Dual T cell receptor/chimeric antigen receptor engineered NK-92 cells targeting the HPV16 E6 oncoprotein and the tumor-associated antigen L1CAM exhibit enhanced cytotoxicity and specificity against tumor cells.

The human papillomavirus type 16 (HPV16) causes a large fraction of genital and oropharyngeal carcinomas. To maintain the transformed state, the tumor cells must continuously synthesize the E6 and E7 viral oncoproteins, which makes them tumor-specific antigens. Indeed, specific T cell responses against them have been well documented and CD8+ T cells engineered to express T cell receptors (TCRs) that recognize epitopes of E6 or E7 have been tested in clinical studies with promising results, yet with limited clinical success. Using CD8+ T cells from peripheral blood of healthy donors, we have identified two novel TCRs reactive to an unexplored E618-26 epitope. These TCRs showed limited standalone cytotoxicity against E618-26-HLA-A*02:01-presenting tumor cells. However, a single-signaling domain chimeric antigen receptor (ssdCAR) targeting L1CAM, a cell adhesion protein frequently overexpressed in HPV16-induced cancer, prompted a synergistic effect that significantly enhanced the cytotoxic capacity of NK-92/CD3/CD8 cells armored with both TCR and ssdCAR when both receptors simultaneously engaged their respective targets, as shown by live microscopy of 2-D and 3-D co-cultures. Thus, virus-specific TCRs from the CD8+ T cell repertoire of healthy donors can be combined with a suitable ssdCAR to enhance the cytotoxic capacity of the effector cells and, indirectly, their specificity.

RG2-VLP: a Vaccine Designed to Broadly Protect against Anogenital and Skin Human Papillomaviruses Causing Human Cancer.

The family of human papillomaviruses (HPV) includes over 400 genotypes. Genus α genotypes generally infect the anogenital mucosa, and a subset of these HPV are a necessary, but not sufficient, cause of cervical cancer. Of the 13 high-risk (HR) and 11 intermediate-risk (IR) HPV associated with cervical cancer, genotypes 16 and 18 cause 50% and 20% of cases, respectively, whereas HPV16 dominates in other anogenital and oropharyngeal cancers. A plethora of ßHPVs are associated with cutaneous squamous cell carcinoma (CSCC), especially in sun-exposed skin sites of epidermodysplasia verruciformis (EV), AIDS, and immunosuppressed patients. Licensed L1 virus-like particle (VLP) vaccines, such as Gardasil 9, target a subset of αHPV but no ßHPV. To comprehensively target both α- and ßHPVs, we developed a two-component VLP vaccine, RG2-VLP, in which L2 protective epitopes derived from a conserved αHPV epitope (amino acids 17 to 36 of HPV16 L2) and a consensus ßHPV sequence in the same region are displayed within the DE loop of HPV16 and HPV18 L1 VLP, respectively. Unlike vaccination with Gardasil 9, vaccination of wild-type and EV model mice (Tmc6Δ/Δ or Tmc8Δ/Δ) with RG2-VLP induced robust L2-specific antibody titers and protected against ß-type HPV5. RG2-VLP protected rabbits against 17 αHPV, including those not covered by Gardasil 9. HPV16- and HPV18-specific neutralizing antibody responses were similar between RG2-VLP- and Gardasil 9-vaccinated animals. However, only transfer of RG2-VLP antiserum effectively protected naive mice from challenge with all ßHPVs tested. Taken together, these observations suggest RG2-VLP's potential as a broad-spectrum vaccine to prevent αHPV-driven anogenital, oropharyngeal, and ßHPV-associated cutaneous cancers. IMPORTANCE Licensed preventive HPV vaccines are composed of VLPs derived by expression of major capsid protein L1. They confer protection generally restricted to infection by the αHPVs targeted by the up-to-9-valent vaccine, and their associated anogenital cancers and genital warts, but do not target ßHPV that are associated with CSCC in EV and immunocompromised patients. We describe the development of a two-antigen vaccine protective in animal models against known oncogenic αHPVs as well as diverse ßHPVs by incorporation into HPV16 and HPV18 L1 VLP of 20-amino-acid conserved protective epitopes derived from minor capsid protein L2.

ISA101 and nivolumab for HPV-16+ cancer: updated clinical efficacy and immune correlates of response.

