Welcome to the IPVC 2023 Conference Program Scheduling
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SINGLE-CELL TRANSCRIPTIONAL PROFILING REVEALS A DIVERSE IMMUNE LANDSCAPE IN HUMAN PAPILLOMAVIRUS 6 OR 11-DRIVEN RECURRENT RESPIRATORY PAPILLOMAS
Abstract
Introduction
Immune constituency within HPV 6 or 11-related recurrent respiratory papillomas (RRP) is incompletely understood.
Methods
We performed single-cell transcriptomics and single-cell T cell receptor (TCR) sequencing on 13 papilloma specimens obtained from adult patients with RRP.
Results
Bioinformatic study of the myeloid cell compartment revealed multiple distinct clusters of neutrophilic and monocytic cells, including several that express high levels of immunosuppression-associated transcripts TREM2 and ARG1. Small populations of FOXP3, IL2RA and CTLA4 positive CD4 regulatory T cells were identified. Other CD4 T cell populations harbored expression profiles indicative of skewing away from anti-tumor Th1 helper function and toward production of Th2-related cytokines. Study of CD8 T cells revealed a broad diversity of transcriptional phenotypes, with some populations expressing high levels of exhaustion markers TOX, PDCD1, and LAG3 and the tissue retention marker CD103, all previously associated with tumor-antigen specificity in studies of malignancy. Overall, expression of activation markers IFNG, PRF and GZMs was low in most CD8 and CD4 clusters, indicating a lack of strong active HPV-specific T cell immunity and possibly local immunosuppression. TCRs from clonotypes within two distinct exhausted, CD103+ CD8 clusters were reconstructed and cloned into expression vectors to allow determination of antigen-specificity. Using a panel of in silico predicted HLA class I binding HPV6 and 11 epitopes and autologous antigen presenting cells, co-culture experiments are underway to identify and validate a library of HPV6 and 11-specific TCRs.
Conclusions
Our single cell analysis of RRP reveals immune-related cellular diversity and transcriptional phenotypes implying the presence of immunosuppressive myeloid cells and regulatory CD4 T cells. Experimental validation of the antigen-specificity of TCRs from papilloma infiltrating CD8 T cells is underway to determine if the inability of CD8 TIL to detect and eliminate HPV infected epithelial cells is due to lack of HPV antigen-specificity or local immunosuppression.
OROPHARYNGEAL TUMOR CELLS INDUCE COX-2 EXPRESSION IN PERIPHERAL BLOOD MONOCYTES BY SECRETION OF IL-1ALPHA
Abstract
Introduction
Introduction: Oropharyngeal squamous cell cancer (OPC) accounts for 3% of all cancers and greater than 1.5% of all cancer deaths in the United States, with marked treatment-associated morbidity in survivors. More than 80% of OPC is caused by HPV-16. Tumors induced by HPV have been linked to impaired immune functions in the local tumor microenvironment, with less focus on the effects these tumors have on systemic immune responses, especially innate responses.
Methods
Methods: PGE2 and IL-1 levels were measured by ELISA. COX-2 mRNA was determined by RT-Q-PCR, and COX-2 protein measured by Western Blot. RNA-SEQ was performed with Next-seq 550 (Ilumina) and analyzed with Partek software.
Results
Results: Plasma levels of PGE2 are elevated in OPC patients (p<0.0001) and decrease post-treatment. HPV-16+ OPC patients have higher plasma PGE2 levels than HPV- patients (p<0.01). COX-2 mRNA is elevated in patients’ monocytes compared to monocytes from controls (p<0.01). Conditioned media from primary cultures of OPC biopsies and tumor cell lines can induce COX-2 in monocytes, with induction significantly greater in patient’s monocytes than controls (p=0.002). We identified IL-1α expression by these tumors, and confirmed the presence of IL-1α, but not IL-1β, in the conditioned medium. Finally, both Anakinra, and rilonacept significantly block the effect of conditioned medium on monocytes, confirming that IL-1α is a major paracrine factor secreted by oropharyngeal tumors and tumor cell lines.
Conclusions
Conclusions: IL-1α secreted by oropharyngeal tumors activates monocytes to produce PGE2, a known immunosuppressant, suggesting that there is an enhanced immune-mediated feed-back on the tumors. Studies are ongoing to characterize additional pro-tumor immune modulators made by monocytes in response to tumor-conditioned medium. This should identify novel targets for immune intervention.
