Development of assays and immune correlates to analyze efficacy of the novel HPV mRNA vaccines

다국어 초록 (Multilingual Abstract)

Development of assays and immune correlates to analyze efficacy of the novel HPV mRNA vaccines Hyogyeong Hwang Dept. of Health Sciences and Technology Graduate School of Gachon University Human papillomavirus (HPV) comprises more than 100 subtypes, and persistent infection with certain high-risk HPV types can progress to malignancy. Although commercial vaccines such as Cervarix™ (GSK) and GARDASIL® (MSD) are available, domestic vaccination rates remain low due to high costs and supply instability. To address these limitations, this study aimed to develop mRNA vaccine targeting the major capsid protein L1 of HPV and to establish standardized assays and immune correlates of protection for evaluating vaccine efficacy. To this end, VLP ELISA and pseudovirus- based neutralization assay (PBNA) were standardized, and a mouse model of pseudovirus (PsV) infection was established. Quantitative assays for measuring binding and neutralizing antibodies were developed using reference standards, and PsV infection in mice was quantitatively validated using in vivo imaging system (IVIS). Based on GARDASIL® 9, protective threshold was determined, and the corresponding antibody levels were yielding IgG titer 1.0 IU/mL and NT50 (50% neutralizing titer) 0.07 IU/mL as proposed immune correlates of protection. Upon application of the assays to sera from individuals vaccinated with GARDASIL® 9, antibody levels were comparable to those observed in vaccinated mice, demonstrating the clinical applicability of the established assays. For new vaccine development, HPV16 L1 mRNA was synthesized and formulated into lipid nanoparticles (LNPs) to produce a vaccine candidate, and its immunogenicity was evaluated using the established assays. mRNA vaccine induced IgG and neutralizing antibody responses comparable to those elicited by GARDASIL® 9 and demonstrated significant protective effects in the PsV infection mouse model. Furthermore, cellular immune responses marked by IFN-γ and TNF-α secretion were observed, suggesting a complementary protective mechanism alongside humoral immunity. In conclusion, this study established assays and immune correlates for assessing the efficacy of the novel HPV mRNA vaccine, providing a foundational framework that may facilitate future domestic HPV vaccine development and its clinical application.
Keywords: Human Papillomavirus, efficacy evaluation assays, immune correlates of protection, mRNA vaccine

국문 초록 (Abstract)

HPV (Human Papillomavirus)는 100가지가 넘는 아형이 존재하며, 일부 고위험군 아형의 지속 감염은 악성 종양을 유발할 수 있다. 현재 CervarixTM (GSK)와 GARDASIL® (MSD)과 같은 백신이 상용화되어 있으나, 고가의 접종 비용과 공급 불안정으로 인해 국내 접종률은 낮은 수준에 머물러 있다. 이에 본 연구는 HPV의 주요 캡시드 단백질인 L1을 표적으로 한 mRNA 백신을 개발하고, 이를 평가하기 위해 표준화된 평가법과 면역지표를 제시하고자 했다. 이를 위해 VLP ELISA 및 pseudovirus-based neutralization assay (PBNA)를 표준화하고, pseudovirus (PsV) 감염 마우스 모델을 구축하였다. 표준물질을 이용하여 결합항체 및 중화항체의 정량법을 확립하였으며, IVIS 이미징을 통해 마우스에서 PsV 감염 여부를 정량적으로 검증하였다. 또한, GARDASIL® 9를 기준으로 보호 역치를 설정하고, 해당 역치에 대응하는 항체가를 IgG 1.0 IU/mL, NT50 0.07 IU/mL로 산정하여 면역지표로 제시하였다. 확립된 평가법을 GARDASIL® 9 접종한 사람의 혈청에 적용한 결과, 마우스 혈청의 항체가와 유사한 수준으로 관찰되었다. 이는 본 평가법의 임상적 적용 가능성을 확인하였다. 새로운 HPV 백신 개발을 위해 HPV16 L1 mRNA를 합성하고 LNP에 제형화하여 백신 후보를 제작하였으며, 확립한 평가법을 이용해 면역원성을 분석하였다. 그 결과, 본 백신은 GARDASIL® 9과 유사한 수준의 IgG 및 중화항체 반응을 유도하였고, PsV 감염 모델에서도 유의한 보호 효과를 보였다. 또한 IFN-γ, TNF-α 분비를 포함한 세포성 면역 반응이 유도되어 체액성 면역을 보완하는 보호 기전 가능성을 시사하였다. 종합적으로, 본 연구는 새로운 HPV mRNA 백신의 효능 평가를 위한 평가법과 면역지표를 확립하였으며, 향후 국내 HPV 백신 개발 및 임상 적용을 위한 기초 자료로 활용될 수 있다는 점에서 의의를 가진다.
주요어: Human Papillomavirus, 평가법, 면역지표, mRNA 백신

목차 (Table of Contents)

• 1. Abstract 1
• 2. Contents 3
• 2.1 List of Tables 6
• 2.2 List of Figures 7
• 3. Introduction 8
• 4. Materials & Methods 12
• 4.1 Ethics, animals, immunization and infection model
• 4.1.1 Ethics statement 12
• 4.1.2 Mice and immunization 12
• 4.1.3 HPV pseudovirus (PsV) infection model 13
• 4.2 mRNA vaccine preparation
• 4.2.1 mRNA construct design. 14
• 4.2.2 In vitro transcription (IVT) and purification 16
• 4.2.3 Denaturing agarose gel electrophoresis 16
• 4.2.4 Dot blot 17
• 4.2.5 LNP encapsulation 17
• 4.3 In vitro expression analysis
• 4.3.1 Cell transfection 18
• 4.3.2 Western blot 19
• 4.4 Antibody-based assays
• 4.4.1 VLP ELISA 20
• 4.4.2 Pseudovirus-based neutralization assay (PBNA) 23
• 4.5 Immune response profiling
• 4.5.1 Splenocyte preparation 26
• 4.5.2 Intracellular cytokine staining 26
• 4.6 Statistical analysis 27
• 5. Results 28
• 5.1 HPV PsV-based mouse infection model 28
• 5.2 Establishment of immune correlates of protection for HPV vaccines 31
• 5.3 Evaluation of HPV mRNA vaccine efficacy
• 5.3.1 Construction of HPV16 mRNA vaccine 34
• 5.3.2 Humoral immune responses 36
• 5.3.3 Cellular immune responses 40
• 5.3.4 In vivo protection 43
• 5.4 Clinical application of VLP ELISA and PBNA 44
• 6. Discussion 46
• 7. Reference 51
• 8. 국문 초록 58