3084 Background: Belzupacap sarotalocan (bel-sar) is a novel virus like-drug conjugate in clinical stage development for multiple solid tumors. The virus-like particle component of the drug is derived from the papillomavirus and specifically binds to heparan sulfate proteoglycans (HSPGs) and chondroitin sulfate proteoglycans (CSPGs) with unique sulfation modifications on the surface of cancer cells. Upon activation with near-infrared light, cell associated bel-sar causes immediate cell death and induces an anti-tumor immune response, with a single administration leading to elimination of solid tumors accompanied by long-term protective immunity in a number of animal tumor models. Cancer stem cells (CSCs) represent a distinct subpopulation of tumor cells that possess stem-like characteristics, including self-renewal. These cells are thought to drive disease persistence and are intrinsically more resistant to conventional cancer treatment, such as chemotherapy, radiotherapy, and immunotherapy. CSC-mediated resistance remains a major obstacle to achieving durable clinical responses and the elimination of this cell population is hypothesized to be essential for long-lasting tumor control. Methods: We developed and characterized a panel of human-derived CSC models (bladder, breast, oropharyngeal, prostate cancers, glioblastoma, uveal melanoma) using a SORE6 lentivirus reporter in which stem-like transcription factors SOX2 and OCT4 drive GFP expression to identify CSC subpopulations. Bel-sar binding and potency was compared between CSC and the more differentiated non-cancer stem cells (nCSC) populations. Additionally, chemotherapy resistant tumor lines were established from the CSC panel and were similarly tested for bel-sar targeting and cytotoxicity. Results: CSC populations were validated by SOX2/OCT4 co-expression, CSC surface marker expression (CD24, CD44, EpCAM-1, CXCR4), ALDH activity, and tumorsphere formation. Bel-sar was capable of binding CSCs with equal or enhanced efficiency as compared to nCSCs through cell surface HSPGs and CSPGs as demonstrated by heparin and chondroitin sulfate inhibition. Additionally, bel-sar was able to kill CSCs and nCSCs with equal efficiency in vitro. Following chemotherapy-induced enrichment of CSCs, bel-sar maintained its therapeutic effect on chemotherapy-resistant CSC populations, reinforcing previous data demonstrating complete responses in animal models. Conclusions: This study highlights bel-sar’s distinctive capacity to target and eradicate CSCs across diverse tumor types. These results support bel-sar’s clinical development for local tumor control and suggest bel-sar treatment may be effective against recurring or chemotherapy-resistant tumors.

The commercial prophylactic vaccines for human papillomavirus (HPV) are subunit vaccines composed of spontaneously assembled virus-like particles formed from 360 copies of the L1 major virion protein. The vaccines have exceeded expectations in immunogenicity, clinical trials efficacy, and effectiveness in national immunisation programmes. Booster doses are not required to generate these outcomes. In this Review, we discuss the immunological and virological considerations that can help with explaining the high potency of HPV vaccines. These considerations provide biological plausibility for HPV vaccines being the first subunit vaccines to confer high-level, long-term protection after a single priming dose, support projections of eliminating vaccine-targeted types in populations with achievable coverage rates in adolescents, and offer insights for developing future antimicrobial vaccines.

Herpes simplex virus 2 (HSV-2) is associated with genital ulcers, neonatal encephalitis, increased risk of HIV infection, and dementia. There is no licensed HSV-2 vaccine. We developed nanoparticles displaying the HSV-2 attachment protein gD and fusion mediation protein complex gH/gL. Immunization of mice and non-human primates elicited high levels of neutralizing antibodies. Vaccination conferred robust protection in mice, preventing disease and nearly eliminating infection and shedding following HSV-2 challenge. While gD induced high neutralizing antibody titers, gH/gL contributed substantially to protection despite lower neutralization titers. Instead, gH/gL immunization generated strong fusion-blocking responses which were an important correlate of protection, showing that standard neutralization assays incompletely capture the importance of fusion-blocking activity. These findings demonstrate that targeting both HSV-2 attachment and fusion elicit complementary mechanisms for protection from infection and that neutralizing antibody alone may be insufficient for protection. Overall, these results present an innovative strategy for an HSV-2 vaccine.

