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The aim of this study was to classify the diversity of anal HPV and non-HPV sexually transmitted infections (STIs) and compare the concordance between anal and genital infections in HIV-infected and uninfected women living in the Tapajós region, Amazon, Brazil. A cross-sectional study was performed with 112 HIV-uninfected and 41 HIV-infected nonindigenous women. Anal and cervical scrapings were collected and analyzed for HPV, Chlamydia trachomatis (CT), Neisseria gonorrheae (NG), Trichomonas vaginalis (TV), Mycoplasma genitalium (MG), and Human alphaherpesvirus 2 (HSV-2). The Kappa test evaluated the concordance between anal and genital infections. The overall prevalence of anal HPV infection was 31.3% in HIV-uninfected and 97.6% in HIV-infected women. The most frequent anal high-risk HPV (hrHPV) types were HPV18 and HPV16 in HIV-uninfected women and HPV51, HPV59, HPV31, and HPV58 in HIV-infected women. Anal HPV75 Betapapillomavirus was also identified. Anal non-HPV STIs were identified in 13.0% of all participants. The concordance analysis was fair for CT, MG, and HSV-2, almost perfect agreement for NG, moderate for HPV, and variable for the most frequent anal hrHPV types. Thus, a high prevalence of anal HPV infection with moderate and fair concordance between anal and genital HPV and non-HPV STIs was observed in our study.

Vaccination of patients against neoantigens expressed in concurrent tumors, recurrent tumors, or tumors developing in individuals at risk of cancer is posing major challenges in terms of which antigens to target and is limited to patients expressing neoantigens in their tumors. Here, we describe a vaccination strategy against antigens that were induced in tumor cells by downregulation of the peptide transporter associated with antigen processing (TAP). Vaccination against TAP downregulation–induced antigens was more effective than vaccination against mutation-derived neoantigens, was devoid of measurable toxicity, and inhibited the growth of concurrent and future tumors in models of recurrence and premalignant disease. Human CD8+ T cells stimulated with TAPlow dendritic cells elicited a polyclonal T-cell response that recognized tumor cells with experimentally reduced TAP expression. Vaccination against TAP downregulation–induced antigens overcomes the main limitations of vaccinating against mostly unique tumor-resident neoantigens and could represent a simpler vaccination strategy that will be applicable to most patients with cancer.

Supplementary Figures and Table

Supplemental Figure 1

Supplemental Table 1

High-risk human papillomavirus-associated cancers express viral oncoproteins (e.g., E6 and E7) that induce and maintain the malignant phenotype. The viral origin of these proteins makes them attractive targets for development of a therapeutic vaccine. Camelid-derived single-domain antibody fragments (nanobodies or VHHs) that recognize cell surface proteins on antigen-presenting cells (APC) can serve as targeted delivery vehicles for antigens attached to them. Such VHHs were shown to induce CD4+ and CD8+ T-cell responses against model antigens conjugated to them via sortase, but antitumor responses had not yet been investigated. Here, we tested the ability of an anti-CD11b VHH (VHHCD11b) to target APCs and serve as the basis for a therapeutic vaccine to induce CD8+ T-cell responses against HPV+ tumors. Mice immunized with VHHCD11b conjugated to an H-2Db-restricted immunodominant E7 epitope (E749-57) had more E7-specific CD8+ T cells compared with those immunized with E749-57 peptide alone. These CD8+ T cells acted prophylactically and conferred protection against a subsequent challenge with HPV E7-expressing tumor cells. In a therapeutic setting, VHHCD11b-E749-57 vaccination resulted in greater numbers of CD8+ tumor–infiltrating lymphocytes compared with mice receiving E749-57 peptide alone in HPV+ tumor-bearing mice, as measured by in vivo noninvasive VHH-based immune-positron emission tomography (immunoPET), which correlated with tumor regression and survival outcome. Together, these results demonstrate that VHHs can serve as a therapeutic cancer vaccine platform for HPV-induced cancers. Cancer Immunol Res; 6(7); 870–80. ©2018 AACR.

Determinants of parental HPV vaccine hesitancy, including medical mistrust and exposure to negative vaccine information, are understudied in racial/ethnic minority communities where vaccine uptake is low. We conducted a cross-sectional survey (March 2021) among parents of adolescents, ages 9–17 years, from an academic enrichment program serving low-income, first-generation, underrepresented minority families in Los Angeles to understand determinants of parental HPV vaccine hesitancy. Parents completed self-administered surveys, including a 9-item HPV vaccine hesitancy scale, in either English, Spanish, or Chinese. Logistic regression was used to identify individual and interpersonal factors associated with parental hesitancy and adolescent HPV vaccination. One-fifth of parents (n = 357) reported high HPV vaccine hesitancy and > 50% reported concerns about safety or side effects. High medical mistrust was associated with high parental HPV vaccine hesitancy (adjusted-OR 1.69, 95% CI: 1.13, 2.37). Community-tailored and multilevel strategies to increase vaccine confidence are needed to improve HPV and other adolescent vaccinations.

