The next horizon in precision oncology: Proteogenomics to inform cancer diagnosis and treatment.

When it comes to precision oncology, proteogenomics may provide better prospects to the clinical characterization of tumors, help make a more accurate diagnosis of cancer, and improve treatment for patients with cancer. This perspective describes the significant contributions of The Cancer Genome Atlas and the Clinical Proteomic Tumor Analysis Consortium to precision oncology and makes the case that proteogenomics needs to be fully integrated into clinical trials and patient care in order for precision oncology to deliver the right cancer treatment to the right patient at the right dose and at the right time.

The COVID-19 Serology Studies Workshop: Recommendations and Challenges.

The development, validation, and appropriate application of serological assays to detect antibodies to SARS-CoV-2 are essential to determining seroprevalence of this virus in the United States and globally and in guiding government leadership and the private sector on back-to-work policies. An interagency working group of the US Department of Health and Human Services convened a virtual workshop to identify knowledge gaps and key outstanding scientific issues and to develop strategies to fill them. Key outcomes of the workshop included recommendations for (1) advancing serology assays as a tool to better understand SARS-CoV-2 infection and (2) conducting crucial serology field studies to advance an understanding of immunity to SARS-CoV-2, leading to protection and duration of protection, including the correlation between serological test results and risk of reinfection.

Harnessing anti-cytomegalovirus immunity for local immunotherapy against solid tumors.

Tumor infiltration by T cells profoundly affects cancer progression and responses to immunotherapy. However, the tumor immunosuppressive microenvironment can impair the induction, trafficking, and local activity of antitumor T cells. Here, we investigated whether intratumoral injection of virus-derived peptide epitopes could activate preexisting antiviral T cell responses locally and promote antitumor responses or antigen spreading. We focused on a mouse model of cytomegalovirus (CMV), a highly prevalent human infection that induces vigorous and durable T cell responses. Mice persistently infected with murine CMV (MCMV) were challenged with lung (TC-1), colon (MC-38), or melanoma (B16-F10) tumor cells. Intratumoral injection of MCMV-derived T cell epitopes triggered in situ and systemic expansion of their cognate, MCMV-specific CD4+ or CD8+ T cells. The MCMV CD8+ T cell epitopes injected alone provoked arrest of tumor growth and some durable remissions. Intratumoral injection of MCMV CD4+ T cell epitopes with polyinosinic acid:polycytidylic acid (pI:C) preferentially elicited tumor antigen-specific CD8+ T cells, promoted tumor clearance, and conferred long-term protection against tumor rechallenge. Notably, secondary proliferation of MCMV-specific CD8+ T cells correlated with better tumor control. Importantly, intratumoral injection of MCMV-derived CD8+ T cell-peptide epitopes alone or CD4+ T cell-peptide epitopes with pI:C induced potent adaptive and innate immune activation of the tumor microenvironment. Thus, CMV-derived peptide epitopes, delivered intratumorally, act as cytotoxic and immunotherapeutic agents to promote immediate tumor control and long-term antitumor immunity that could be used as a stand-alone therapy. The tumor antigen-agnostic nature of this approach makes it applicable across a broad range of solid tumors regardless of their origin.

The Effect of Advances in Lung-Cancer Treatment on Population Mortality.

BACKGROUND: Lung cancer is made up of distinct subtypes, including non-small-cell lung cancer (NSCLC) and small-cell lung cancer (SCLC). Although overall mortality from lung cancer has been declining in the United States, little is known about mortality trends according to cancer subtype at the population level because death certificates do not record subtype information. METHODS: Using data from Surveillance, Epidemiology, and End Results (SEER) areas, we assessed lung-cancer mortality and linked deaths from lung cancer to incident cases in SEER cancer registries. This allowed us to evaluate population-level mortality trends attributed to specific subtypes (incidence-based mortality). We also evaluated lung-cancer incidence and survival according to cancer subtype, sex, and calendar year. Joinpoint software was used to assess changes in incidence and trends in incidence-based mortality. RESULTS: Mortality from NSCLC decreased even faster than the incidence of this subtype, and this decrease was associated with a substantial improvement in survival over time that corresponded to the timing of approval of targeted therapy. Among men, incidence-based mortality from NSCLC decreased 6.3% annually from 2013 through 2016, whereas the incidence decreased 3.1% annually from 2008 through 2016. Corresponding lung cancer-specific survival improved from 26% among men with NSCLC that was diagnosed in 2001 to 35% among those in whom it was diagnosed in 2014. This improvement in survival was found across all races and ethnic groups. Similar patterns were found among women with NSCLC. In contrast, mortality from SCLC declined almost entirely as a result of declining incidence, with no improvement in survival. This result correlates with limited treatment advances for SCLC in the time frame we examined. CONCLUSIONS: Population-level mortality from NSCLC in the United States fell sharply from 2013 to 2016, and survival after diagnosis improved substantially. Our analysis suggests that a reduction in incidence along with treatment advances - particularly approvals for and use of targeted therapies - is likely to explain the reduction in mortality observed during this period.