BACKGROUND: The combination of ISA101, a human papilloma virus (HPV) 16 peptide vaccine, and nivolumab showed a promising response rate of 33% in patients with incurable HPV-16+ cancer. Here we report long-term clinical outcomes and immune correlates of response. METHODS: Patients with advanced HPV-16+ cancer and less than two prior regimens for recurrence were enrolled to receive ISA101 (100 µg/peptide) on days 1, 22, and 50 and nivolumab 3 mg/kg every 2 weeks beginning day 8 for up to 1 year. Baseline tumor samples were stained with multiplex immunofluorescence for programmed death-ligand 1 (PD-L1), programmed cell death protein-1 (PD-1), CD3, CD8, CD68, and pan-cytokeratin in a single panel and scanned with the Vectra 3.0 multispectral microscope. Whole transcriptome analysis of baseline tumors was performed with Affymetrix Clariom D arrays. Differential gene expression analysis was performed on responders versus non-responders. RESULTS: Twenty-four patients were followed for a median of 46.5 months (95% CI, 46.0 months to not reached (NR)). The median duration of response was 11.2 months (95% CI, 8.51 months to NR); three out of eight (38%) patients with objective response were without progression at 3 years. The median and 3-year overall survival were 15.3 months (95% CI, 10.6 months to 27.2 months) and 12.5% (95% CI, 4.3% to 36%), respectively. The scores for activated T cells ((CD3+PD-1+)+(CD3+CD8+PD-1+)), activated cytotoxic T cells (CD3+CD8+PD-1+), and total macrophage ((CD68+PD-L1-)+(CD68+PD-L1+)) in tumor were directly correlated with clinical response (p<0.05) and depth of response with the two complete response patients having the highest degree of CD8+ T cells. Gene expression analysis revealed differential regulation of 357 genes (≥1.25 fold) in non-responders versus responders (p<0.05). Higher expression of immune response, inflammatory response and interferon-signaling pathway genes were correlated with clinical response (p<0.05). CONCLUSIONS: Efficacy of ISA101 and nivolumab remains promising in long-term follow-up. Increased infiltration by PD-1+ T cells and macrophages was predictive of response. Enrichment in gene sets associated with interferon-γ response and immune infiltration strongly predicted response to therapy. A randomized trial is ongoing to test this strategy and to further explore correlates of immune response with combined nivolumab and ISA101, versus nivolumab alone. TRIAL REGISTRATION NUMBER: NCT02426892.

Immunogenicity and safety of the human papillomavirus vaccine in young survivors of cancer in the USA: a single-arm, open-label, phase 2, non-inferiority trial.

BACKGROUND: Young survivors of cancer are at increased risk for cancers that are related to human papillomavirus (HPV), primarily caused by oncogenic HPV types 16 and 18. We aimed to examine the immunogenicity and safety of the three-dose series of HPV vaccine in young survivors of cancer. METHODS: We conducted an investigator-initiated, phase 2, single-arm, open-label, non-inferiority trial at five National Cancer Institute-designated comprehensive cancer centres in the USA. Eligible participants were survivors of cancer who were HPV vaccine-naive, were aged 9-26 years, in remission, and had completed cancer therapy between 1 and 5 years previously. Participants received three intramuscular doses of either quadrivalent HPV vaccine (HPV4; enrolments on or before March 1, 2016) or nonavalent HPV vaccine (HPV9; enrolments after March 1, 2016) over 6 months (on day 1, at month 2, and at month 6). We also obtained data from published clinical trials assessing safety and immunogenicity of HPV4 and HPV9 in 9-26-year-olds from the general population, as a comparator group. The primary endpoint was antibody response against HPV types 16 and 18 at month 7 in the per-protocol population. A response was deemed non-inferior if the lower bound of the multiplicity-adjusted 95% CI was greater than 0·5 for the ratio of anti-HPV-16 and anti-HPV-18 geometric mean titres (GMTs) in survivors of cancer versus the general population. Responses were examined separately in male and female participants by age group (ie, 9-15 years and 16-26 years). Safety was assessed in all participants who received at least one vaccine dose and for whom safety data were available. This study is registered with ClinicalTrials.gov, NCT01492582. This trial is now completed. FINDINGS: Between Feb 18, 2013, and June 22, 2018, we enrolled 453 survivors of cancer, of whom 436 received one or more vaccine doses: 203 (47%) participants had survived leukaemia, 185 (42%) were female, and 280 (64%) were non-Hispanic white. Mean age at first dose was 15·6 years (SD 4·6). 378 (83%) of 453 participants had evaluable immunogenicity data; main reasons for exclusion from per-protocol analysis were to loss to follow-up, patient reasons, and medical reasons. Data were also obtained from 26 486 general population controls. The ratio of mean GMT for anti-HPV types 16 and 18 in survivors of cancer versus the general population was more than 1 for all subgroups (ie, aged 9-15 years, aged 16-26 years, male, and female groups) in both vaccine cohorts (ranging from 1·64 [95% CI 1·12-2·18] for anti-HPV type 16 in female participants aged 9-15 years who received HPV9, to 4·77 [2·48-7·18] for anti-HPV type 18 in male participants aged 16-26 years who received HPV4). Non-inferiority criteria were met within each age and sex subgroup, except against HPV type 18 in female participants aged 16-26 years receiving HPV9 (4·30 [0·00-9·05]). Adverse events were reported by 237 (54%) of 435 participants; injection site pain was most common (174 [40%] participants). One serious adverse event (ie, erythema nodosum) was possibly related to vaccine (HPV9; 16-26 year female cohort). INTERPRETATION: Immunogenicity and safety of HPV vaccine three-dose series in survivors of cancer is similar to that in the general population, providing evidence for use in this clinically vulnerable population. FUNDING: US National Cancer Institute, Merck, Sharp & Dohme, and American Lebanese Syrian Associated Charities.