Figure S6 shows the association of circulating cytokine levels with objective clinical response.

Purpose: The goal of this study was to investigate the therapeutic potential of a novel immunotherapy strategy resulting in immunity to localized or metastatic human papillomavirus 16–transformed murine tumors.Experimental Design: Animals bearing E7-expressing tumors were coimmunized by lymph node injection with E7 49-57 antigen and TLR3-ligand (synthetic dsRNA). Immune responses were measured by flow cytometry and antitumor efficacy was evaluated by tumor size and survival. In situ cytotoxicity assays and identification of tumor-infiltrating lymphocytes and T regulatory cells were used to assess the mechanisms of treatment resistance in bulky disease. Chemotherapy with cyclophosphamide was explored to augment immunotherapy in late-stage disease.Results: In therapeutic and prophylactic settings, immunization resulted in a considerable expansion of E7 49-57 antigen–specific T lymphocytes in the range of 1/10 CD8+ T cells. The resulting immunity was effective in suppressing disease progression and mortality in a pulmonary metastatic disease model. Therapeutic immunization resulted in control of isolated tumors up to a certain volume, and correlated with antitumor immune responses measured in blood. In situ analysis showed that within bulky tumors, T-cell function was affected by negative regulatory mechanisms linked to an increase in T regulatory cells and could be overcome by cyclophosphamide treatment in conjunction with immunization.Conclusions: This study highlights a novel cancer immunotherapy platform with potential for translatability to the clinic and suggests its potential usefulness for controlling metastatic disease, solid tumors of limited size, or larger tumors when combined with cytotoxic agents that reduce the number of tumor-infiltrating T regulatory cells. (Clin Cancer Res 2009;15(19):6167–76)

Purpose:A phase I clinical trial (GOG-9929) examined the safety and efficacy of adjuvant immune-modulation therapy with the checkpoint inhibitor ipilimumab [anti–CTL antigen-4 (anti–CTLA-4)] following chemoradiation therapy (CRT) for newly diagnosed node-positive human papillomavirus (HPV)-related cervical cancer. To better understand the mechanism of action and to identify predictive biomarkers, immunologic and viral correlates were assessed before, during, and after treatment.Patients and Methods:Twenty-one patients who received CRT and ≥2 doses of ipilimumab and 5 patients who received CRT only were evaluable for translational endpoints. Circulating T-cell subsets were evaluated by multiparameter flow cytometry. Cytokines were evaluated by multiplex ELISA. HPV-specific T cells were evaluated in a subset of patients by IFNγ ELISpot.Results:Expression of the activation markers ICOS and PD-1 significantly increased on T-cell subsets following CRT and were sustained or increased following ipilimumab treatment. Combined CRT/ipilimumab treatment resulted in a significant expansion of both central and effector memory T-cell populations. Genotype-specific E6/E7-specific T-cell responses increased post-CRT in 1 of 8 HPV16+ patients and in 2 of 3 HPV18+ patients. Elevation in levels of tumor-promoting circulating cytokines (TNFα, IL6, IL8) post-CRT was significantly associated with worse progression-free survival.Conclusions:Our data indicate that CRT alone and combined with ipilimumab immunotherapy show immune-modulating activity in women with locally advanced cervical cancer and may be a promising therapeutic option for the enhancement of antitumor immune cell function after primary CRT for this population at high risk for recurrence and metastasis. Several key immune biomarkers were identified that were associated with clinical response.