Chondroitin Sulfate Proteoglycans Are De Facto Cellular Receptors for Human Papillomavirus 16 under High Serum Conditions.

Human papillomaviruses (HPVs) are nonenveloped double-stranded DNA viruses that utilize heparan sulfate proteoglycans (HSPGs) as initial attachment factors prior to cell entry and infection. While extensively characterized, the selective interaction between HPV and HSPGs is generally studied using standard in vitro conditions, which fail to account for the effects that media additives, such as fetal bovine serum (FBS), can have on viral binding. As environmental conditions and growth factors associated with wound healing are thought to play a role in natural HPV infection, we sought to investigate the effects that serum or platelet extracts could have on the binding and infectivity of HPV. Here, we demonstrate that high concentrations of FBS and human serum greatly inhibit HPV16 binding, and that for FBS, this effect results from the obstruction of cell surface HSPGs by serum-derived heparin-binding proteins (HBPs). Surprisingly, we found that under these conditions, HPV particles utilize 6O-sulfated chondroitin sulfate proteoglycans (CSPGs) as initial binding receptors prior to infection. These findings were corroborated by small interfering RNA (siRNA)-mediated knockdown experiments, as well as through a cancer cell line screen, where we identified a strong association between viral binding in high serum and the expression of chondroitin sulfate biosynthesis genes. Furthermore, HPV binding in the presence of human platelet lysate also demonstrated an increased dependance on CSPGs, suggesting a possible role for these receptor proteoglycans in active wound healing environments. Overall, this work highlights the significant influence that serum/platelet factors can have on virus binding and identifies CSPGs as alternative cell attachment receptors for HPV. IMPORTANCE Heparan sulfate proteoglycans (HSPGs) have previously been identified as primary attachment factors for the initial binding of human papillomaviruses (HPVs) prior to infection. Here, we demonstrate that in vitro, HPV binding to HSPGs is strongly dependent on the surrounding experimental conditions, including the concentration of fetal bovine serum (FBS). We found that high concentrations of FBS can block HSPG-binding sites and cause a dependence on 6O-sulfated chondroitin sulfate proteoglycans (CSPGs) as alternative initial viral receptors. Further, we demonstrate that use of a human-derived alternative to FBS, human platelet lysate, also occludes HSPG-dependent binding, causing a shift toward CSPGs for viral attachment. As HPV infection of basal epithelial cells is thought to occur at sites of microtrauma with exposure to high serum levels and platelet factors, these unexpected findings highlight a possible role for CSPGs as important cellular receptors for the binding and infectivity of HPV in vivo.

Precancerous cervical lesions caused by non-vaccine-preventable HPV types after vaccination with the bivalent AS04-adjuvanted HPV vaccine: an analysis of the long-term follow-up study from the randomised Costa Rica HPV Vaccine Trial.