Human papillomavirus variants are classified into lineages based on their whole genome sequence, but the impact of lineage variation on the structure or function of encoded proteins is unclear. We used a global panel of lineage-specific natural infection sera to assess antibody-binding specificity for lineage-specific L1L2 antigens, and we used these data to create relational antigenic maps. Merging these data with neutralizing antibody data demonstrated similar spatial geometry and revealed dependency on a limited number of amino residues on the capsid surface. These data inform the degree of lineage specificity within the natural infection humoral immune response to oncogenic human papillomaviruses.

Intratumoral (IT) immunotherapy can stimulate the tumor microenvironment and enhance anti-tumor immunity. We investigated IT delivery of three licensed viral vaccines—Shingrix (VZV shingles), Gardasil-9 (HPV), and Spikevax (SARS-CoV-2)—in prevaccinated mice using the murine tumor model TC-1, which expresses HPV16 oncogenes E6 and E7. Shingrix IT injection often induced tumor regression and resistance to secondary challenge. Injecting a VZV glycoprotein E (gE)-derived MHC-II-restricted peptide with polyI:C also led to durable remission, highlighting the role of gE-specific CD4+ T cells. While Gardasil-9 IT injection alone was ineffective, combining a HPV L1-derived MHC-I-restricted peptide with polyI:C or Shingrix enhanced tumor regression. Both approaches elicited CD8+ T cells against the E7 tumor viral oncoprotein. Tumor microenvironment analysis revealed remodeling of the myeloid compartment, significant induction of IFN-γ, TNF-α, and CXCL9 and broad gene expression reprograming. In a dual-flank model, IT injection of Shingrix with an MHC-I-restricted E7 tumor-specific peptide eliminated primary and non-injected tumors. Finally, Spikevax IT injection showed modest tumor growth delay, while improved control was observed with a SARS-CoV-2 spike-derived MHC-I-restricted peptide and polyI:C. These results demonstrate the potential of licensed vaccines as promising platforms for IT immunotherapy, either alone or combined with vaccine- or tumor-derived MHC-I-restricted peptide epitopes.

BACKGROUND:Multidose human papillomavirus (HPV) vaccination is efficacious, yet the vaccine has been underused globally. Emerging data suggest that a single dose may provide protection. Whether a single dose of HPV vaccine would provide similar protection to two doses is uncertain. METHODS:In this trial, we assessed whether one dose of an HPV vaccine was noninferior to two doses. Girls 12 to 16 years of age were randomly assigned, in a 1:1:1:1 ratio, to receive one or two doses of a bivalent HPV vaccine or one or two doses of a nonavalent HPV vaccine. The primary end point was new HPV type 16 or 18 infection occurring from month 12 to month 60 and persisting for at least 6 months. The prespecified noninferiority margin was 1.25 infections per 100 participants. We also assessed vaccine effectiveness by comparing HPV16 or HPV18 infection among the trial participants with that among girls and women enrolled in a nonrandomized survey. RESULTS:A total of 20,330 participants were enrolled and underwent randomization, and 3005 unvaccinated participants were enrolled in the survey. The noninferiority analysis showed that one vaccine dose was noninferior to two doses in preventing HPV16 or HPV18 infection. The rate difference between one and two doses of the bivalent vaccine was -0.13 infections per 100 participants (95% confidence interval [CI], -0.45 to 0.15; P<0.001 for noninferiority), and the difference between one and two doses of the nonavalent vaccine was 0.21 infections per 100 participants (95% CI, -0.09 to 0.51; P<0.001 for noninferiority). The vaccine effectiveness was at least 97% in each of the four trial groups. No safety concerns were identified. CONCLUSIONS:One dose of either a bivalent or nonavalent HPV vaccine provided protection against HPV16 or HPV18 infection and was not inferior to two doses. (Funded by the National Cancer Institute and others; ESCUDDO ClinicalTrials.gov number, NCT03180034.).