Supplementary Data from Lymph Node–Targeted Immunotherapy Mediates Potent Immunity Resulting in Regression of Isolated or Metastatic Human Papillomavirus–Transformed Tumors

Prostate stem cell antigen (PSCA) is an attractive antigen to target using therapeutic vaccines because of its overexpression in prostate cancer, especially in metastatic tissues, and its limited expression in other organs. Our studies offer the first evidence that a PSCA-based vaccine can induce long-term protection against prostate cancer development in prostate cancer–prone transgenic adenocarcinoma mouse prostate (TRAMP) mice. Eight-week-old TRAMP mice displaying prostate intraepithelial neoplasia were vaccinated with a heterologous prime/boost strategy consisting of gene gun–delivered PSCA-cDNA followed by Venezuelan equine encephalitis virus replicons encoding PSCA. Our results show the induction of an immune response against a newly defined PSCA epitope that is mediated primarily by CD8 T cells. The prostates of PSCA-vaccinated mice were infiltrated by CD4-positive, CD8-positive, CD11b-positive, and CD11c-positive cells. Vaccination induced MHC class I expression and cytokine production [IFN-γ, tumor necrosis factor-α, interleukin 2 (IL-2), IL-4, and IL-5] within prostate tumors. This tumor microenvironment correlated with low Gleason scores and weak PSCA staining on tumor cells present in hyperplastic zones and in areas that contained focal and well-differentiated adenocarcinomas. PSCA-vaccinated TRAMP mice had a 90% survival rate at 12 months of age. In contrast, all control mice had succumbed to prostate cancer or had heavy tumor loads. Crucially, this long-term protective immune response was not associated with any measurable induction of autoimmunity. The possibility of inducing long-term protection against prostate cancer by vaccination at the earliest signs of its development has the potential to cause a dramatic paradigm shift in the treatment of this disease. [Cancer Res 2008;68(3):861–9]

Supplementary Figure 1 Legend from In vivo Effects of Vaccination with Six-Transmembrane Epithelial Antigen of the Prostate: A Candidate Antigen for Treating Prostate Cancer

Supplementary Figure 1 from In vivo Effects of Vaccination with Six-Transmembrane Epithelial Antigen of the Prostate: A Candidate Antigen for Treating Prostate Cancer

Supplementary Figure Legends 1-2 from Prostate Stem Cell Antigen Vaccination Induces a Long-term Protective Immune Response against Prostate Cancer in the Absence of Autoimmunity

Supplementary Figures 1-2 from Prostate Stem Cell Antigen Vaccination Induces a Long-term Protective Immune Response against Prostate Cancer in the Absence of Autoimmunity

Therapeutic vaccine studies should be designed to elicit durable, high magnitude, and efficacious T cell responses, all of which can be impacted by the choice of the vaccination schedule. Here, we compare different prime-boost intervals (PBI) in a human papillomavirus (HPV) model using a HPV16E7E6 Venezuelan equine encephalitis virus replicon particle (VRP) vaccination to address the optimal boosting schedule, quality of immune response, and overall in vivo efficacy. Six different vaccine regimens were tested with each group receiving booster vaccinations at different time intervals. Analysis of T-cell responses demonstrated a significant HPV16 E7 specific CD8+ T cell response with at minimum a one-week PBI between antigen re-exposure. Significant E7-specific in vivo cytotoxicity was also observed with longer PBIs. Additionally, longer PBIs led to an enhanced memory recall response to tumor challenge, which correlated with differential expansion of T cell memory subsets. Our findings imply that when using alphavirus vector platforms as a vaccination strategy, a one-week PBI is sufficient to induce high magnitude effector T cells with potent anti-tumor activity. However, longer PBIs lead to enhanced long-term protective anti-tumor immunity. These findings have implications for therapeutic vaccine clinical trials in which shorter intervals of prime-boost regimens may lead to suboptimal durable immune responses.

While intramuscular vaccination with HPV16 antigens has been shown to induce robust tumor-specific immune responses in the periphery, this has led to limited efficacy in the clinic. One potential explanation is the lack of sufficient infiltration of antigen-specific T cells into the tumor. The few T cells that make their way into the tumor are met with a plethora of negative signals that suppress their activity and abrogate local T cell expansion. To overcome this limitation of intramuscular cancer vaccines, we have developed an approach using in situ vaccination with a combination of immune modulators and tumor antigens, all encoded by mRNAs and delivered using nanoparticles. In this study, we show the superior efficacy and immunogenicity of a locally delivered mRNA cancer vaccine over that of an intramuscular HPV16 cancer vaccine. Both routes of administration result in robust induction of antigen-specific T cells, but local administration of mRNA-encoded antigens and immune modulators also resulted in enhanced infiltration of immune cells into the tumor, local Th1/M1 cytokine induction, and more-complete tumor clearance. Specifically, we found that the inclusion of the immunomodulators IL-12 and TNFSF14/LIGHT enabled tumor clearance in a murine tumor model. While tumors treated with mRNA-encoded HPV16 antigen alone induced robust systemic antigen-specific responses, minimal levels of local T cell expansion and infiltration, proinflammatory cytokine induction, and tumor control were observed. By contrast, tumors treated with mRNA-encoded HPV16 antigen, IL-12p70, and TNFSF14 completely cleared large established HPV16 expressing tumors with long-lasting recurrence free survival and immune memory. This observation was replicated in in a second murine tumor model, confirming the superior potency of locally delivered mRNA cancer vaccines in combination with immunomodulators. Consequently, we are developing a locally administrated mRNA therapy for the treatment of patients with cervical intraepithelial neoplasia (CIN). Citation Format: Weiqun Liu, Diane M. Da Silva, Edward E. Lemmens, Colin J. McKinlay, Sangeeta Nath, Meredith L. Leong, Samuel Deutsch, W. Martin Kast, Ole Audun W. Haabeth. Local delivery of mRNA immunotherapy encoding HPV16 antigen, IL-12, and LIGHT/TNFSF14 results in superior immunogenicity and tumor clearance in a murine model of HPV16-driven cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5163.