BACKGROUND: In women vaccinated against human papillomavirus (HPV), reductions in cervical disease and related procedures results in more women having intact transformation zones, potentially increasing the risk of cervical lesions caused by non-vaccine-preventable HPV types, a phenomenon termed clinical unmasking. We aimed to evaluate HPV vaccine efficacy against cervical intraepithelial neoplasia grade 2 or worse (CIN2+) and cervical intraepithelial neoplasia grade 3 or worse (CIN3+) attributed to non-preventable HPV types in the long-term follow-up phase of the Costa Rica HPV Vaccine Trial (CVT). METHODS: CVT was a randomised, double-blind, community-based trial done in Costa Rica. Eligible participants were women aged 18-25 years who were in general good health. Participants were randomly assigned (1:1) to receive an HPV 16 and 18 AS04-adjuvanted vaccine or control hepatitis A vaccine, using a blocked randomisation method (permuted block sizes of 14, 16, and 18). Vaccines in both groups were administered intramuscularly with 0·5 mL doses at 0, 1, and 6 months. Masking of vaccine allocation was maintained throughout the 4-year randomised trial phase, after which participants in the hepatitis A virus vaccine control group were provided the HPV vaccine and exited the study; a screening-only, unvaccinated control group was enrolled. The unvaccinated control group and HPV vaccine group were followed up for 7 years, during which treatment allocation was not masked. One of the prespecified primary endpoints for the long-term follow-up phase was precancers associated with HPV types not prevented by the vaccine, defined as histologically confirmed incident CIN2+ events or CIN3+ events attributed to any HPV type except HPV 16, 18, 31, 33, and 45. Our primary analytical period was years 7-11. Primary analyses were in all participants with at least one follow-up visit and excluded participants with a previous endpoint (ie, modified intention-to-treat cohort). Safety endpoints have been reported elsewhere. This trial is registered with ClinicalTrials.gov, NCT00128661 and NCT00867464. The randomised, masked trial phase is completed; an unmasked subset of women in the HPV-vaccinated group is under active investigation. FINDINGS: Between June 28, 2004, and Dec 21, 2005, 7466 participants were enrolled (HPV vaccine group n=3727 and hepatitis A virus vaccine control group n=3739). Between March 30, 2009, and July 5, 2012, 2836 women enrolled in the new unvaccinated control group. The primary analytical cohort (years 7 to 11) included 2767 participants in the HPV vaccine group and 2563 in the unvaccinated group for the CIN2+ events endpoint assessment and 2826 participants in the HPV vaccine group and 2592 in the unvaccinated control group for the CIN3+ events endpoint assessment. Median follow-up during years 7 to 11 for women included for the CIN2+ events analysis was 52·8 months (IQR 44·0 to 60·7) for the HPV vaccine group and 49·8 months (42·0 to 56·9) for the unvaccinated control group. During years 7 to 11, clinical unmasking was observed with a negative vaccine efficacy against CIN2+ events attributed to non-preventable HPV types (-71·2% [95% CI -164·0 to -12·5]), with 9·2 (95% CI 2·1 to 15·6) additional CIN2+ events attributed to non-preventable HPV types per 1000 HPV-vaccinated participants versus HPV-unvaccinated participants. 27·0 (95% CI 14·2 to 39·9) fewer CIN2+ events irrespective of HPV type per 1000 vaccinated participants were observed during 11 years of follow-up. Vaccine efficacy against CIN3+ events attributed to non-preventable HPV types during years 7 to 11 was -135·0% (95% CI -329·8 to -33·5), with 8·3 (3·0 to 12·8) additional CIN3+ events attributed to non-preventable HPV types per 1000 vaccinated participants versus unvaccinated participants. INTERPRETATION: Higher rates of CIN2+ events and CIN3+ events due to non-preventable HPV types in vaccinated versus unvaccinated participants suggests clinical unmasking could attenuate long-term reductions in high-grade disease following successful implementation of HPV vaccination programmes in screened populations. Importantly, the net benefit of vaccination remains considerable; therefore, HPV vaccination should still be prioritised as primary prevention for cervical cancer. FUNDING: National Cancer Institute and National Institutes of Health Office of Research on Women's Health. TRANSLATION: For the Spanish translation of the abstract see Supplementary Materials section.

Antibodies to Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) in All of Us Research Program Participants, 2 January to 18 March 2020.

BACKGROUND: With limited severe acute respiratory syndrome coronavirus (SARS-CoV-2) testing capacity in the United States at the start of the epidemic (January-March 2020), testing was focused on symptomatic patients with a travel history throughout February, obscuring the picture of SARS-CoV-2 seeding and community transmission. We sought to identify individuals with SARS-CoV-2 antibodies in the early weeks of the US epidemic. METHODS: All of Us study participants in all 50 US states provided blood specimens during study visits from 2 January to 18 March 2020. Participants were considered seropositive if they tested positive for SARS-CoV-2 immunoglobulin G (IgG) antibodies with the Abbott Architect SARS-CoV-2 IgG enzyme-linked immunosorbent assay (ELISA) and the EUROIMMUN SARS-CoV-2 ELISA in a sequential testing algorithm. The sensitivity and specificity of these ELISAs and the net sensitivity and specificity of the sequential testing algorithm were estimated, along with 95% confidence intervals (CIs). RESULTS: The estimated sensitivities of the Abbott and EUROIMMUN assays were 100% (107 of 107 [95% CI: 96.6%-100%]) and 90.7% (97 of 107 [83.5%-95.4%]), respectively, and the estimated specificities were 99.5% (995 of 1000 [98.8%-99.8%]) and 99.7% (997 of 1000 [99.1%-99.9%]), respectively. The net sensitivity and specificity of our sequential testing algorithm were 90.7% (97 of 107 [95% CI: 83.5%-95.4%]) and 100.0% (1000 of 1000 [99.6%-100%]), respectively. Of the 24 079 study participants with blood specimens from 2 January to 18 March 2020, 9 were seropositive, 7 before the first confirmed case in the states of Illinois, Massachusetts, Wisconsin, Pennsylvania, and Mississippi. CONCLUSIONS: Our findings identified SARS-CoV-2 infections weeks before the first recognized cases in 5 US states.

Selection, Characterization, Calibration, and Distribution of the U.S. Serology Standard for Anti-SARS-CoV-2 Antibody Detection.