REDIRECTING PRE-EXISTING VIRAL IMMUNITY TOWARDS CANCERS VIA MURINE PAPILLOMAVIRUS T=1 VIRUS-INSPIRED PARTICLES (VIPS)
Abstract
Introduction
VerImmune is developing “Anti-tumor Immune Redirection” (AIR). This involves redirecting immune memory from viral infections to destroy tumor cells. Our strategy utilizes a T=1 icosahedral structure assembled from murine papillomavirus L1 capsid proteins. The assembled structure is termed 'Virus-inspired Particles' (ViPs) to differentiate them from conventional papillomavirus T=7 virus-like particles (VLPs) or Pseudovirions (PsVs). Here we show both in vitro and in vivo how ViPs can serve as a novel delivery system that redirects viral CD8+ T-cells to target non-virally related tumors for destruction
Methods
Tumor HSPG specific binding of our murine ViP was assessed against a panel of murine and human cancer cell lines in vitro. Cytotoxicity against these same murine and human cancer cell lines was next evaluated using co-culture T-cell assays. Demonstration of in vivo anti-tumor activity of the murine ViP via AIR was performed utilizing B16.F10 tumor bearing mice C57/BL6 mice harboring HPV16 E7 pre-existing immunity. Finally, to assess if murine ViPs are affected by pre-existing HPV or MusPV neutralizing antibodies, murine ViPs were mixed with these antibodies prior to binding and cytotoxicity assays.
Results
Characterization of ViPs reveals distinct biophysical differences from VLPs or PsVs. However, ViPs retain specificity to tumors via HSPG similar to HPV VLPs. In CD8+ T-cell co-culture assays, ViPs exerted specific HPV16 E7 and/or CMV pp65 “NLV” cytotoxic CD8+ T-cells respectively to kill non-antigenically related tumors. Pre-mixing of ViPs with either Gardasil-9 or mouse papillomavirus specific neutralizing sera did not block binding or cytotoxicity of the ViPs in vitro. Finally, in vivo anti-tumor activity against murine B16.F10 which does not contain any HPV E7 antigen was demonstrated.
Conclusions
Our results demonstrate the potential of AIR as a tumor antigen–agnostic immunotherapeutic. Additionally, the specificity of ViPs to tumor HSPGs makes AIR potentially applicable across many solid tumors regardless of their origin.
IN-DEPTH ASSESSMENT OF INNATE AND ADAPTIVE RESPONSES TO NINE-VALENT HPV VACCINE IN HUMANS
Abstract
Introduction
It is not known how human papillomavirus (HPV) vaccines stimulate the immune system to generate potent and durable antibody responses, but this information is valuable to vaccinology.
Methods
We are conducting a pilot cohort study to evaluate in-depth innate immune, B-, and CD4+ T-cell responses to the nine-valent HPV vaccine (9vHPV). Participants include 16 men and women, 18-45 years of age, who have never received an HPV vaccine, have no history of HPV disease, and are seronegative for all 9vHPV types in a validated M9ELISA (Panicker et al. J. Immunol. Methods 2021). Our goals are to identify innate immune pathways activated by the vaccine and determine if early gene expression signatures of vaccine-activated B- and CD4+ T-cells in secondary lymphoid tissues predict their fate in peripheral and primary lymphoid memory compartments. To assess peripheral and secondary lymphoid B- and CD4+ T-cell responses, blood samples and lymph node aspirates will be collected before and after each 9vHPV dose. Participants will also have bone marrow aspirates collected two years after the first 9vHPV dose, when HPV-specific antibody titers have plateaued, to identify long-lived HPV-specific plasma cells. We will determine the magnitude of vaccine-elicited B-cell, plasma cell, and CD4+ T-cell responses and conduct single cell RNAseq on these cells to determine gene expression signatures and paired antibody sequences for clonal fate mapping.
Results
We have screened 14 participants, 43% (n=6) were seronegative for all 9vHPV types by M9ELISA. The rest screen-failed, with one failing safety lab criteria and seven being seropositive for one or more HPV types. Baseline lymph node aspirates yielded 0.21-3.8x106 lymphocytes. Study design and current demographic and laboratory data (screen results, FNA yields pre- and post-vaccination) will be presented.
Conclusions
Our study is 37.5% enrolled and our results will advance critical human tissue immunology studies and vaccine development efforts for other pathogens.
RG2-VLP: A VACCINE DESIGNED TO BROADLY PROTECT AGAINST ANOGENITAL AND SKIN HUMAN PAPILLOMAVIRUSES CAUSING HUMAN CANCER
Abstract
Introduction
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 βHPV that are associated with CSCC in EV and immunocompromised patients. We describe the development of a two-antigen vaccine, RG2-VLP which is both protective in animal models against known oncogenic αHPVs as well as diverse βHPVs by incorporation into HPV16 and HPV18 L1 VLP of 20-amino-acid conserved protective epitopes derived from minor capsid protein L2.