Many cancer immunotherapies require the strong activation of the T-cell mediated adaptive immune response to elicit their full anti-tumor potential. Antigen processing and presentation is a complex, multistep process that can be significantly enhanced via antigen modification, leading to better antigen-specific immune responses. Using mRNA, we can express exogenous antigens inside the cell and facilitate the subcellular localization of these antigens to enhance antigen processing and presentation. In this work, we introduced endolysosomal trafficking domains from CD1d molecules to drive subcellular localization of antigenic proteins to the late endosomes (LE) to enhance class I and class II antigen loading. CD1d are molecules that naturally traffic between the membrane and the late endosomes, where antigens are processed and loaded to both class II and class I molecules in a TAP-independent manner. We compared the immunogenicity of a novel endolysosomal trafficking domain, human CD1d, coupled to a chimeric HPV16 E6 and E7 (HPV16E6E7-hCD1d) antigen to antigen designs intended to be either a) secreted (Sec-HPV16E6E7), b) retained in the endoplasmic reticulum (ER) by an ER retention signal KDEL (HPV16E6E7-KDEL) or c) unmodified antigen sequence (HPV16E6E7). To ensure intracellular translation and expression, all antigens were encoded and delivered as mRNAs. The immunogenicity of each antigen design was assessed in HPV16 positive cervical intraepithelial neoplasia (CIN) donor derived PBMCs and healthy controls. Using confocal microscopy, we observed that the hCD1d trafficking domain directed the translated E6E7 protein into late endosomes, which was not observed for any of the other antigen designs. Importantly, antigen localization to late endosomes resulted in significantly improved immunogenicity in vitro as well as in vivo. We observed a consistent increase in numbers of activated antigen-specific T cells across multiple CIN donor PBMCs treated with HPV16E6E7-hCD1d compared to the other 3 designs, as measured by Interferon gamma secretion, surface marker expression, and T-cell proliferation. To assess the generalizability of this approach, we also compared immunogenicity in human donor PBMCs using the human cytomegalovirus antigen pp65, and observed similar results. Lastly, the immunogenicity of each antigen design was tested in vivo by intramuscular injection of nanoparticle formulated mRNAs in C56/Bl6 mice, and inclusion of the murine analogue (mCD1d) resulted in significantly increased generation of HPV16-specific T cell responses. Based on these findings, we are developing a novel mRNA-based cancer therapeutic that includes the addition of hCD1d trafficking domain in the HPV16-specific antigen component. Citation Format: Weiqun Liu, Daniel Fernandez, Colin J. McKinlay, Daniel O. Frimannsson, Meredith L. Leong, W. Martin Kast, Samuel Deutsch, Ole Audun W. Haabeth. Modification of mRNA-encoded HPV16 antigens to include endolysosomal trafficking domains drives cross-presentation and results in superior in vivo and ex vivo antigen-specific responses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 690.