Human papilloma virus infection of uterine cervix and spectrum of cervical pathology in human immunodeficiency virus/AIDS.

BACKGROUND: Human papilloma virus (HPV) is one of the most common causes of sexually transmitted viral diseases worldwide. High-risk HPV types such as HPV16 and 18 are known to cause cervical dysplasia and carcinoma. In human immunodeficiency virus (HIV)-positive individual, chance of HPV coinfection and risk of cervical dysplasia/carcinoma have been found to be significantly more than in HIV-negative individuals. AIM: In this institution-based, cross-sectional, observational study, we aim to find out the relationship of HPV infection of the uterine cervix with cervical dysplasia and neoplasia in HIV-infected/AIDS patients. MATERIALS AND METHODS: Conventional Pap smears were taken from HIV-infected individuals admitted in the department of gynecology and obstetrics and reported by the Bethesda system. A second sample was sent to the virology unit of ICMR for detection and typing of HPV. Control samples were taken from HIV-negative individuals. RESULTS: Fifty HIV-positive patients were included in this study. On cervical Pap smear examination, 32 cases were cytologically benign and 18 cases showed atypical cytomorphology. Twenty-four cases were HPV positive, among which 16 were cytologically atypical and 8 were benign. HPV 16 was the most common subtype (50%) followed by HPV 18 (37.5%) and others (12.5%) in HIV-positive patients. Chance of cervical dysplasia increased with age independent of HIV infection and with progressive lower CD4 count. Koilocytosis was a significant predictor of HPV infection. Majority of patients were asymptomatic. Peak incidence of HPV infection occurred in reproductive age group (20-40 years). The association between HIV and HPV coinfection (P = 0.002) and between HPV infection and cytology atypia (P < 0.0001) was statistically significant. CONCLUSION: Present study highlights the necessity of routine cervical Pap smear screening in HIV infected reproductive age-group women. Early detection enables dysplasia to revert or be effectively managed.

Vaccine efficacy against persistent human papillomavirus (HPV) 16/18 infection at 10 years after one, two, and three doses of quadrivalent HPV vaccine in girls in India: a multicentre, prospective, cohort study.

BACKGROUND: A randomised trial designed to compare three and two doses of quadrivalent human papillomavirus (HPV) vaccine in adolescent girls in India was converted to a cohort study after suspension of HPV vaccination in trials by the Indian Government. In this Article, the revised aim of the cohort study was to compare vaccine efficacy of single dose to that of three and two doses in protecting against persistent HPV 16 and 18 infection at 10 years post vaccination. METHODS: In the randomised trial, unmarried girls aged 10-18 years were recruited from nine centres across India and randomly assigned to either two doses or three doses of the quadrivalent HPV vaccine (Gardasil [Merck Sharp & Dohme, Whitehouse Station, NJ, USA]; 0·5 mL administered intramuscularly). After suspension of recruitment and vaccination, the study became a longitudinal, prospective cohort study by default, and participants were allocated to four cohorts on the basis of the number vaccine doses received per protocol: the two-dose cohort (received vaccine on days 1 and 180 or later), three-dose cohort (days 1, 60, and 180 or later), two-dose default cohort (days 1 and 60 or later), and the single-dose default cohort. Participants were followed up yearly. Cervical specimens were collected from participants 18 months after marriage or 6 months after first childbirth, whichever was earlier, to assess incident and persistent HPV infections. Married participants were screened for cervical cancer as they reached 25 years of age. Unvaccinated women age-matched to the married vaccinated participants were recruited to serve as controls. Vaccine efficacy against persistent HPV 16 and 18 infections (the primary endpoint) was analysed for single-dose recipients and compared with that in two-dose and three-dose recipients after adjusting for imbalance in the distribution of potential confounders between the unvaccinated and vaccinated cohorts. This trial is registered with ISRCTN, ISRCTN98283094, and ClinicalTrials.gov, NCT00923702. FINDINGS: Vaccinated participants were recruited between Sept 1, 2009, and April 8, 2010 (date of vaccination suspension), and followed up over a median duration of 9·0 years (IQR 8·2-9·6). 4348 participants had three doses, 4980 had two doses (0 and 6 months), and 4949 had a single dose. Vaccine efficacy against persistent HPV 16 and 18 infection among participants evaluable for the endpoint was 95·4% (95% CI 85·0-99·9) in the single-dose default cohort (2135 women assessed), 93·1% (77·3-99·8) in the two-dose cohort (1452 women assessed), and 93·3% (77·5-99·7) in three-dose recipients (1460 women assessed). INTERPRETATION: A single dose of HPV vaccine provides similar protection against persistent infection from HPV 16 and 18, the genotypes responsible for nearly 70% of cervical cancers, to that provided by two or three doses. FUNDING: Bill & Melinda Gates Foundation.