Methods
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 βHPV sequence in the same region are displayed within the DE loop of HPV16 and HPV18 L1 VLP, respectively. We vaccinated rabbits and as well as FVB mice, including TMC6 or TMC8 knockouts as models of EV, to study the immunogenicity of the RG2-VLP vaccines across a wide variety of HPV genotypes. Longevity of RG2-VLP responses compared to Gardasil-9 over 1 year was also studied.
Results
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 β-types. RG2-VLP also 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 even after one year. However, only transfer of RG2-VLP antiserum effectively protected naive mice from challenge with all βHPVs tested.
Conclusions
Our observations suggest RG2-VLP's potential as a broad-spectrum vaccine to prevent αHPV-driven anogenital, oropharyngeal, and βHPV-associated cutaneous cancers. The duration of both 16/18 L1-VLP as well as RG1 specific responses by RG2-VLP also support long term immunogenecity.
INTRA-TUMOR IMMUNOTHERAPY OF HPV-ASSOCIATED CANCER.
Abstract
Introduction
In situ vaccination (ISV) is becoming a therapeutic strategy to stimulate and diversify anti-tumor immune responses. Ano-genital and part of oropharyngeal cancers (OPC) are HPV-associated. Since OPC therapy is burdened by comorbidities and high recurrence rates, new approaches like immunotherapy are needed. Both genital and oropharyngeal cancers are easily accessible for ISV. Our previous studies showed several HPV DNA vaccines were able to affect tumor growth in mouse models. Similar results were achieved by antibodies (intrabobies) in single-chain format (scFvs) against HPV16 E6 and E7 proteins. Present study was developed to determine if ISV may enhance immunotherapy effectiveness.
Methods
Several pre-clinical mouse models of HPV-associated cancer in subcutaneous or oral orthotopic localizations were utilized. Two DNA plasmids expressing anti-HPV16 E6 or E7 intrabodies were delivered intra-tumor, respectively. PGIPss-L2-E7 and E7-Sap (Italian and International Patent) DNA vaccines were utilized for ISV. PGIPss-L2-E7 vaccine is based on fusion HPV16 antigens (L2 and E7) linked to signal sequence (ss) of polygalacturonase-inhibiting protein (PGIPss) from Phaseolus vulgaris. E7-Sap vaccine is a fusion construct of a non-toxic saporin mutant (SAP) with harmless version of HPV16 E7 gene.
Results
Part of these results were already presented at 6th Meeting on Emerging Issues in Oncogenic Virus Research (June 2022 –Italy). We showed that both anti-HPV16 E6 and HPV16 E7 scFvs affected tumor progression in all experimental mouse models increasing apoptosis within the tumor. Systemic administration of PGIPss-L2-E7 was able to decrease tumor growth and additional intra-tumor delivery of DNA vaccines improved tumor growth control and induced strong apoptosis in neoplastic cells
Conclusions
ISV with HPV DNA vaccines as well as with intrabodies are effective in increasing antitumor responses. This activity seems to rely on driving tumor cells towards the apoptotic pathway. This scenario could open new perspectives of therapy by introducing ISV in immunotherapy scheduling.
NEXT GENERATION L2-BASED HPV VACCINES CROSS-PROTECT AGAINST CUTANEOUS PAPILLOMAVIRUS INFECTION AND TUMOR DEVELOPMENT
Abstract
Introduction
Licensed L1-VLP-based vaccines have been a great success in reducing anogenital cancers, but are limited regarding cross-protection against human papillomavirus (HPV) types not covered by the vaccine. Next generation vaccines induce broad cross-protection against highly conserved sequences of L2. We tested two novel L2-based HPV vaccine candidates, HPV16 RG1-VLP and CUT-PANHPVAX, in the preclinical model Mastomys coucha. These animals are naturally infected with the cutaneous Mastomys natalensis papillomavirus (MnPV), which induces cutaneous tumors.
Methods
In an exploratory setting, virus-free animals were vaccinated with HPV16 RG1-VLP, CUT-PANHPVAX, MnPV-VLPs (positive control) or PBS (negative control) prior to experimental MnPV-infection. Seroconversion against L2 peptides, presence of (cross-)neutralizing antibodies, viral load at the infection site and occurrence of skin tumors were examined. Microscopical examinations and immunofluorescent stainings were used to check the skin even for premalignant changes.