Purpose: A phase I clinical trial (GOG-9929) examined the safety and efficacy of adjuvant immune-modulation therapy with the checkpoint inhibitor ipilimumab [anti–CTL antigen-4 (anti–CTLA-4)] following chemoradiation therapy (CRT) for newly diagnosed node-positive human papillomavirus (HPV)-related cervical cancer. To better understand the mechanism of action and to identify predictive biomarkers, immunologic and viral correlates were assessed before, during, and after treatment. Patients and Methods: Twenty-one patients who received CRT and ≥2 doses of ipilimumab and 5 patients who received CRT only were evaluable for translational endpoints. Circulating T-cell subsets were evaluated by multiparameter flow cytometry. Cytokines were evaluated by multiplex ELISA. HPV-specific T cells were evaluated in a subset of patients by IFNγ ELISpot. Results: Expression of the activation markers ICOS and PD-1 significantly increased on T-cell subsets following CRT and were sustained or increased following ipilimumab treatment. Combined CRT/ipilimumab treatment resulted in a significant expansion of both central and effector memory T-cell populations. Genotype-specific E6/E7-specific T-cell responses increased post-CRT in 1 of 8 HPV16+ patients and in 2 of 3 HPV18+ patients. Elevation in levels of tumor-promoting circulating cytokines (TNFα, IL6, IL8) post-CRT was significantly associated with worse progression-free survival. Conclusions: Our data indicate that CRT alone and combined with ipilimumab immunotherapy show immune-modulating activity in women with locally advanced cervical cancer and may be a promising therapeutic option for the enhancement of antitumor immune cell function after primary CRT for this population at high risk for recurrence and metastasis. Several key immune biomarkers were identified that were associated with clinical response.

Cervical cancer constitutes a significant health burden for women globally. While most patients with early-stage disease can be cured with radical surgery or chemoradiotherapy, patients with high-risk locally advanced disease or with recurrent/metastatic disease have a poor prognosis with standard treatments. Immunotherapies are a rational treatment for this HPV-driven cancer that commonly expresses programmed cell death ligand-1. Before 2021, pembrolizumab was the only United States Food and Drug Administration-approved immunotherapy in cervical cancer, specifically for the second-line recurrent or metastatic (r/m) setting. In late 2021, the antibody-drug conjugate tisotumab vedotin was approved for second-line r/m cervical cancer and pembrolizumab combined with chemotherapy ± bevacizumab was approved for first-line r/m disease based on results from KEYNOTE-826. Moreover, with at least 2 dozen additional immunotherapy clinical trials in the second-line and first-line r/m setting, as well as in locally advanced disease, the treatment landscape for cervical cancer may eventually encounter a potential paradigm shift. Pivotal trials of immunotherapies for cervical cancer that were recently approved or with the potential for regulatory consideration through 2024 are reviewed. As immunotherapy has the opportunity to establish new standards of care in the treatment of cervical cancers, new biomarkers to identify the ideal patient populations for these therapies may also become important. However, issues with access, affordability, and compliance in low- and middle-income countries are anticipated.

Persistent human papillomavirus (HPV) infection is causally linked to the development of several human cancers, including cervical, vulvar, vaginal, anal, penile, and oropharyngeal cancers. To address the need for a therapeutic vaccine against HPV-associated diseases, here we test and compare the immunogenicity and therapeutic efficacy of a bacterial exotoxin fusion protein covalently linked to the HPV16 E7 oncoprotein adjuvanted with CpG or GPI-0100 in the C3.43 preclinical HPV16-transformed tumor model. We show that TVGV-1 protein vaccine adjuvanted with either CpG or GPI-0100 adjuvant induces a high frequency of E7-specific CD8(+) T cells, and both adjuvants are able to assist the immune response in inducing polyfunctional cytokine-secreting lytic T cells that show therapeutic efficacy against well-established C3.43 tumors. CpG-adjuvanted TVGV-1 resulted in higher frequencies of IFNγ secreting and degranulating E7-specific T cells compared to GPI-0100-adjuvanted TVGV-1, resulting in marginally increased in vivo efficacy. Despite minor differences in immune response outcomes, we consider both CpG ODN and GPI-0100 to be promising vaccine adjuvants to increase the immunogenicity and therapeutic efficacy of the TVGV-1 protein for HPV16-driven cancers.