The SARS-CoV-2 pandemic resulted in a demand for highly specific and sensitive serological testing to evaluate seroprevalence and antiviral immune responses to infection and vaccines. Hence, there was an urgent need for a serology standard to harmonize results across different natural history and vaccine studies. The Frederick National Laboratory for Cancer Research (FNLCR) generated a U.S. serology standard for SARS-CoV-2 serology assays and subsequently calibrated it to the WHO international standard (National Institute for Biological Standards and Control [NIBSC] code 20/136) (WHO IS). The development included a collaborative study to evaluate the suitability of the U.S. serology standard as a calibrator for SARS-CoV-2 serology assays. The eight laboratories participating in the study tested a total of 17 assays, which included commercial and in-house-derived binding antibody assays, as well as neutralization assays. Notably, the use of the U.S. serology standard to normalize results led to a reduction in the inter-assay coefficient of variation (CV) for IgM levels (pre-normalization range, 370.6% to 1,026.7%, and post-normalization range, 52.8% to 242.3%) and a reduction in the inter-assay CV for IgG levels (pre-normalization range, 3,416.3% to 6,160.8%, and post-normalization range, 41.6% to 134.6%). The following results were assigned to the U.S. serology standard following calibration against the WHO IS: 246 binding antibody units (BAU)/mL for Spike IgM, 764 BAU/mL for Spike IgG, 1,037 BAU/mL for Nucleocapsid IgM, 681 BAU/mL for Nucleocapsid IgG assays, and 813 neutralizing international units (IU)/mL for neutralization assays. The U.S. serology standard has been made publicly available as a resource to the scientific community around the globe to help harmonize results between laboratories.

The tumor suppressor activity of DLC1 requires the interaction of its START domain with Phosphatidylserine, PLCD1, and Caveolin-1.

BACKGROUND: DLC1, a tumor suppressor gene that is downregulated in many cancer types by genetic and nongenetic mechanisms, encodes a protein whose RhoGAP and scaffolding activities contribute to its tumor suppressor functions. The role of the DLC1 START (StAR-related lipid transfer; DLC1-START) domain, other than its binding to Caveolin-1, is poorly understood. In other START domains, a key function is that they bind lipids, but the putative lipid ligand for DLC1-START is unknown. METHODS: Lipid overlay assays and Phosphatidylserine (PS)-pull down assays confirmed the binding of DLC1-START to PS. Co-immunoprecipitation studies demonstrated the interaction between DLC1-START and Phospholipase C delta 1 (PLCD1) or Caveolin-1, and the contribution of PS to those interactions. Rho-GTP, cell proliferation, cell migration, and/or anchorage-independent growth assays were used to investigate the contribution of PS and PLCD1, or the implications of TCGA cancer-associated DLC1-START mutants, to DLC1 functions. Co-immunoprecipitations and PS-pull down assays were used to investigate the molecular mechanisms underlying the impaired functions of DLC1-START mutants. A structural model of DLC1-START was also built to better understand the structural implications of the cancer-associated mutations in DLC1-START. RESULTS: We identified PS as the lipid ligand for DLC1-START and determined that DLC1-START also binds PLCD1 protein in addition to Caveolin-1. PS binding contributes to the interaction of DLC1 with Caveolin-1 and with PLCD1. The importance of these activities for tumorigenesis is supported by our analysis of 7 cancer-associated DLC1-START mutants, each of which has reduced tumor suppressor function but retains wildtype RhoGAP activity. Our structural model of DLC1-START indicates the mutants perturb different elements within the structure, which is correlated with our experimental findings that the mutants are heterogenous with regard to the deficiency of their binding properties. Some have reduced PS binding, others reduced PLCD1 and Caveolin-1 binding, and others are deficient for all of these properties. CONCLUSION: These observations highlight the importance of DLC1-START for the tumor suppressor function of DLC1 that is RhoGAP-independent. They also expand the versatility of START domains, as DLC1-START is the first found to bind PS, which promotes the binding to other proteins.

An Introduction to Virus Infections and Human Cancer.

Approximately, 1.4 million virus-induced cancers occur annually, representing roughly 10% of the worldwide cancer burden, with the majority (> 85%) occurring in the lower- and middle-income countries. The viruses associated with the greatest number of cancer cases are human papillomaviruses (HPVs), which cause cervical cancer and several other epithelial malignancies, and hepatitis viruses HBV and HCV, which are responsible for the majority of hepatocellular cancer. Other oncoviruses include Epstein-Barr virus (EBV), Kaposi's sarcoma herpesvirus (KSHV), human T-cell leukemia virus (HTLV-I), and Merkel cell polyoma virus (MCPyV). These oncoviruses include various classes of DNA and RNA viruses and induce cancer by a variety of mechanisms. However, cancers develop in a minority of infected individuals and almost always after chronic infection of many year's duration. Identification of the oncoviruses has provided critical insights in human carcinogenesis and led to several interventions that may reduce the risk of developing the tumors they induce. These interventions include preventive vaccines against HBV and HPV, screening for persistent HPV and HCV infections, antivirals for the treatment of chronic HBV and HCV infection, and screening the blood supply for the presence of HBV and HCV. Further efforts to identify additional oncogenic viruses in human cancers and new insights into etiology and pathogenesis of virally induced cancers would likely lead to new approaches for prophylactic and therapeutic interventions.