Supplemental Table 1. 15 leading causes of cancer death in the U.S. in 2019. Supplemental table 2. Trends in total cancer mortality rates, incidence rates and relative survival in the U.S. Supplemental table 3. Trends in lung cancer mortality rates, incidence rates and relative survival in the U.S. Supplemental table 4. Trends in colorectal cancer mortality rates, incidence rates and relative survival in the U.S. Supplemental table 5. Trends in pancreatic cancer mortality rates, incidence rates and relative survival in the U.S. Supplemental table 6. Trends in female breast cancer mortality rates, incidence rates and relative survival in the U.S. Supplemental table 7. Trends in prostate cancer mortality rates, incidence rates and relative survival in the U.S. Supplemental table 8. Trends in liver/IHBD cancer mortality rates, incidence rates and relative survival in the U.S. Supplemental table 9. Trends in cancer mortality rates in the U.S., 2000-2019 for selected sites. Supplemental Table 10. US breast cancer incidence-based mortality*. Supplemental Table 11. U.S. breast cancer incidence-based mortality by age at death. Supplemental Figure 1. Liver cancer incidence (1992-2019), survival (2000-2019) and mortality (2000-2019 projected through 2047). This figure excludes cases of intrahepatic bile duct cancer.

Background: New approaches to control HPV infections and prevent progression to cervical precancer are needed. We investigate the probability of viral clearance and progression to cervical precancer in women infected with HPV16/18 without evidence of precancer at study enrollment to inform research efforts targeted at reducing cervical cancer. Methods: We included 530 women aged 18-25 who tested HPV16/18 DNA-positive and did not have cytological evidence of high-grade-squamous-intraepithelial-lesion (HSIL) at enrollment in CVT. At each visit, clinicians collected cervical cells for cytology and HPV-DNA testing. Those with abnormal cytology were referred to colposcopy, biopsy, and treatment as needed. We estimated the probability of HPV clearance (loss of detection) and progression to intraepithelial neoplasia grades 2 or 3 or worse (CIN2+, CIN3+) based on histological findings by expert pathologists over 4-years of follow-up. Results: At enrollment, there were 550 prevalently detected HPV16 and/or HPV18 infections among 530 women without cytologic HSIL. Corresponding probabilities of HPV16 and HPV18 clearance were 68.5 % (95 %CI 63.6 %-73.0 %) and 85.0 % (78.6 %-90.1 %) by 24-months and 82.1 % (78.0 %-85.7 %) and 90.2 % (84.7 %-94.2 %) by 48-months after initial detection. Risk of clearance of prevalently detected HPV16 and HPV18 infections decreased with increasing age by 12-, 24-, 36-, and 48-months after initial detection (p(trend)-values >= 0.31). Among the same 530 women, by 24-months and 48-months of follow-up, the corresponding probabilities of progression to CIN2+ were 5.5 % (3.8 %-7.7 %) and 13.0 % (10.4 %-16.1 %), and to CIN3+ were 2.6 % (1.5 %-4.3 %) 7.5 % (5.5 %-10.0 %). Probability of progression was 2-5 times higher in women with HPV16 infections than with HPV18. Risk of progression to CIN2+ and CIN3+ increased with increasing age during the follow-up period (p(trend)-values >= 0.11). Conclusions: Among young adult women without evidence of HSIL by cytology, clearance of prevalently detected HPV16/18 infection is a common event, and progression to precancer occurs infrequently but in a sizeable proportion of those with prevalent infection.