Diversity in Human Leukocyte Antigen (HLA) genes has been associated with risk of several diseases, including Non-Hodgkin’s lymphoma and ulcerative colitis. Reduced diversity at HLA loci may adversely affect the host’s ability to recognize foreign antigens and tumor neoantigens, and subsequently, increase disease burden. To better understand the role of inherited HLA diversity in colorectal cancer (CRC) risk, we utilized data from a population-based study of 10,347 participants (5,574 CRC cases and 4,773 healthy controls) from the Molecular Epidemiology of Colorectal Cancer Study (MECC). Germline DNA samples were genotyped using genome-wide arrays, and HLA Class I and II four-digit resolution alleles were imputed using SNP2HLA and a reference panel of 5,225 individuals from the Type 1 Diabetes Genetics Consortium. Heterozygosity and homozygosity at each HLA locus and the number of homozygous genotypes at class I loci (A, B, C) and class II loci (DQB1, DRB1, DPB1) were quantified. To examine the joint effect of Class I and Class II loci, we combined the total number of homozygotes for all loci and categorized into 3 groups: heterozygotes at all loci, 1 to 4 homozygotes, or 5 or more homozygotes. Logistic regression was used to estimate the risk of CRC associated with HLA locus homozygosity. Individuals with heterozygous genotypes for all loci served as the reference category, and analyses were adjusted for sex, age, genotyping platform, and global ancestry. Individuals with homozygous genotypes at all 3 Class I genes had an increased risk of CRC when compared to those with heterozygous genotypes at all Class I loci (OR: 1.34; 95% CI: 1.02-1.76, P = 0.033; Ptrend = 0.039). A similar association was observed for Class II loci, with an OR of 1.32 (95% CI: 1.05-1.65, P = 0.015; Ptrend = 0.157). For HLA Class I and II combined, individuals with five or more homozygous genotypes at HLA class I or II loci were at higher risk for developing CRC (OR: 1.84, 95% CI: 1.24-2.73, P = 0.0023; Ptrend = 0.015), when compared to those with all heterozygous genotypes. Our findings support a heterozygote advantage at HLA class I and II loci as a protective factor for CRC. This indicates an important role for HLA genetic variability in the etiology of CRC potentially operating through a mechanism of decreased diversity of tumor neoantigens that can be displayed to the adaptive immune system. Citation Format: Ya-Yu Tsai, Chenxu Qu, Joseph D. Bonner, Rebeca Sanz-Pamplona, Sidney Lindsey, Marilena Melas, Kevin J. McDonnell, Gregory E. Idos, Christopher P. Walker, Kevin K. Tsang, Diane M. Da Silva, Ferran Moratalla, Asaf Maoz, Hedy S. Rennert, W. Martin Kast, Joel K. Greenson, Victor Moreno, Gad Rennert, Stephen B. Gruber, Stephanie L. Schmit. Heterozygote advantage at HLA class I and II loci and colorectal cancer risk [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5874.

Vaccination of patients against neoantigens expressed in concurrent tumors, recurrent tumors, or tumors developing in individuals at risk of cancer is posing major challenges in terms of which antigens to target and is limited to patients expressing neoantigens in their tumors. Here, we describe a vaccination strategy against antigens that were induced in tumor cells by downregulation of the peptide transporter associated with antigen processing (TAP). Vaccination against TAP downregulation–induced antigens was more effective than vaccination against mutation-derived neoantigens, was devoid of measurable toxicity, and inhibited the growth of concurrent and future tumors in models of recurrence and premalignant disease. Human CD8+ T cells stimulated with TAPlow dendritic cells elicited a polyclonal T-cell response that recognized tumor cells with experimentally reduced TAP expression. Vaccination against TAP downregulation–induced antigens overcomes the main limitations of vaccinating against mostly unique tumor-resident neoantigens and could represent a simpler vaccination strategy that will be applicable to most patients with cancer.

High-risk human papillomavirus-associated cancers express viral oncoproteins (e.g., E6 and E7) that induce and maintain the malignant phenotype. The viral origin of these proteins makes them attractive targets for development of a therapeutic vaccine. Camelid-derived single-domain antibody fragments (nanobodies or VHHs) that recognize cell surface proteins on antigen-presenting cells (APC) can serve as targeted delivery vehicles for antigens attached to them. Such VHHs were shown to induce CD4+ and CD8+ T-cell responses against model antigens conjugated to them via sortase, but antitumor responses had not yet been investigated. Here, we tested the ability of an anti-CD11b VHH (VHHCD11b) to target APCs and serve as the basis for a therapeutic vaccine to induce CD8+ T-cell responses against HPV+ tumors. Mice immunized with VHHCD11b conjugated to an H-2Db-restricted immunodominant E7 epitope (E749-57) had more E7-specific CD8+ T cells compared with those immunized with E749-57 peptide alone. These CD8+ T cells acted prophylactically and conferred protection against a subsequent challenge with HPV E7-expressing tumor cells. In a therapeutic setting, VHHCD11b-E749-57 vaccination resulted in greater numbers of CD8+ tumor–infiltrating lymphocytes compared with mice receiving E749-57 peptide alone in HPV+ tumor-bearing mice, as measured by in vivo noninvasive VHH-based immune-positron emission tomography (immunoPET), which correlated with tumor regression and survival outcome. Together, these results demonstrate that VHHs can serve as a therapeutic cancer vaccine platform for HPV-induced cancers. Cancer Immunol Res; 6(7); 870–80. ©2018 AACR.