A Prime-Pull-Amplify Vaccination Strategy To Maximize Induction of Circulating and Genital-Resident Intraepithelial CD8+ Memory T Cells.

Recent insight into the mechanisms of induction of tissue-resident memory (TRM) CD8+ T cells (CD8+ TRM) enables the development of novel vaccine strategies against sexually transmitted infections. To maximize both systemic and genital intraepithelial CD8+ T cells against vaccine Ags, we assessed combinations of i.m. and intravaginal routes in heterologous prime-boost immunization regimens with unrelated viral vectors. Only i.m. prime followed by intravaginal boost induced concomitant strong systemic and intraepithelial genital-resident CD8+ T cell responses. Intravaginal boost with vectors expressing vaccine Ags was far superior to intravaginal instillation of CXCR3 chemokine receptor ligands or TLR 3, 7, and 9 agonists to recruit and increase the pool of cervicovaginal CD8+ TRM Transient Ag presentation increased trafficking of cognate and bystander circulating activated, but not naive, CD8+ T cells into the genital tract and induced in situ proliferation and differentiation of cognate CD8+ TRM Secondary genital CD8+ TRM were induced in the absence of CD4+ T cell help and shared a similar TCR repertoire with systemic CD8+ T cells. This prime-pull-amplify approach elicited systemic and genital CD8+ T cell responses against high-risk human papillomavirus type 16 E7 oncoprotein and conferred CD8-mediated protection to a vaccinia virus genital challenge. These results underscore the importance of the delivery route of nonreplicating vectors in prime-boost immunization to shape the tissue distribution of CD8+ T cell responses. In this context, the importance of local Ag presentation to elicit genital CD8+ TRM provides a rationale to develop novel vaccines against sexually transmitted infections and to treat human papillomavirus neoplasia.

Efficacy of the bivalent HPV vaccine against HPV 16/18-associated precancer: long-term follow-up results from the Costa Rica Vaccine Trial.

BACKGROUND: Oncogenic human papillomavirus (HPV) infections cause most cases of cervical cancer. Here, we report long-term follow-up results for the Costa Rica Vaccine Trial (publicly funded and initiated before licensure of the HPV vaccines), with the aim of assessing the efficacy of the bivalent HPV vaccine for preventing HPV 16/18-associated cervical intraepithelial neoplasia grade 2 or worse (CIN2+). METHODS: Women aged 18-25 years were enrolled in a randomised, double-blind, controlled trial in Costa Rica, between June 28, 2004, and Dec 21, 2005, designed to assess the efficacy of a bivalent vaccine for the prevention of infection with HPV 16/18 and associated precancerous lesions at the cervix. Participants were randomly assigned (1:1) to receive an HPV 16/18 AS04-adjuvanted vaccine or control hepatitis A vaccine. Vaccines were administered intramuscularly in three 0·5 mL doses at 0, 1, and 6 months and participants were followed up annually for 4 years. After the blinded phase, women in the HPV vaccine group were invited to enrol in the long-term follow-up study, which extended follow-up for 7 additional years. The control group received HPV vaccine and was replaced with a new unvaccinated control group. Women were followed up every 2 years until year 11. Investigators and patients were aware of treatment allocation for the follow-up phase. At each visit, clinicians collected cervical cells from sexually active women for cytology and HPV testing. Women with abnormal cytology were referred to colposcopy, biopsy, and treatment as needed. Women with negative results at the last screening visit (year 11) exited the long-term follow-up study. The analytical cohort for vaccine efficacy included women who were HPV 16/18 DNA-negative at vaccination. The primary outcome of this analysis was defined as histopathologically confirmed CIN2+ or cervical intraepithelial neoplasia grade 3 or worse associated with HPV 16/18 cervical infection detected at colposcopy referral. We calculated vaccine efficacy by year and cumulatively. This long-term follow-up study is registered with ClinicalTrials.gov, NCT00867464. FINDINGS: 7466 women were enrolled in the Costa Rica Vaccine Trial; 3727 received the HPV vaccine and 3739 received the control vaccine. Between March 30, 2009, and July 5, 2012, 2635 women in the HPV vaccine group and 2836 women in the new unvaccinated control group were enrolled in the long-term follow-up study. 2635 women in the HPV vaccine group and 2677 women in the control group were included in the analysis cohort for years 0-4, and 2073 women from the HPV vaccine group and 2530 women from the new unvaccinated control group were included in the analysis cohort for years 7-11. Median follow-up time for the HPV group was 11·1 years (IQR 9·1-11·7), 4·6 years (4·3-5·3) for the original control group, and 6·2 years (5·5-6·9) for the new unvaccinated control group. At year 11, vaccine efficacy against incident HPV 16/18-associated CIN2+ was 100% (95% CI 89·2-100·0); 34 (1·5%) of 2233 unvaccinated women had a CIN2+ outcome compared with none of 1913 women in the HPV group. Cumulative vaccine efficacy against HPV 16/18-associated CIN2+ over the 11-year period was 97·4% (95% CI 88·0-99·6). Similar protection was observed against HPV 16/18-associated CIN3-specifically at year 11, vaccine efficacy was 100% (95% CI 78·8-100·0) and cumulative vaccine efficacy was 94·9% (73·7-99·4). During the long-term follow-up, no serious adverse events occurred that were deemed related to the HPV vaccine. The most common grade 3 or worse serious adverse events were pregnancy, puerperium, and perinatal conditions (in 255 [10%] of 2530 women in the unvaccinated control group and 201 [10%] of 2073 women in the HPV vaccine group). Four women in the unvaccinated control group and three in the HPV vaccine group died; no deaths were deemed to be related to the HPV vaccine. INTERPRETATION: The bivalent HPV vaccine has high efficacy against HPV 16/18-associated precancer for more than a decade after initial vaccination, supporting the notion that invasive cervical cancer is preventable. FUNDING: US National Cancer Institute.

