted in PBST were added at 100 μL/well and incubated at 37 °C for 2 hours. Mouse IgG secondary antibody (SouthernBiotech, Birmingham, USA) was diluted 1:5,000, while mouse IgG1 and IgG2a secondary antibodies (Cell Signaling Technology, Danvers, USA) and human secondary antibody (Cell Signaling Technology, Danvers, USA) were diluted 1:3,000. After five PBS washes, 100 μL/well of TMB substrate solution was added and incubated at room temperature in the dark for 15 minutes. The reaction was terminated with 50 μL/well of TMB stop solution, and absorbance was measured at 450 nm using a microplate reader. Data were acquired using SoftMax Pro v5.4.1. For quantification of binding antibody levels, the standard serum was assigned concentration of 1,000 AU/mL, and serial dilutions were used to generate standard curve. Standard curve was fitted using a sigmoidal, four-parameter logistic (4PL) model with X defined as log(concentration) in GraphPad Prism v8.0.2. Sample antibody concentrations were calculated by interpolating the corresponding OD values from the fitted standard curve and expressed as AU/mL. 2.10 Pseudovirus-based neutralization assay HEK293TT cells were seeded in 96-well flat-bottom plates at 3×10 4 cells/well in 100 μL of culture media (DMEM supplemented with 10% FBS, 1% NEAA, 1% Glutamax, 1% antibiotic-antimycotic) and incubated for 5 hours at 37 °C with 5% CO 2 . Serum samples were diluted 1:40 in 8-point, 4-fold series, while standard serum (10/140, NIBSC, Potters Bar, UK) was diluted 1:10 in 8-point, 4-fold series. PsV was diluted in media to 300 TCID50/well. Serum and PsV mixtures (1:4, v/v) were incubated at 4 °C for 1 hour, then added to HEK293TT cells. Following 72 hours of incubation at 37 °C, 5% CO 2 , 100 μL of media was removed and replaced with 100 μL of ONE-Glo luciferin (Promega, Madison, USA) diluted 1:3 in distilled water. Plates were incubated at room temperature for 3 minutes, resuspended 10–15 times to detach cells, and 150 μL transferred to 96-well flat-bottom black plates, avoiding bubbles. Luciferase signals were measured using luminometer (measurement interval, 0.3 s). Neutralization curves were fitted using four-parameter logistic (4PL) nonlinear regression model with variable slope, and IC50 values were calculated from the fitted curves using GraphPad Prism v8.0.2. Neutralization (%) was calculated using the following formula. IU/mL values were calculated using the same standard curve-based method described for ELISA, with antibody concentrations expressed in IU/mL rather than AU/mL. 2.11 Intracellular cytokine staining Mouse splenocytes were prepared by crushing spleens through 70 μm cell strainers and washed with PBS. Cells were centrifuged at 1600 rpm for 5 minutes, resuspended, and RBCs lysed with pre-warmed lysis buffer at 37 °C for 5 minutes. Cells were resuspended in RPMI with 10% FBS and counted. For antigen-specific intracellular cytokine analysis, splenocytes (1×10 6 cells/well) were stimulated with HPV16 L1 peptides (PM-HPV16-L1, JPT Peptide Technologies, Berlin, Germany) 1 μg/mL and Golgi-plug 1 μL/mL for 6 hours at 37 °C with 5% CO 2 . Cells were stained for surface markers CD4-FITC and CD8-PE (BD Biosciences, Franklin Lakes, USA), followed by intracellular cytokines IFNγ-BV421 (BioLegend, San Diego, USA), TNFα-APC-Cy7, and IL-2-PerCP-Cy5.5 (BD Biosciences, Franklin Lakes, USA) using Cytofix/Cytoperm™ Plus kit (BD Biosciences, Franklin Lakes, USA). All antibodies were diluted at 1:100, and staining was performed at 4 °C for 30 minutes. Data were acquired using BD LSRII and analyzed with FlowJo v10 (BD Biosciences, Franklin Lakes, USA). 2.12 Enzyme-linked immunospot Mouse IFN-γ ELISpot assays were performed according to the manufacturer’s instructions (R&D Systems, Minnesota, USA). Briefly, ELISpot plates were pre-equilibrated by adding 200 μL/well of RPMI supplemented with 10% FBS and 1% antibiotic–antimycotic solution and incubating at room temperature for 20 minutes. After removal of the medium, mouse splenocytes were seeded at 1×10 5 cells/well in 50 μL and stimulated with HPV16 L1 peptides (JPT Peptide Technologies, Berlin, Germany) 1 μg/mL at 37 °C in 5% CO 2 for 20 hours. Following incubation, plates were washed four times with 300 μL/well of wash buffer. After the final wash, plates were inverted and tapped dry on paper towels. Streptavidin-AP concentrate A (100 μL/well) was added and incubated overnight at 4 °C. Plates were then washed four times with wash buffer, followed by the addition of BCIP/NBT substrate (100 μL/well). Plates were incubated for 1 hours at room temperature in the dark. After terminating the reaction, plates were washed four times with distilled water. Plates were inverted to remove residual liquid and the plastic underdrain was removed. After complete air-drying at room temperature, spots were analyzed using an ELISpot reader (AID autoimmun diagnostika gmbh, Straβberg, Germany). For analysis, unstimu
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