Germline determinants of humoral immune response to HPV-16 protect against oropharyngeal cancer.

Although several oropharyngeal cancer (OPC) susceptibility loci have been identified, most previous studies lacked detailed information on human papillomavirus (HPV) status. We conduct a genome-wide analysis by HPV16 serology status in 4,002 oral cancer cases (OPC and oral cavity cancer (OCC)) and 5,256 controls. We detect four susceptibility loci pointing to a distinct genetic predisposition by HPV status. Our most notable finding in the HLA region, that is now confirmed to be specific of HPV(+)OPC risk, reveal two independent loci with strong protective effects, one refining the previously reported HLA class II haplotype association. Antibody levels against HPV16 viral proteins strongly implicate the protective HLA variants as major determinants of humoral response against L1 capsid protein or E6 oncoprotein suggesting a natural immune response against HPV(+)OPC promoted by HLA variants. This indicates that therapeutic vaccines that target E6 and attenuate viral response after established HPV infections might protect against HPV(+)OPC.

Expansion of Human Papillomavirus-Specific T Cells in Periphery and Cervix in a Therapeutic Vaccine Recipient Whose Cervical High-Grade Squamous Intraepithelial Lesion Regressed.

Advances in high-throughput sequencing have revolutionized the manner with which we can study T cell responses. We describe a woman who received a human papillomavirus (HPV) therapeutic vaccine called PepCan, and experienced complete resolution of her cervical high-grade squamous intraepithelial lesion. By performing bulk T cell receptor (TCR) ß deep sequencing of peripheral blood mononuclear cells before and after 4 vaccinations, 70 putatively vaccine-specific clonotypes were identified for being significantly increased using a beta-binomial model. In order to verify the vaccine-specificity of these clonotypes, T cells with specificity to a region, HPV 16 E6 91-115, previously identified to be vaccine-induced using an interferon-γ enzyme-linked immunospot assay, were sorted and analyzed using single-cell RNA-seq and TCR sequencing. HPV specificity in 60 of the 70 clonotypes identified to be vaccine-specific was demonstrated. TCR ß bulk sequencing of the cervical liquid-based cytology samples and cervical formalin-fixed paraffin-embedded samples before and after 4 vaccinations demonstrated the presence of these HPV-specific T cells in the cervix. Combining traditional and cutting-edge immunomonitoring techniques enabled us to demonstrate expansion of HPV-antigen specific T cells not only in the periphery but also in the cervix. Such an approach should be useful as a novel approach to assess vaccine-specific responses in various anatomical areas.

Effectiveness of various human papillomavirus vaccination strategies: A community randomized trial in Finland.

INTRODUCTION: We conducted a community-randomized trial (NCTBLINDED) in Finland to assess gender-neutral and girls-only vaccination strategies with the AS04-adjuvanted human papillomavirus (HPV)-16/18 (AS04-HPV-16/18)vaccine. METHODS: Girls and boys (12-15 years) were invited. We randomized 33 communities (1:1:1 ratio): Arm A: 90% of randomly selected girls and boys received AS04-HPV-16/18 vaccine and 10% received hepatitis B vaccine (HBV); Arm B: 90% of randomly selected girls received AS04-HPV-16/18 vaccine, 10% of girls received HBV, and all boys received HBV; Arm C: all participants received HBV. Effectiveness measurements against prevalence of HPV-16/18 cervical infection were estimated in girls at 18.5 years. The main measures were: (1) overall effectiveness comparing Arms A or B, regardless of vaccination status, vs Arm C; (2) total effectiveness comparing AS04-HPV-16/18 vaccinated girls in pooled Arms A/B vs Arm C; (3) indirect effectiveness (herd effect) comparing girls receiving HBV or unvaccinated in Arm A vs Arm C. Co-primary objectives were overall effectiveness following gender-neutral or girls-only vaccination. RESULTS: Of 80,272 adolescents invited, 34,412 were enrolled. Overall effectiveness was 23.8% (95% confidence interval: -19.0, 51.1; P = 0.232) with gender-neutral vaccination. Following girls-only vaccination, overall effectiveness was 49.6% (20.1, 68.2; P = 0.004). Total effectiveness was over 90% regardless of vaccination strategy. No herd effect was found. Immunogenicity of the AS04-HPV-16/18 vaccine was high in both sexes. CONCLUSIONS: This study illustrates the difficulty in conducting community randomized trials. It is not plausible that vaccinating boys would reduce overall effectiveness, and the apparent lack of herd effect was unexpected given findings from other studies. This analysis was likely confounded by several factors but confirms the vaccine's high total effectiveness as in clinical trials.