Human Papillomavirus 16 Capsids Mediate Nuclear Entry during Infection.

Infectious human papillomavirus 16 (HPV16) L1/L2 pseudovirions were found to remain largely intact during vesicular transport to the nucleus. By electron microscopy, capsids with a diameter of 50 nm were clearly visible within small vesicles attached to mitotic chromosomes and to a lesser extent within interphase nuclei, implying nuclear disassembly. By confocal analysis, it was determined that nuclear entry of assembled L1 is dependent upon the presence of the minor capsid protein, L2, but independent of encapsidated DNA. We also demonstrate that L1 nuclear localization and mitotic chromosome association can occur in vivo in the murine cervicovaginal challenge model of HPV16 infection. These findings challenge the prevailing concepts of PV uncoating and disassembly. More generally, they document that a largely intact viral capsid can enter the nucleus within a transport vesicle, establishing a novel mechanism by which a virus accesses the nuclear cellular machinery.IMPORTANCE Papillomaviruses (PVs) comprise a large family of nonenveloped DNA viruses that include HPV16, among other oncogenic types, the causative agents of cervical cancer. Delivery of the viral DNA into the host cell nucleus is necessary for establishment of infection. This was thought to occur via a subviral complex following uncoating of the larger viral capsid. In this study, we demonstrate that little disassembly of the PV capsid occurs prior to nuclear delivery. These surprising data reveal a previously unrecognized viral strategy to access the nuclear replication machinery. Understanding viral entry mechanisms not only increases our appreciation of basic cell biological pathways but also may lead to more effective antiviral interventions.

Adenovirus vector-based prime-boost vaccination via heterologous routes induces cervicovaginal CD8+ T cell responses against HPV16 oncoproteins.

Recent advances in immunotherapy against cancer underscore the importance of T lymphocytes and tumor microenvironment, but few vaccines targeting cancer have been approved likely due in part to the dearth of common tumor antigens, insufficient immunogenicity and the evolution of immune evasion mechanisms during the progression to malignancy. Human papillomaviruses (HPVs) are the primary etiologic agents of cervical cancer and progression from persistent HPV-infection to cervical intraepithelial lesions and eventually cancer requires persistent expression of the oncoproteins E6 and E7. This offers the opportunity to specifically target these virus-specific antigens for vaccine-induced clearance of infected cells before cancers develop. Here we have evaluated the immunogenicity of Adenovirus Types 26 and 35 derived vectors expressing a fusion of HPV16 E6 and E7 oncoproteins after intramuscular (IM) and/or intravaginal (Ivag) immunization in mice. The adenovirus vectors were shown to transduce an intact cervicovaginal epithelium. IM prime followed by Ivag boost maximized the induction and trafficking of HPV-specific CD8+ T cells producing IFN-γ and TNF-α to the cervicovaginal tract. Importantly, the cervicovaginal CD8+ T cells expressed CD69 and CD103; hallmarks of intraepithelial tissue-resident memory CD8+ T cells. This prime-boost strategy targeting heterologous locations also induced circulating HPV-specific CD8+ T cell responses. Our study prompts further evaluation of Ivag immunization with adenoviral vectors expressing modified E6 and E7 antigens for therapeutic vaccination against persistent HPV infection and cervical intraepithelial neoplasia.

Interferon Gamma Prevents Infectious Entry of Human Papillomavirus 16 via an L2-Dependent Mechanism.

