This study reports follow-up rates for abnormal Pap test results among inpatients screened through the Johns Hopkins Cervical Cancer Screening Program (CCSP). Between 2018 and 2023, 997 patients underwent screening in the CCSP. Of that total, 20.7% (206/997) had abnormal Pap tests warranting further care, and of these individuals, 54.5% did not follow-up. Individuals with a history of homelessness were significantly less likely to follow up (OR: 0.35). Those who had documented assistance for transportation were more likely to return for care (OR: 2.98), especially amongst individuals with a history of homelessness (OR: 4.84). The findings highlight potential targeted interventions based on housing and transportation assistance. Such interventions have been added to our inpatient protocol, with the goal of improving care continuity and effective screening.

Systematic whole-protein screening and comprehensive profiling of antigen-specific CD4+ T cells are crucial for advancing vaccine design and cancer immunotherapies, yet remain technically challenging. Here, we present a high-throughput platform that utilizes large-scale class II single-chain trimer libraries to detect antigen-specific CD4+ T cells, while simultaneously profiling their antigen specificity, TCRα/β sequences, MHC restriction, whole transcriptomes, and patient/timepoint origins at single-cell resolution. Upon rigorous platform validation, we screened the full SARS-CoV-2 spike receptor binding domain in a longitudinal cohort of 22 participants, identifying 2,188 antigen-specific CD4+ T cells and showing key metrics defining the immunogenicity of class II-restricted viral antigens. We further extended the platform to whole-protein screening of HPV-16 E6/E7 in a cohort of precancerous patients, indicating HPV-specific CD4 TCRs that, upon extensive characterization, demonstrate strong therapeutic potential. By integrating high-throughput antigen screening with high-dimensional, multi-modal cellular characterization, our approach provides detailed insight into CD4+ T cell immunity, potentially guiding vaccine design and next-generation TCR-based cancer immunotherapies.

Background The identification of cancer-specific T cell receptor (TCR) sequences is paramount to the advancement of cancer immunotherapies. Recent studies and clinical trials have shown that monoclonal T cell therapy is prone to immune evasion of cancer cells by loss of HLA heterozygosity and low antigen heterogeneity. Cocktail T cell therapy which comprises of TCRs corresponding to multiple HLAs and antigens has been proposed to improve the efficacy of adoptive cell transfer therapy. In addition to CD8+ cytotoxic T cells, neoantigen-specific CD4+ T cells, while identified as important for immunotherapy-induced anti-tumor responses, remain a largely untapped therapeutic resources due to the challenging nature of identification and isolation. Hence, a rapid and high-throughput discovery of both CD8+ and CD4+ TCRs against multiples Class I and II HLAs and cancer antigens is an urgent need. We engineered peptide-bound major histocompatibility complex (pMHC) proteins as capture agents for cancer-specific T cells. The design of these single-chain-trimers (SCTs) enables high-throughput multiplexing for identification and isolation of cancer-targeting CD4+ and CD8+ T cells from multiple patients against large panels of cancer antigens. We applied the technology to identify CD8+ and CD4+ TCRs against oncogenic proteins E6 and E7 from HPV-16, which is the leading cause of cervical cancer. Methods A panel of 200+ Class I SCTs and 100+ Class II SCTs were designed and expressed in a high-throughput platform. PBMCs from precancerous HPV-16+ patients with cervical lesions were collected and enriched with CD8+ and CD4+ T cells. A large pool of 200+ Class I SCT tetramer pool with barcode as antigen identifier was used to capture cancer-specific CD8+ T cells. A computational analysis pipeline was established to pair TCR α and β. HLA-matching cognate antigen was assigned to each TCR pair after UMI count correction and noise removal. The antigen-specific TCRs are subsequently sequenced, validated for functionality, and analyzed for therapeutic applications. Results We identified 43 CD8+ TCR pairs against E6 and E7 oncoproteins from HPV-16 and they are in progress for pre-clinical validation. Conclusions The SCT platform enables rapid identification of cancer-specific CD+ and CD4+ T cells and allows detailed characterization of anti-tumor T cells for which alternative solutions are extremely limited. We applied the technology to PBMCs extracted from HPV-16 related precancerous patients in a clinical trial and discovered cancer-specific TCRs. In summary, the application of the SCT technology is of high value to the fundamental and clinical immune-oncology studies.

Cervical cancer is a major cause of morbidity and mortality globally with a disproportionate impact on women in low- and middle-income countries. In 2021, the World Health Organization (WHO) called for increased vaccination, screening, and treatment to eliminate cervical cancer. However, even with widespread rollout of human papillomavirus (HPV) prophylactic vaccines, millions of women who previously acquired HPV infections will remain at risk for progression to cancer for decades to come. The development and licensing of an affordable, accessible therapeutic HPV vaccine, designed to clear or control carcinogenic HPV and/or to induce regression precancer could significantly contribute to the elimination efforts, particularly benefiting those who missed out on the prophylactic vaccine. One barrier to development of such vaccines is clarity around the regulatory pathway for licensure. In Washington, D.C. on September 12-13, 2023, a meeting was convened to provide input and guidance on trial design with associated ethical and regulatory considerations. This report summarizes the discussion and conclusions from the meeting. Expert presentation topics included the current state of research, potential regulatory challenges, WHO preferred product characteristics, modeling results of impact of vaccine implementation, epidemiology and natural history of HPV infection, immune responses related to viral clearance and/or precancer regression including potential biomarkers, and ethical considerations. Panel discussions were held to explore specific trial design recommendations to support the licensure process for two vaccine indications: (1) treatment of prevalent HPV infection or (2) treatment of cervical precancers. Discussion covered inclusion/exclusion criteria, study endpoints, sample size and power, safety, study length, and additional data needed, which are reported here. Further research of HPV natural history is needed to address identified gaps in regulatory guidance, especially for therapeutic vaccines intended to treat existing HPV infections.