Anti-HPV16 Antibody Titers Prior to an Incident Cervical HPV16/31 Infection.

The goal of this study was to investigate the serological titers of circulating antibodies against human papillomavirus (HPV) type 16 (anti-HPV16) prior to the detection of an incident HPV16 or HPV31 infection amongst vaccinated participants. Patients were selected from a prospective post-HPV vaccine longitudinal cohort at Mount Sinai Adolescent Health Center in Manhattan, NY. We performed a nested case-control study of 43 cases with incident detection of cervical HPV16 (n = 26) or HPV31 (n = 17) DNA who had completed the full set of immunizations of the quadrivalent HPV vaccine (4vHPV). Two control individuals whom had received three doses of the vaccine (HPV16/31-negative) were selected per case, matched on age at the first dose of vaccination and follow-up time in the study: a random control, and a high-risk control that was in the upper quartile of a sexual risk behavior score. We conducted an enzyme-linked immunosorbent assay (ELISA) for the detection of immunoglobulin G (IgG) antibodies specific to anti-HPV16 virus-like particles (VLPs). The results suggest that the average log antibody titers were higher among high-risk controls than the HPV16/31 incident cases and the randomly selected controls. We show a prospective association between anti-HPV16 VLP titers and the acquisition of an HPV16/31 incident infection post-receiving three doses of 4vHPV vaccine.

HPV-16 E6/E7 DNA tattoo vaccination using genetically optimized vaccines elicit clinical and immunological responses in patients with usual vulvar intraepithelial neoplasia (uVIN): a phase I/II clinical trial.

BACKGROUND: Usual vulvar intraepithelial neoplasia (uVIN) is a premalignancy caused by persistent infection with high-risk types of human papillomavirus (HPV), mainly type 16. Even though different treatment modalities are available (eg, surgical excision, laser evaporation or topical application of imiquimod), these treatments can be mutilating, patients often have recurrences and 2%-8% of patients develop vulvar carcinoma. Therefore, immunotherapeutic strategies targeting the pivotal oncogenic HPV proteins E6 and E7 are being explored to repress carcinogenesis. METHOD: In this phase I/II clinical trial, 14 patients with HPV16+ uVIN were treated with a genetically enhanced DNA vaccine targeting E6 and E7. Safety, clinical responses and immunogenicity were assessed. Patients received four intradermal HPV-16 E6/E7 DNA tattoo vaccinations, with a 2-week interval, alternating between both upper legs. Biopsies of the uVIN lesions were taken at screening and +3 months after last vaccination. Digital photography of the vulva was performed at every check-up until 12 months of follow-up for measurement of the lesions. HPV16-specific T-cell responses were measured in blood over time in ex vivo reactivity assays. RESULTS: Vaccinations were well tolerated, although one grade 3 suspected unexpected serious adverse reaction was observed. Clinical responses were observed in 6/14 (43%) patients, with 2 complete responses and 4 partial responses (PR). 5/14 patients showed HPV-specific T-cell responses in blood, measured in ex vivo reactivity assays. Notably, all five patients with HPV-specific T-cell responses had a clinical response. CONCLUSIONS: Our results indicate that HPV-16 E6/E7 DNA tattoo vaccination is a biologically active and safe treatment strategy in patients with uVIN, and suggest that T-cell reactivity against the HPV oncogenes is associated with clinical benefit. TRIAL REGISTRATION NUMBER: NTR4607.

Enhancing immunogenicity of HPV16 E7 DNA vaccine by conjugating codon-optimized GM-CSF to HPV16 E7 DNA.