The quantity and quality of immune responses in colorectal cancers (CRC) are widely variable and have important clinical, therapeutic, and prognostic implications. We studied clinical and epidemiologic factors that might influence T-cell quantity and clonality within colorectal adenocarcinomas to better understand the drivers of diverse immune responses. Incident cases of CRC from the Molecular Epidemiology of Colorectal Cancer Study (MECC) were interviewed, and 6,006 cases had complete epidemiologic data. Archived tumor blocks were retrieved from 3,865 (64.4%) cases, and all were reviewed by a single expert pathologist who quantified TILS/hpf. Sufficient tissue for macrodissection and measurement of TCR abundance and clonality using the immunoSEQ assay (Adaptive Biotechnologies) was available and completed for 2,750 cases. Logistic regression, negative binomial regression and linear regression models were used to evaluate potential associations between clinical and epidemiologic variables for: TILS/hpf, TCR abundance, and T-cell clonality. The stage distribution was representative of cancer incidence in the population, and the MSI-H phenotype was observed in 14.2% of cases. Clinical, pathologic, and epidemiologic variables including aspirin, alcohol, diet, hormone use, physical activity, smoking, and statins were assessed in relation to immune measures. Among other findings, >5 years of statins (p Citation Format: Stephen B. Gruber, Joseph D. Bonner, Sidney S. Lindsey, Ya-Yu Tsai, Rebeca Sanz-Pamplona, M. Henar Alonso, Marilena Melas, Hedy S. Rennert, Kevin J. McDonnell, Gregory E. Idos, Christopher P. Walker, W. Martin Kast, Diane Da Silva, Harlan S. Robins, Joel K. Greenson, Victor Moreno, Stephanie L. Schmit, Gad Rennert. Clinical and epidemiologic predictors of clonal immune responses in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2737.

Immune responses to colorectal cancer (CRC) are important prognostic factors as measured by either tumor infiltrating lymphocytes per high powered field or the presence of Crohn's like lymphoid reaction. New technologies to quantify specific features of immune responses include immunoSeq (Adaptive Biotechnologies), which accurately quantifies T-cell receptor (TCR) abundance and T-cell receptor clonality. Here we measured the independent contributions of TCR abundance and TCR clonality to 5-year CRC-specific survival in the Molecular Epidemiology of Colorectal Cancer (MECC) study. The MECC study consented, interviewed, and followed 6,006 incident cases of CRC diagnosed between March 31, 1998 and July 1, 2017 (median follow-up time = 6.9 years). Archived paraffin blocks were retrieved, and 2,750 cases had sufficient tissue and adequate DNA extraction to perform the immunoSeq assay. Analyses were restricted to 1,625 samples with more than 100 T-cells to permit accurate quantification of TCR abundance and TCR clonality as measured by Simpson Clonality Index. TCR abundance was log2 transformed for analyses, and Simpson Clonality Index was log2-transformed and z-scale normalized separately by assay version. In unadjusted models, dichotomized log2 transformed TCR abundance (Hazard Ratio (HR) = 0.57, 95% confidence interval (CI) 0.47-0.71, p=9.77E-08) and dichotomized TCR clonality (HR = 0.77, 95% CI 0.63-0.94, p=0.01) were each statistically significantly associated with 5-year CRC-specific survival. In a model with adjustment for age, sex, microsatellite instability, stage and assay version, both dichotomized log2 transformed TCR abundance (HR = 0.61, 95% CI, 0.49-0.75, p=3.03E-06), and dichotomized TCR clonality (HR = 0.81, 95% CI, 0.66-0.99, p=0.048) were associated with improved 5-year disease-specific survival. Conclusion: TCR abundance and TCR clonality are independent prognostic indicators in CRC. Citation Format: Joseph Bonner, Sephanie L. Schmit, Sidney S. Lindsey, Ya-Yu Tsai, Rebeca Sanz-Pamplona, M. Henar Alonso, Marilena Melas, Hedy S. Rennert, Kevin J. McDonnell, Gregory Idos, Christopher P. Walker, W. Martin Kast, Diane Da Silva, Harlan S. Robins, Joel K. Greenson, Victor Moreno, Gad Rennert, Stephen B. Gruber. T-cell abundance, clonality and disease specific survival in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 835.