Supplementary Figure 2 from Gene Expression Analysis of Preinvasive and Invasive Cervical Squamous Cell Carcinomas Identifies HOXC10 as a Key Mediator of Invasion

BACKGROUND:Ablation or surgical excision is the typical treatment of anal high-grade squamous intraepithelial lesions (HSIL). Recurrences are common due to the persistence of underlying human papillomavirus (HPV) infection. Additional well-tolerated and effective non-surgical options for HPV-associated anal disease are needed. METHODS:This 3+3 dose escalation Phase I clinical trial evaluated the safety and tolerability of artesunate suppositories in the treatment of patients with biopsy-proven HSIL. RESULTS:The maximal tolerated dose was 400 mg, administered in 3 cycles. All adverse events associated with the use 200- and 400-mg artesunate suppositories were Grade 1. At the 600-mg dose, patients experienced clinically significant nausea. CONCLUSION:Artesunate suppositories are a safe treatment option for anal HSIL.

Supplemental Figure 2. Characterization of tumor infiltrating T cells following treatment with systemic DNA vaccination and locally applied imiquimod

Comparison of HPV16 E7-specific CD8+ T cells induced by HPV16 E7 long peptide after either intramuscular or intracervicovaginal vaccination

Supplementary Figure 1 from Gene Expression Analysis of Preinvasive and Invasive Cervical Squamous Cell Carcinomas Identifies HOXC10 as a Key Mediator of Invasion

Supplementary Figure 7:Detection of CD103 and s1p1 expression by HPV16 E7-specific CD8+ T cells after intracervicovaginal vaccination Supplementary Figure 8: Comparison of HPV16 E7-specific CD8+ T cells induced by pNGVL4a-Sig/E7(detox)/HSP70 intracervicovaginal vaccination followed by either intracervicovaginal or subcutaneous TA-HPV boost

Characterization of MAdCAM-1 expression in cervicovaginal tissue after vaccination

Supplementary Table 1 from Gene Expression Analysis of Preinvasive and Invasive Cervical Squamous Cell Carcinomas Identifies <i>HOXC10</i> as a Key Mediator of Invasion

Supplementary Table 3 from Gene Expression Analysis of Preinvasive and Invasive Cervical Squamous Cell Carcinomas Identifies HOXC10 as a Key Mediator of Invasion

Supplementary Figure 1 from Epigallocatechin-3-Gallate Enhances CD8+ T Cell–Mediated Antitumor Immunity Induced by DNA Vaccination

Supplementary Figure Legends

Supplementary Legends for Figures 1-3 from Gene Expression Analysis of Preinvasive and Invasive Cervical Squamous Cell Carcinomas Identifies HOXC10 as a Key Mediator of Invasion

Local and systemic immune responses produced by different vaccination routes

OBJECTIVE:To evaluate the safety, tolerability, and efficacy of topical artesunate ointment for treatment of biopsy-confirmed Human papillomavirus (HPV)-associated Vulvar intraepithelial neoplasia (VIN) 2/3. METHODS:Participants were enrolled on a prospective, IRB-approved, dose-escalation phase I trial testing either 1, 2 or 3 treatment cycles (5 days), every other week, as applicable. Clinical assessments were completed prior to each dose cycle and included exam and review of adverse event (AE) diary cards. HPV testing and colposcopy was completed at 15 and 28 weeks. AEs were assessed according to CTCAE 4.0 criteria. Complete responders (CR) underwent biopsy of the treated site at the 28-weeks while partial (PR) and non (NR)-responders underwent surgical resection or biopsy and ablation. RESULTS:Fifteen patients consented to and began treatment. Per-protocol assessments were completed in 100% at 15- and 80% at 28-weeks. All patients completed prescribed cycles with no grade 3 or 4 AEs. Vulvovaginal burning/ was the most common AE occurring in 93.3%. AEs were grade 2 in 23.7% and included vulvovaginal pruritus (n = 3), swelling (n = 3) and candidiasis (n = 2). The highest ORR was in the 3-cycle group (88.9% with 55.6% CR). HPV-16 was detected either alone (46.7%) or with other subtypes (33.3%) in 80% of lesions and 5 of 8 (62.5%) with CR had complete viral clearance. CONCLUSIONS:Topical artesunate for treatment of high-grade VIN shows high tolerability, low toxicity and evidence for clinical response in this initial small series. The safety and observed responses support further study in a Phase II trial.