OBJECTIVE: To generate immunity against human papillomavirus (HPV), the use of a recombinant DNA vaccine to carry an appropriate target gene is a promising and cost-effective approach. Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a potent immunomodulatory cytokine that enhances the efficacy of vaccines by promoting the development and prolongation of humoral and cellular immunity. In this study, we linked codon-optimized GM-CSF (cGM-CSF) to the HPV16 E7 sequence as fused protein and evaluated the immunogenic potential of this DNA vaccine. MATERIALS AND METHODS: We have demonstrated that cGM-CSF enhanced immunity against tumor challenges by generating and promoting the proliferation of HPV16 E7-specific CD8+ T cells, which secrete IFN-γ in the murine model. In this study, we aimed to evaluate the immunogenic potential of DNA vaccine that constructed by linking codon-optimized GM-CSF to HPV16 E7 sequence in the animal model. We study the half-life of RNA decay and cellular location of HPV16 E7 by Q-PCR and Western blot. We also assess immune response in the animal model by flow cytometry and ELISA. RESULTS: The cGM-CSF-E7 sequence increased and extended the expression of E7 mRNA, in comparison with the E7 sequence alone. Mice vaccinated with the cGM-CSF-E7 DNA vaccine exhibited a slower rate of tumor growth than those vaccinated with the unconjugated E7 DNA vaccine. We also found that the CD4 and CD8+ T cells from these mice showed strong secretion of IFN-γ. CONCLUSION: Through in vivo antibody depletion experiments, we demonstrated that both CD4+ and CD8+ T cells play an important role in the suppression of tumor growth.

Combination immunotherapy with two attenuated Listeria strains carrying shuffled HPV-16 E6E7 protein causes tumor regression in a mouse tumor model.

Cervical cancer continues to impose a heavy burden worldwide, and human papilloma virus (HPV) infection, especially persistent infection with type 16 (HPV-16), is known to be the primary etiological factor. Therapeutic vaccines are urgently needed because prophylactic vaccines are ineffective at clearing pre-existing HPV infection. Here, two recombinant Listeria strains (LMΔ-E6E7 & LIΔ-E6E7) with deletions of the actA and plcB genes, expressing the shuffled HPV-16 E6E7 protein were constructed. The strains were delivered into the spleen and liver by intravenous inoculation, induced antigen-specific cellular immunity and were eliminated completely from the internal organs several days later. Intravenously treating with single strain for three times, or with both strains alternately for three times significantly reduced the tumor size and prolonged the survival time of model mice. Combination immunotherapy with two strains seemed more effective than immunotherapy with single strain in that it enhanced the survival of the mice, and the LMΔ-E6E7-prime-LIΔ-E6E7-boost strategy showed significant stronger efficacy than single treatment with the LIΔ-E6E7 strain. The antitumor effect of this treatment might due to its ability to increase the proportion of CD8+ T cells and reduce the proportion of T regulatory cells (Tregs) in the intratumoral milieu. This is the first report regarding Listeria ivanovii-based therapeutic vaccine candidate against cervical cancer. Most importantly we are the first to confirm that combination therapy with two different recombinant Listeria strains has a more satisfactory antitumor effect than administration of a single strain. Thus, we propose a novel prime-boost treatment strategy.

Human papillomavirus seroprevalence in pregnant women following gender-neutral and girls-only vaccination programs in Finland: A cross-sectional cohort analysis following a cluster randomized trial.

BACKGROUND: Cervical cancer elimination through human papillomavirus (HPV) vaccination programs requires the attainment of herd effect. Due to its uniquely high basic reproduction number, the vaccination coverage required to achieve herd effect against HPV type 16 exceeds what is attainable in most populations. We have compared how gender-neutral and girls-only vaccination strategies create herd effect against HPV16 under moderate vaccination coverage achieved in a population-based, community-randomized trial. METHODS AND FINDINGS: In 2007-2010, the 1992-1995 birth cohorts of 33 Finnish communities were randomized to receive gender-neutral HPV vaccination (Arm A), girls-only HPV vaccination (Arm B), or no HPV vaccination (Arm C) (11 communities per trial arm). HPV16/18/31/33/35/45 seroprevalence differences between the pre-vaccination era (2005-2010) and post-vaccination era (2011-2016) were compared between all 8,022 unvaccinated women <23 years old and resident in the 33 communities during 2005-2016 (2,657, 2,691, and 2,674 in Arms A, B, and C, respectively). Post- versus pre-vaccination-era HPV seroprevalence ratios (PRs) were compared by arm. Possible outcome misclassification was quantified via probabilistic bias analysis. An HPV16 and HPV18 seroprevalence reduction was observed post-vaccination in the gender-neutral vaccination arm in the entire study population (PR16 = 0.64, 95% CI 0.10-0.85; PR18 = 0.72, 95% CI 0.22-0.96) and for HPV16 also in the herpes simplex virus type 2 seropositive core group (PR16 = 0.64, 95% CI 0.50-0.81). Observed reductions in HPV31/33/35/45 seroprevalence (PR31/33/35/45 = 0.88, 95% CI 0.81-0.97) were replicated in Arm C (PR31/33/35/45 = 0.79, 95% CI 0.69-0.90). CONCLUSIONS: In this study we only observed herd effect against HPV16/18 after gender-neutral vaccination with moderate vaccination coverage. With only moderate vaccination coverage, a gender-neutral vaccination strategy can facilitate the control of even HPV16. Our findings may have limited transportability to other vaccination coverage levels. TRIAL REGISTRATION: ClinicalTrials.gov number NCT00534638, https://clinicaltrials.gov/ct2/show/NCT00534638.