Background Anal intraepithelial neoplasia is associated with high-risk human papillomavirus (hrHPV) as a precursor to anal cancer. However, factors other than hrHPV are likely to be involved and further study of cofactors is required because of the possibility of syndemic interactions. Methods Three hundred and fourteen patients underwent 457 operations. Histopathology and hrHPV testing using the Digene Hybrid Capture 2 (HC 2) method were performed. Demographic factors and sexually transmissible infections (STIs) were recorded. Results Results showed that hrHPV alone was associated with HSIL (OR = 4.65, p < 0.001). None of the other STIs were alone associated with HSIL but amplification of risk was found when hrHPV infection occurred with HIV (OR = 11.1); syphilis (OR = 5.58); HSV 2 (OR = 7.85); gonorrhoea (OR = 6.45) and some other infections. Conclusions These results suggest that hrHPV is a sufficient cause of anal HSIL. Seropositivity for HIV, HSV 2, T. pallidum, HBV and HCV and a history of gonorrhoea or chlamydia exert a powerful amplifying factor increasing the risk of HSIL above the risk with hrHPV alone. Other co-factors which are associated with an increased risk of HSIL are increased age, male gender, MSM behaviour and self-reported history of more than 50 sexual partners. This pattern of disease in patients with warts is characteristic of a syndemic with potential serious increased risk of anal carcinoma., Highlights • High-risk HPV is a necessary and sufficient cause of progression from LSIL to HSIL. • HIV, HSV 2, HBV and HCV are associated with an amplified risk of hrHPV induced HSIL. • Gonorrhoea, chlamydia, and syphilis are associated with increased odds HSIL. • HSIL shows syndemic interaction patterns with STIs and behavioural/social factors.

Objective: The aim of this study was to evaluate secretory leukocyte protease inhibitor (SLPI) expression in anal biopsies from HIVpositive (HIV+) individuals, and compare that to anal intraepithelial neoplasia (AIN) diagnoses and human papillomavirus (HPV) status. Design: This is a cross-sectional study of a cohort of 54 HIV+ (31 males and 23 females) from an AIDS clinic in Rio de Janeiro, Brazil. Methods: The study material consisted of anorectal tissue biopsies obtained from HIV+ subjects, which were used to construct tissue microarray paraffin blocks for immunohistochemical analysis of SLPI expression. Biopsies were evaluated by an expert pathologist and classified as low-grade AIN1, high-grade AIN2/3, or normal squamous epithelium. In addition, DNA from the biopsies was extracted and analyzed for the presence of low- or high-risk HPV DNA. Results: Histologically, normal squamous epithelium from the anorectal region showed strong positive SLPI staining in 17/20 (85%) samples. In comparison, 9/17 (53%) dysplastic squamous epithelial samples from AIN1 patients showed strong SLPI staining, and only 5/17 (29%) samples from AIN2/3 patients exhibited strong SPLI staining, which both were significantly fewer than those from normal tissue (P = 0.005). Furthermore, there was a significantly higher proportion of samples in which oncogenic high-risk HPV genotypes were detected in low SLPI-expressing tissues than that in tissues with high SLPI expression (P = 0.040). Conclusions: Taken together these results suggest that low SLPI expression is associated with high-risk HPV infections in the development of AIN.

Human papillomavirus (HPV) entry into epithelial cells is independent of canonical endocytic pathways. Upon interaction with host cells, HPV establishes infection by traversing through an endocytic pathway that is clathrin- and caveolin-independent, but dependent on the annexin A2/S100A10 heterotetramer (A2t). We examined the contribution of monomeric annexin A2 (AnxA2) vs. A2t in HPV infection and endocytosis, and further characterized the role of these molecules in protein trafficking. We specifically show that cell surface A2t is not required for HPV attachment, and in the absence of A2t virion internalization remains clathrin-independent. Without A2t, viral progression from early endosomes to multivesicular endosomes is significantly inhibited, capsid uncoating is dramatically reduced, and lysosomal degradation of HPV is accelerated. Furthermore, we present evidence that AnxA2 forms a complex with CD63, a known mediator of HPV trafficking. Overall, the observed reduction in infection is less significant in the absence of S100A10 alone compared to full A2t, supporting an independent role for monomeric AnxA2. More broadly, we show that successful infection by multiple oncogenic HPV types is dependent on A2t. These findings suggest that A2t is a central mediator of high-risk HPV intracellular trafficking post-entry and pre-viral uncoating.

Table S1. Progression free survival among patients with epithelial ovarian cancer. Table S2 Overall survival among patients with epithelial ovarian cancer. Figure S1 Figure of failure free survival among patients with epithelial ovarian cancer by Sp17 serum concentration. Figure S2 Figure of overall survival among patients with epithelial ovarian cancer by Sp17 serum concentration. (PDF 359 kb)