In this study, we report that gamma interferon (IFN-γ) treatment, but not IFN-α, -ß, or -λ treatment, dramatically decreased infection of human papillomavirus 16 (HPV16) pseudovirus (PsV). In a survey of 20 additional HPV and animal papillomavirus types, we found that many, but not all, PsV types were also inhibited by IFN-γ. Microscopic and biochemical analyses of HPV16 PsV determined that the antiviral effect was exerted at the level of endosomal processing of the incoming capsid and depended on the JAK2/STAT1 pathway. In contrast to infection in the absence of IFN-γ, where L1 proteolytic products are produced during endosomal capsid processing and L2/DNA complexes segregate from L1 in the late endosome and travel to the nucleus, IFN-γ treatment led to decreased L1 proteolysis and retention of L2 and the viral genome in the late endosome/lysosome. PsV sensitivity or resistance to IFN-γ treatment was mapped to the L2 protein, as determined with infectious hybrid PsV, in which the L1 protein was derived from an IFN-γ-sensitive HPV type and the L2 protein from an IFN-γ-insensitive type or vice versa.IMPORTANCE A subset of HPV are the causative agents of many human cancers, most notably cervical cancer. This work describes the inhibition of infection of multiple HPV types, including oncogenic types, by treatment with IFN-γ, an antiviral cytokine that is released from stimulated immune cells. Exposure of cells to IFN-γ has been shown to trigger the expression of proteins with broad antiviral effector functions, most of which act to prevent viral transcription or translation. Interestingly, in this study, we show that infection is blocked at the early step of virus entry into the host cell by retention of the minor capsid protein, L2, and the viral genome instead of trafficking into the nucleus. Thus, a novel antiviral mechanism for IFN-γ has been revealed.

A Cell-Free Assembly System for Generating Infectious Human Papillomavirus 16 Capsids Implicates a Size Discrimination Mechanism for Preferential Viral Genome Packaging.

UNLABELLED: We have established a cell-free in vitro system to study human papillomavirus type 16 (HPV16) assembly, a poorly understood process. L1/L2 capsomers, obtained from the disassembly of virus-like particles (VLPs), were incubated with nuclear extracts to provide access to the range of cellular proteins that would be available during assembly within the host cell. Incorporation of a reporter plasmid "pseudogenome" was dependent on the presence of both nuclear extract and ATP. Unexpectedly, L1/L2 VLPs that were not disassembled prior to incubation with a reassembly mixture containing nuclear extract also encapsidated a reporter plasmid. As with HPV pseudoviruses (PsV) generated intracellularly, infection by cell-free particles assembled in vitro required the presence of L2 and was susceptible to the same biochemical inhibitors, implying the cell-free assembled particles use the infectious pathway previously described for HPV16 produced in cell culture. Using biochemical and electron microscopy analyses, we observed that, in the presence of nuclear extract, intact VLPs partially disassemble, providing a mechanistic explanation to how the exogenous plasmid was packaged by these particles. Further, we provide evidence that capsids containing an <8-kb pseudogenome are resistant to the disassembly/reassembly reaction. Our results suggest a novel size discrimination mechanism for papillomavirus genome packaging in which particles undergo iterative rounds of disassembly/reassembly, seemingly sampling DNA until a suitably sized DNA is encountered, resulting in the formation of a stable virion structure. IMPORTANCE: Little is known about papillomavirus assembly biology due to the difficulties in propagating virus in vitro. The cell-free assembly method established in this paper reveals a new mechanism for viral genome packaging and will provide a tractable system for further dissecting papillomavirus assembly. The knowledge gained will increase our understanding of virus-host interactions, help to identify new targets for antiviral therapy, and allow for the development of new gene delivery systems based on in vitro-generated papillomavirus vectors.

Human papillomavirus capsids preferentially bind and infect tumor cells.

We previously determined that human papillomavirus (HPV) virus-like particles (VLPs) and pseudovirions (PsV) did not, respectively, bind to or infect intact epithelium of the cervicovaginal tract. However, they strongly bound heparan sulfate proteoglycans (HSPG) on the basement membrane of disrupted epithelium and infected the keratinocytes that subsequently entered the disrupted site. We here report that HPV capsids (VLP and PsV) have the same restricted tropism for a wide variety of disrupted epithelial and mesothelial tissues, whereas intact tissues remain resistant to binding. However, the HPV capsids directly bind and infect most tumor-derived cell lines in vitro and have analogous tumor-specific properties in vivo, after local or intravenous injection, using orthotopic models for human ovarian and lung cancer, respectively. The pseudovirions also specifically infected implanted primary human ovarian tumors. Heparin and ι-carrageenan blocked binding and infection of all tumor lines tested, implying that tumor cell binding is HSPG-dependent. A survey using a panel of modified heparins indicates that N-sulfation and, to a lesser degree, O-6 sulfation of the surface HSPG on the tumors are important for HPV binding. Therefore, it appears that tumor cells consistently evolve HSPG modification patterns that mimic the pattern normally found on the basement membrane but not on the apical surfaces of normal epithelial or mesothelial cells. Consequently, appropriately modified HPV VLPs and/or PsV could be useful reagents to detect and potentially treat a remarkably broad spectrum of cancers.