Investigating the association between serum human papillomavirus type 16 E7 antibodies and risk of head and neck cancer.

Human papillomavirus (HPV) is recognized as a major cause of oropharyngeal cancer (OPC) in Western countries. Less is known regarding its contribution to the OPC occurring in Asia. The current study aimed to investigate the association between antibody responses to HPV16 E7 and head and neck cancer (HNC) risk in a hospital-based case-control study conducted in Taiwan with 693 HNC cases and 1,035 controls. A positive association was observed between seropositivity to HPV16 E7 and OPC risk, whereas no significant association was found in the non-OPC cases. The increased OPC risk associated with seropositivity to HPV16 E7 was more significant among nonbetel quid or noncigarette users. Seropositivity to HPV16 E7 showed moderate agreement with P16 expression in OPC. OPC patients that were seropositive to HPV16 E7 or p16 positive were more highly educated and less likely to use alcohol, betel quids, and cigarettes compared to HPV16 E7 seronegative or p16 negative OPC patients. Furthermore, patients with p16 positive OPC were more likely to be women compared to patients with p16 negative OPC, likely owing to the low prevalence of alcohol, betel quid, and cigarette users among women. Overall, this study suggested that similar to Western countries, HPV may also be an important risk factor of OPC in Taiwan. With the declining consumption of betel quids and cigarettes in Taiwan, a higher percentage of OPC cases in Taiwan will be attributed to HPV in the future. Public health measures, including HPV vaccination, need to be implemented to prevent the occurrence of HPV-positive OPC.

Induction of Therapeutic Protection in an HPV16-Associated Mouse Tumor Model Through Targeting the Human Papillomavirus-16 E5 Protein to Dendritic Cells.

HPV E5 is an oncoprotein mainly expressed in premalignant lesions, which makes it an important target for a vaccine to prevent or cure cervical cancer (CC). In this study, we evaluated whether E5 targeted to DEC-205, present in dendritic cells (DCs), could induce a therapeutic protection against HPV16-induced tumor cells in a mouse model. The HPV-16 E5 (16E5) protein was cross-linked to a monoclonal antibody (mAb) specific to mouse DEC-205 (anti-DEC-205:16E5) or to an isotype control mAb (isotype:16E5). Rotavirus VP6 was cross-linked to the mouse anti-DEC-205 mAb (anti-DEC-205:VP6) as a non-specific antigen control. BALB/c mice were inoculated subcutaneously (s.c.) with the 16E5-expressing BMK-16/myc tumor cells, and 7 and 14 days later the mice were immunized s.c. with the conjugates, free 16E5 or PBS in the presence of adjuvant. Tumor growth was monitored to evaluate protection. A strong protective immune response against the tumor cells was induced when the mice were inoculated with the anti-DEC-205:16E5 conjugate, since 70% of the mice controlled the tumor growth and survived, whereas the remaining 30% developed tumors and died by day 72. In contrast, 100% of the mice in the control groups died by day 30. The anti-DEC-205:16E5 conjugate was found to induce 16E5-specific memory T cells, with a Th1/Th17 profile. Both CD4+ and CD8+ T cells contributed to the observed protection. Finally, treating mice that had developed tumors with an anti-PD-1 mAb, delayed the tumor growth for more than 20 days. These results show that targeting 16E5 to DEC-205, alone or combined with an immune checkpoint blockade, could be a promising protocol for the treatment of the early stages of HPV-associated cancer.

Characterization of recombinant gorilla adenovirus HPV therapeutic vaccine PRGN-2009.

There are approximately 44,000 cases of human papillomavirus-associated (HPV-associated) cancer each year in the United States, most commonly caused by HPV types 16 and 18. Prophylactic vaccines successfully prevent healthy people from acquiring HPV infections via HPV-specific antibodies. In order to treat established HPV-associated malignancies, however, new therapies are necessary. Multiple recombinant gorilla adenovirus HPV vaccine constructs were evaluated in NSG-ß2m-/- peripheral blood mononuclear cell-humanized mice bearing SiHa, a human HPV16+ cervical tumor, and/or in the syngeneic HPV16+ TC-1 model. PRGN-2009 is a therapeutic gorilla adenovirus HPV vaccine containing multiple cytotoxic T cell epitopes of the viral oncoproteins HPV 16/18 E6 and E7, including T cell enhancer agonist epitopes. PRGN-2009 treatment reduced tumor volume and increased CD8+ and CD4+ T cells in the tumor microenvironment of humanized mice bearing the human cervical tumor SiHa. PRGN-2009 monotherapy in the syngeneic TC-1 model also reduced tumor volumes and weights, generated high levels of HPV16 E6-specific T cells, and increased multifunctional CD8+ and CD4+ T cells in the tumor microenvironment. These studies provide the first evaluation to our knowledge of a therapeutic gorilla adenovirus HPV vaccine, PRGN-2009, showing promising preclinical antitumor efficacy and induction of HPV-specific T cells, along with the rationale for its evaluation in clinical trials.