Human papillomavirus (HPV) 16 is transported in a retrograde fashion from the cell surface to the Golgi apparatus. Prior to mitosis, the virus loses association with the Golgi and, following nuclear envelope breakdown, is found associated with the condensed mitotic chromatin. The intervening steps have not been well defined. It was previously demonstrated that the virus is transported to the mitotic chromosomes in vesicles. Here, we describe the role of the endoplasmic reticulum (ER) in the post-Golgi trafficking and the importance of the ER-generated coat protein complex II (COPII) anterograde trafficking pathway in HPV infection. HPV pseudovirus (PsV) colocalized with COPII components and silencing of this pathway inhibited HPV infection. Additionally, the inner COPII coat protein, Sec24b, could be biochemically isolated in association with HPV capsid proteins. This study provides insight into the mechanism of post-Golgi HPV trafficking.

HPV vaccines exhibit high type-specific and antibody-mediated protection against anogenital infection, even after a single dose. Complete and long-term “sterilizing” immunity against incident infection appears to be established in most HPV vaccinees, suggesting that not only are persistent levels of virus-inhibiting antibodies routinely generated but that they are also exceptionally potent at preventing infection. The process of HPV infection is unusually protracted at several steps, including slow internalization after the virions bind to the cell surface. This observation prompted us to comprehensively evaluate the ability of neutralizing antibodies to prevent infection subsequent to HPV pseudovirion attachment to cells. Using sera and memory B cell-derived monoclonal antibodies from Gardasil-vaccinated women, we observed almost complete post-attachment neutralization of HPV16 pseudovirion infection of HaCaT cells three hours after attachment, even when vaccinees’ sera were diluted 250-fold, with a gradual loss of activity up to 18 h. Unexpectedly, three distinct mechanisms of post-attachment neutralization were discovered, capsid shedding from the cell surface, capsid retention on the cell surface, and rapid capsid degradation after internalization.

The use of mouse papillomavirus (MmuPV1) to study infections, disease outcomes, and vaccine strategies in mice has greatly enhanced our understanding of human papillomavirus. However, as with other species-specific infectious agents used as models for human disease, such studies may pose a risk to facilities that house large numbers of the model agent's natural host, especially when the full natural history of the infection is uncertain. In this study, we describe our recent experience showing that containment of MmuPV1 can be difficult, and that its use in research facilities may cause unexpected, long-lasting environmental contamination. Following the identification of symptomatic index cases of MmuPV1 in nude mice, we identified widespread contamination of an ∼10,000 cage facility, including MmuPV1 infection in mice of varying strains and immunocompetencies. Concerningly, many years separated the experimental use of MmuPV1 in the facility and our subsequent identification of index cases. We report our methods to identify, survey, and eliminate MmuPV1 from the facility, and the evolution of decontamination procedures that proved successful.