Results
Besides vaccine-specific seroconversion against HPV16 RG1 and CUT-PANHPVAX, the animals also developed cross-reactive antibodies against MnPV L2, which were cross-neutralizing MnPV pseudovirions in vitro. Like for the MnPV-VLP control group, both L2-based vaccines conferred in vivo protection since after experimental infection, viral loads in plucked hair were lower when compared to mock-vaccinated controls. Importantly, the formation of neutralizing antibodies, whether directed against L1-VLPs or L2, was able to prevent skin tumor formation. Consequently, while 83% of animals from the PBS group developed skin tumors, only 33% of the HPV16 RG1-VLP group, 17% CUT-PANHPVAX and 0% of those from the VLP group did so. Protected animals not even showed microscopical signs of MnPV infection in the skin.
Conclusions
For the first time, our study shows the proof-of-principle of two next generation L2-based vaccines that are currently entering clinical trials in an infection model with its genuine PV. Even across different PV genera, these L2-based vaccines have a promising efficacy to protect against HPV-induced skin tumors.
REPURPOSING ANTIVIRAL VACCINES FOR LOCAL IMMUNOTHERAPY AGAINST SOLID TUMORS
Abstract
Introduction
Local immunotherapy against solid tumors is considered a viable approach to stimulate the tumor immune microenvironment and to promote anti-tumor immune responses. We have previously shown that intratumoral recall of anti-cytomegalovirus memory CD4+ and CD8+ T cells was effective to control primary tumor growth and to promote epitope spreading in murine preclinical cancer models. Here, we interrogate whether preexisting anti-vaccine immunity induced by licensed subunit vaccines could be leveraged for local cancer immunotherapy in the TC-1 murine tumor model expressing the HPV16 oncogenes E6 and E7.
Methods
We selected Shingrix, a varicella zoster vaccine containing the glycoprotein E (gE) antigen and adjuvant AS01B, and Gardasil-9, a human papillomavirus vaccine containing the L1 virus-like particles and alum adjuvant for their unique ability to induce CD4 and CD8 T cell responses, respectively.
Results
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 the HPV16 E7 tumor antigen, to tumor immune activation and a profound 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 selected MHC-I-restricted L1 minimal peptide epitopes led to complete and durable remissions suggesting efficient tumor control by L1-specific CD8 T cells.
Conclusions
Together our results provide evidence that anti-viral subunit vaccines can be repurposed for local cancer therapy as safer and off-the-shelf agents. Such immunotherapeutics could be broadly implemented and provide greater access to cancer treatment in low-resource settings.
ROLE OF GENETIC DRIFT IN HPV EVOLUTION
Abstract
Introduction
Pervasive purifying selection on non-synonymous substitutions is a hallmark of papillomavirus genome history. However, codon-based evolutionary models do not explain HPV evolution.
Methods
More than a thousand complete genomes representing Alphapapillomavirus types, lineages and SNP variants were examined phylogenetically and interrogated for the number and position of DNA sequence motifs. Non-coding evolutionary signatures were investigated with Principal Components Analyses, Ancestral State Reconstructions and Phylogenetic Independent Contrasts. Natural selection on expressed amino acids was also evaluated with codon models of evolution.
Results
For anciently diverged Alphapapillomavirus types, composition of the 4 nucleotides and 14 established non-coding DNA sequence motifs suggested two groups. Among the more recently diverged highly oncogenic Alphapapillomavirus 9 types, DNA sequence motifs and nucleotide composition were also able to discriminate most viral types and even some lineages. Ancestral state reconstruction and Phylogenetic Independent Contrasts recovered ancient genome alterations, including CpG, APOBEC3, and TLR9 motifs. Features that were common to Alphapapillomavirus types included a predominance of APOBEC3 sites on the non-coding strand and CpG sites within the E2/E4 overlapping open reading frames. Surprisingly, ancestral state reconstruction recovered punctuated changes in the genome of HPV16 resulting in the fewest CpG sites and the largest number of palindromic motifs of all Alphapapillomavirus types. Bayesian Graphic Models indicated that the E6 locus contains a high proportion of amino acid changes that phylogenetically covary with other sites in the genome. Patterns of nonsynonymous substitutions reflected purifying selection as expected.
Conclusions
Whole genome phylogenetic comparative methods do not indicate a single mode of evolution to explain the diversity of Alphapapillomavirus. Implicit in non-recombining genomes is their evolutionary independence after genetic isolation, divergence, and expansion. Each evolutionary analytical method supports the unanticipated conclusion that genetic drift and different evolutionary drivers have structured Alphapapillomavirus genomes in distinct ways during successive epochs, even extending to differences in variant lineages.