AIDS Malignancy Consortium 054: Safety and Immunogenicity of the Quadrivalent Vaccine in Indian Women Living With HIV.

BACKGROUND: Human papillomavirus (HPV)-associated cervical cancer is a leading cause of death among Indian women. Indian women living with HIV (WLWH) may be at especially high risk. The quadrivalent HPV (qHPV) vaccine is effective in prevention of initial infection with HPV-6/11/16/18 in HIV-negative women. Little is known about previous exposure to HPV-6/11/16/18, safety, and immunogenicity of qHPV in Indian WLWH. METHODOLOGY: One hundred fifty WLWH with different CD4 levels and HIV viral load (VL) were vaccinated at 0/2/6 months at CART-CRS-IDMC, Chennai, India. Serology was performed at weeks 0, 28, and 52 for HPV-6/11/16/18 using a competitive Luminex immunoassay and for HPV-16/18 using a pseudovirion-based neutralization assay. RESULTS: Mean age was 30.8 years (range, 19-44 years). 71/87/73/81% of women were naive (sero-negative and DNA-negative) to HPV-6/11/16/18 at baseline, respectively. Among per-protocol women naive to HPV-6/11/16/18 at baseline, 100/99/99/90%, respectively, seroconverted at week 28 and 95/96/98/71% were sero-positive at week 52, respectively. Pseudovirion-based neutralization assay identified more seroconversion to HPV-18 than competitive Luminex immunoassay. There were no significant differences in the proportion seroconverting by baseline or nadir CD4 or HIV VL; however, there was a trend for increased proportion seroconverting to HPV-18 among women with higher baseline CD4 level (P = 0.052). There were no qHPV-related serious adverse events and no change in CD4 level or HIV VL among women on ART. CONCLUSIONS: qHPV vaccine was safe and immunogenic in Indian WLWH. A high proportion were naive to HPV-6/11/16/18 and may benefit from vaccination although many were married and several years post-initiation of sexual activity.

Antigen Nonspecific Induction of Distinct Regulatory T Cell States in Oncogene-Driven Hyperproliferative Skin.

Regulatory T cells (Tregs) are recruited to nonlymphoid tissues in chronic disease, including cancer, and the tissue environment is held to shape the Treg phenotype diversity. Using single-cell RNA sequencing, we examined the transcriptomic and TCR profile of Tregs recruited to hyperproliferative HPV16 E7-expressing transgenic and control nontransgenic murine skin grafts. Tregs were more abundant in E7 transgenic skin grafts than control grafts, without evidence of E7 specificity. E7 transgenic grafts attracted both Klrg1 + Tregs and Il1r2 + Tregs, which were phenotypically distinct but shared a core gene signature with previously described tumor-infiltrating Tregs. Pseudotime trajectory analysis of Tregs of defined TCR clonotypes predicted phenotypic plasticity within the skin and between the skin and draining lymph nodes. Thus, oncogene-induced hyperproliferative skin expressing a single defined non-self-antigen can attract and induce non-Ag-specific Tregs that acquire distinct regulatory phenotypes characterized by specific effector gene signatures.

Inflammation-related adverse reactions following vaccination potentially indicate a stronger immune response.

Concerns about vaccine safety are an important reason for vaccine hesitancy, however, limited information is available on whether common adverse reactions following vaccination affect the immune response. Data from three clinical trials of recombinant vaccines were used in this post hoc analysis to assess the correlation between inflammation-related solicited adverse reactions (ISARs, including local pain, redness, swelling or induration and systematic fever) and immune responses after vaccination. In the phase III trial of the bivalent HPV-16/18 vaccine (Cecolin®), the geometric mean concentrations (GMCs) for IgG anti-HPV-16 and -18 (P<0.001) were significantly higher in participants with any ISAR following vaccination than in those without an ISAR. Local pain, induration, swelling and systemic fever were significantly correlated with higher GMCs for IgG anti-HPV-16 and/or anti-HPV-18, respectively. Furthermore, the analyses of the immunogenicity bridging study of Cecolin® and the phase III trial of a hepatitis E vaccine yielded similar results. Based on these results, we built a scoring model to quantify the inflammation reactions and found that the high score of ISAR indicates the strong vaccine-induced antibody level. In conclusion, this study suggests inflammation-related adverse reactions following vaccination potentially indicate a stronger immune response.