BACKGROUND:In 2022, WHO recommended single-dose human papillomavirus (HPV) vaccination as an alternative schedule to multidose regimens. To provide evidence to support approval of a single-dose indication for the AS04-adjuvanted bivalent HPV vaccine (Cervarix, GlaxoSmithKline), we investigated whether the immune response to a single dose of the bivalent vaccine in girls aged 9-14 years was non-inferior to the immune response to three doses of the quadrivalent HPV vaccine (Gardasil-4, Merck) in women aged 18-25 years, a dose and population combination with demonstrated efficacy. METHODS:This non-randomised, open-label, immunobridging trial enrolled girls aged 9-14 years and women aged 18-25 years in Guanacaste Province, Costa Rica. Healthy girls aged 9-14 years received one dose of bivalent HPV vaccine, whereas healthy women aged 18-25 years received three doses of quadrivalent HPV vaccine at 0, 2, and 6 months. The primary endpoint was geometric mean concentrations (GMCs) of HPV-specific serum antibodies measured by a validated virus-like-particle-based ELISA assay at 36 months. The per-protocol cohort included participants who received the correct number of doses within the predefined vaccination windows, had blood collected at the 36-month study visit for the final analysis, were seronegative at baseline for the specified HPV type, and did not receive additional HPV vaccine doses outside the study. Non-inferiority was declared when the lower bound of the 96% CI for the GMC ratio was greater than or equal to 0·67 for HPV-16 and HPV-18. Seropositivity was a secondary objective. Safety was analysed in the total vaccinated population. This trial is registered with ClinicalTrials.gov, NCT03728881, and is complete. FINDINGS:Between April 1 and Aug 16, 2019, 620 girls and 620 women were enrolled and received their first HPV vaccination. After exclusions, 539 girls and 366 women were HPV-16 seronegative at enrolment and were included in the HPV-16 per-protocol cohort; 523 girls and 373 women were HPV-18 seronegative at enrolment and were included in the HPV-18 per-protocol cohort. At 36 months, the HPV-16 GMC was 21·4 international units (IU)/mL (95% CI 19·7-23·3) in girls in the single-dose bivalent vaccine group and 42·9 IU/mL (95% CI 38·9-47·3) in women in the three-dose quadrivalent vaccine group, resulting in a GMC ratio of 0·50 (96% CI 0·44-0·57); the HPV-18 GMC was 8·0 IU/mL (95% CI 7·4-8·8) in girls in the single-dose bivalent vaccine group and 7·2 IU/mL (95% CI 6·4-8·1) in women in the three-dose quadrivalent vaccine group, resulting in a GMC ratio of 1·11 (96% CI 0·95-1·29). At 36 months, 538 (99·8%, 95% CI 99·1-100) of 539 girls in the single-dose bivalent vaccine group were seropositive for HPV-16 compared with 366 (100%, 99·2-100) of 366 women in the three-dose quadrivalent vaccine group (p=1·00). The proportion of participants who were seropositive for HPV-18 was higher in the single-dose bivalent vaccine group (517 [98·9%, 95% CI 97·6-99·5] of 523 girls) than in the three-dose quadrivalent vaccine group (358 [96·0%, 93·6-97·6] of 373 women; p=0·0065). Two serious adverse events were reported in 620 girls and 13 serious adverse events were reported in 620 women; all serious adverse events were deemed to be unrelated to HPV vaccination. INTERPRETATION:Non-inferior antibody responses for the single-dose bivalent HPV vaccine were seen for HPV-18 but not HPV-16, which would be insufficient evidence to motivate regulatory change, even though seropositivity approached 100% in the follow-up phase and the observed antibody concentrations were similar to protective levels seen in previous trials. Trials that directly evaluate protection afforded by single-dose HPV vaccination against persistent HPV infection will definitively address the level of protection afforded by single-dose HPV vaccination. FUNDING:National Cancer Institute, Cancer Research UK, and the Gates Foundation. TRANSLATION:For the Spanish translation of the abstract see Supplementary Materials section.

B cell responses to membrane-presented antigens appear to be strongly favored over soluble antigens in vivo suggesting that vaccines that mimic membrane-presented antigens may be highly efficacious. We recently demonstrated that human B cell responses to membrane-associated but not to soluble antigens in vitro depended on the expression and activity of the plasma membrane mechanosensitive ion channel, Piezo1. Here, we provide evidence that the efficacy of the current human papillomavirus virus-like particle (HPV VLP) vaccines may be due in part to their inherent ability to mimic Piezo1-dependent membrane presentation of antigens to B cells. We compared HPV-specific human B cell responses to HPV VLPs versus soluble HPV pentameric capsomeres and showed that although both induced calcium responses, only HPV VLP-induced responses were blocked by Piezo1 inhibitors. The kinetics of internalization of HPV-VLP and capsomeres into HPV-specific B cells were similar and neither required Piezo1 function as shown by small interfering RNA (siRNA)-mediated knockdown of Piezo. However, trafficking of HPV-VLPs into intracellular major histocompatibility complex (MHC) class II, lysosomal associated membrane protein 1 (LAMP1)+ antigen-processing compartments was Piezo1-dependent, whereas trafficking of capsomeres was not. In addition, a time course of intracellular trafficking suggested that colocalization of HPV-VLP with MHC classII was more stable over time as compared to capsomeres. Taken together these findings suggest that the ability of HPV-VLP vaccines to mimic Piezo1-dependent membrane antigen presentation may be exploited in the design of highly effective human vaccines.