Topical herpes simplex virus 2 (HSV-2) vaccination with human papillomavirus vectors expressing gB/gD ectodomains induces genital-tissue-resident memory CD8+ T cells and reduces genital disease and viral shedding after HSV-2 challenge.

UNLABELLED: No herpes simplex virus 2 (HSV-2) vaccine has been licensed for use in humans. HSV-2 glycoproteins B (gB) and D (gD) are targets of neutralizing antibodies and T cells, but clinical trials involving intramuscular (i.m.) injection of HSV-2 gB and gD in adjuvants have not been effective. Here we evaluated intravaginal (ivag) genetic immunization of C57BL/6 mice with a replication-defective human papillomavirus pseudovirus (HPV PsV) expressing HSV-2 gB (HPV-gB) or gD (HPV-gD) constructs to target different subcellular compartments. HPV PsV expressing a secreted ectodomain of gB (gBsec) or gD (gDsec), but not PsV expressing a cytoplasmic or membrane-bound form, induced circulating and intravaginal-tissue-resident memory CD8(+) T cells that were able to secrete gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α) as well as moderate levels of serum HSV neutralizing antibodies. Combined immunization with HPV-gBsec and HPV-gDsec (HPV-gBsec/gDsec) vaccines conferred longer survival after vaginal challenge with HSV-2 than immunization with HPV-gBsec or HPV-gDsec alone. HPV-gBsec/gDsec ivag vaccination was associated with a reduced severity of genital lesions and lower levels of viral shedding in the genital tract after HSV-2 challenge. In contrast, intramuscular vaccination with a soluble truncated gD protein (gD2t) in alum and monophosphoryl lipid A (MPL) elicited high neutralizing antibody titers and improved survival but did not reduce genital lesions and viral shedding. Vaccination combining ivag HPV-gBsec/gDsec and i.m. gD2t-alum-MPL improved survival and reduced genital lesions and viral shedding. Finally, high levels of circulating HSV-2-specific CD8(+) T cells, but not serum antibodies, correlated with reduced viral shedding. Taken together, our data underscore the potential of HPV PsV as a platform for a topical mucosal vaccine to control local manifestations of primary HSV-2 infection. IMPORTANCE: Genital herpes is a highly prevalent chronic disease caused by HSV infection. To date, there is no licensed vaccine against HSV infection. This study describes intravaginal vaccination with a nonreplicating HPV-based vector expressing HSV glycoprotein antigens. The data presented in this study underscore the potential of HPV-based vectors as a platform for the induction of genital-tissue-resident memory T cell responses and the control of local manifestations of primary HSV infection.

Strain-specific properties and T cells regulate the susceptibility to papilloma induction by Mus musculus papillomavirus 1.

The immunocytes that regulate papillomavirus infection and lesion development in humans and animals remain largely undefined. We found that immunocompetent mice with varying H-2 haplotypes displayed asymptomatic skin infection that produced L1 when challenged with 6×1010 MusPV1 virions, the recently identified domestic mouse papillomavirus (also designated "MmuPV1"), but were uniformly resistant to MusPV1-induced papillomatosis. Broad immunosuppression with cyclosporin A resulted in variable induction of papillomas after experimental infection with a similar dose, from robust in Cr:ORL SENCAR to none in C57BL/6 mice, with lesional outgrowth correlating with early viral gene expression and partly with reported strain-specific susceptibility to chemical carcinogens, but not with H-2 haplotype. Challenge with 1×1012 virions in the absence of immunosuppression induced small transient papillomas in Cr:ORL SENCAR but not in C57BL/6 mice. Antibody-induced depletion of CD3+ T cells permitted efficient virus replication and papilloma formation in both strains, providing experimental proof for the crucial role of T cells in controlling papillomavirus infection and associated disease. In Cr:ORL SENCAR mice, immunodepletion of either CD4+ or CD8+ T cells was sufficient for efficient infection and papillomatosis, although deletion of one subset did not inhibit the recruitment of the other subset to the infected epithelium. Thus, the functional cooperation of CD4+ and CD8+ T cells is required to protect this strain. In contrast, C57BL/6 mice required depletion of both CD4+ and CD8+ T cells for infection and papillomatosis, and separate CD4 knock-out and CD8 knock-out C57BL/6 were also resistant. Thus, in C57BL/6 mice, either CD4+ or CD8+ T cell-independent mechanisms exist that can protect this particular strain from MusPV1-associated disease. These findings may help to explain the diversity of pathological outcomes in immunocompetent humans after infection with a specific human papillomavirus genotype.