Human Papillomavirus (HPV) type variants have been classified into lineages and sublineages based upon their whole genome sequence. Here we have examined the specificity of antibodies generated following natural infection with lineage variants of oncogenic types (HPV16, 18, 31, 33, 45, 52 and 58) by testing serum samples assembled from existing archives from women residing in Africa, The Americas, Asia or Europe against representative lineage-specific pseudoviruses for each genotype. We have subjected the resulting neutralizing antibody data to antigenic clustering methods and created relational antigenic profiles for each genotype to inform the delineation of lineage-specific serotypes. For most genotypes, there was evidence of differential recognition of lineage-specific antigens and in some cases of a sufficient magnitude to suggest that some lineages should be considered antigenically distinct within their respective genotypes. These data provide compelling evidence for a degree of lineage specificity within the humoral immune response following natural infection with oncogenic HPV.

The AS04-adjuvanted human papillomavirus (HPV)16/18 vaccine, an L1-based vaccine, provides strong vaccine efficacy (VE) against vaccine-targeted type infections, and partial cross-protection to phylogenetically-related types, which may be affected by variant-level heterogeneity. We compared VE against incident HPV31, 33, 35, and 45 detections between lineages and SNPs in the L1 region among 2846 HPV-vaccinated and 5465 HPV-unvaccinated women through 11-years of follow-up in the Costa Rica HPV Vaccine Trial. VE was lower against HPV31-lineage-B (VE=60.7%;95%CI = 23.4%,82.8%) compared to HPV31-lineage-A (VE=94.3%;95%CI = 83.7%,100.0%) (VE-ratio = 0.64;95%CI = 0.25,0.90). Differential VE was observed at several lineage-associated HPV31-L1-SNPs, including a nonsynonymous substitution at position 6372 on the FG-loop, an important neutralization domain. For HPV35, the only SNP-level difference was at position 5939 on the DE-loop, with significant VE against nucleotide-G (VE=65.0%;95%CI = 28.0,87.8) but not for more the common nucleotide-A (VE=7.4%;95%CI = -34.1,36.7). Because of the known heterogeneity in precancer/cancer risk across cross-protected HPV genotype variants by race and region, our results of differential variant-level AS04-adjuvanted HPV16/18 vaccine efficacy has global health implications.

Local immunotherapy against solid tumors is considered a viable approach to stimulate the tumor immune microenvironment and anti-tumor immunity. Here, we interrogate whether preexisting anti-vaccine immunity induced by licensed subunit vaccines could be leveraged for local cancer immunotherapy in the syngeneic murine tumor models. We selected Shingrix, a VZV vaccine containing the glycoprotein E (gE) antigen and adjuvant AS01B, and Gardasil-9, a HPV vaccine containing the L1 virus-like particles and alum because of the CD4 and CD8 T cell responses they respectively induce. Intratumoral injection of Shingrix alone or with immune checkpoint blockade (CTLA-4), in prevaccinated mice delayed tumor growth and often led to complete regression. These responses were associated with the induction of CD8+ T cell responses against tumor associated antigen, to tumor immune activation and alteration of the myeloid compartment. The injection of selected MHC-II-restricted gE minimal peptide epitopes combined with polyI:C also led to durable remission suggesting a contribution of gE-specific CD4 T cells. In contrast, Gardasil-9 i.t. injection did not delay tumor growth or cause tumor rejection which suggests inefficient class I cross-presentation of native VLP in the tumor cells. However, the injection of MHC-I-restricted L1 minimal peptide epitopes led to complete and durable remissions suggesting efficient tumor control by L1-specific CD8 T cells. Our results suggest that anti-viral licensed vaccines can be leverage as a new class of immunotherapeutics for local cancer therapy and intrinsic immunogenicity properties of vaccines should be considered as these effects appeared to be vaccine specific. NIH Intramural program.

BACKGROUND:The Costa Rica HPV Vaccine Trial provided initial evidence that 1 dose of the bivalent human papillomavirus (HPV) vaccine induces stabilizing antibody levels that may provide extended protection against HPV-16/18 infections. We report antibody seropositivity and stability 11 to 16 years after vaccination. METHODS:We invited a random subset of Costa Rica HPV Vaccine Trial participants (n = 398) who had received 3 doses and all women (n = 203) who had received 1 dose at 18 to 25 years of age to follow-up visits 11, 14, and 16 years after vaccination. We calculated HPV-16 and HPV-18 seropositivity and assessed change in enzyme-linked immunosorbent assay antibody levels 11 to 16 years after vaccination among 500 participants. RESULTS:By year 16, 99.4% (95% confidence interval [CI] = 96.8% to 100.0%) and 100.0% (95% CI = 98.9% to 100.0%) of 1-dose and 3-dose recipients, respectively, were HPV-16 seropositive and 98.8% (95% CI = 95.9% to 99.9%) and 100% (95% CI = 98.9% to 100.0%) of 1-dose and 3-dose recipients, respectively, were HPV-18 seropositive. Between years 11 and 16, women who had received 3 doses had a small but statistically significant decrease in the geometric mean concentration for HPV-16 of ‒12.4% (95% CI = ‒16.3% to ‒8.4%) and HPV-18 of ‒13.4% (95% CI = ‒17.2% to ‒9.4%). Among women who had received 1 dose, the decrease was statistically significant for HPV-16 at ‒8.9 (95% CI = ‒14.2% to ‒3.1%) but nonsignificant for HPV-18. Geometric mean concentration ratios of 3:1 dose (year 16) were 3.0 and 2.2 for HPV-16 and HPV-18, respectively. CONCLUSIONS:HPV-16/18 seropositivity remained exceedingly high 16 years after vaccination. Over 5 years, small declines in antibodies were observed. Women should have protection for at least 20 years and likely much longer at the observed rate of decline.

Abstract Local immunotherapy against solid tumors is considered a viable approach to stimulate the tumor microenvironment (TME) and anti-tumor immunity. We assessed in situ vaccination with licensed subunit vaccines to leverage preexisting anti-vaccine immunity in a tumor model expressing HPV16 viral oncogenes E6 and E7. We chose Shingrix, a VZV vaccine containing the glycoprotein E (gE), and Gardasil-9, an HPV vaccine containing the L1 virus-like particles (VLP) to preferentially induce CD4 and CD8 T cell responses, respectively. Intratumoral (IT) injection of Shingrix in prevaccinated mice often led to complete regression and elicited CD8 T cell responses against E7. IT injection of an MHC-II-restricted gE peptide with polyI:C also led to durable remission, suggesting a role for gE-specific CD4 T cells. In contrast, IT injection of Gardasil-9 did not delay tumor growth, but IT injection of an MHC-I-restricted L1 peptide with Shingrix led to complete remissions hence overcoming preexisting antibodies against native HPV VLP. TME analysis showed that IT injection of Shingrix with Gardasil-derived peptide induced IFN-g, TNF-a and CXCL9 and reshaped the myeloid cell infiltrate. Finally, IT injection of Shingrix and the E7 viral neoantigen peptide led to the eradication of all primary injected and abscopal tumors. Our results indicate that Shingrix is a versatile component for in-situ vaccination which can be combined with peptides derived from licensed vaccines or tumor antigens.