Antibodies against high-risk human papillomavirus proteins as markers for noncervical HPV-related cancers in a Black South African population, according to HIV status.

Human papillomavirus (HPV) proteins may elicit antibody responses in the process toward HPV-related malignancy. However, HPV seroepidemiology in noncervical HPV-related cancers remains poorly understood, particularly in populations with a high prevalence of human immunodeficiency virus (HIV). Using a glutathione S-transferase-based multiplex serology assay, antibodies against E6, E7 and L1 proteins of HPV16 and HPV18 were measured in sera of 535 cases of noncervical HPV-related cancers (anal (n = 104), vulval (n = 211), vaginal (n = 49), penile (n = 37) and oropharyngeal (n = 134)) and 6651 non-infection-related cancer controls, from the Johannesburg Cancer Study that recruited Black South African with newly diagnosed cancer between 1995 and 2016. Logistic and Poisson regression models were used to calculate adjusted odds ratios (aOR) and prevalence ratios (aPR) and 95% confidence intervals (CI) in cases versus controls. HPV16 E6 was more strongly associated with noncervical HPV-related cancers than HPV16 L1 or E7, or HPV18 proteins: anal (females (HPV16 E6 aOR = 11.50;95%CI:6.0-22.2), males (aOR = 10.12;95%CI:4.9-20.8), vulval (aOR = 11.69;95%CI:7.9-17.2), vaginal (aOR = 10.26;95%CI:5.0-21), penile (aOR = 18.95;95%CI:8.9-40), and oropharyngeal (females (aOR = 8.95;95%CI:2.9-27.5), males (aOR = 3.49;95%CI:1.8-7.0)) cancers. HPV16-E6 seropositivity ranged from 24.0% to 35.1% in anal, vulval, vaginal and penile cancer but was significantly lower (11.2%) in oropharyngeal cancer. After adjustment for HIV, prevalence of which increased from 22.2% in 1995-2005 to 54.1% in 2010-2016, HPV16 E6 seropositivity increased by period of diagnosis (aPR for 2010-2016 vs. 1995-2006 = 1.84;95%CI:1.1-3.0). Assuming HPV16 E6 seroprevalence reflects HPV attributable fraction, the proportion of certain noncervical-HPV-related cancers caused by HPV is increasing over time in South Africa. This is expected to be driven by the increasing influence of HIV.

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.

Human papillomavirus 68 prevalence and genetic variability based on E6/E7 genes in Sichuan.

HPV68 is a common HR-HPV, its persistent infection is closely related with the occurrence of cervical cancer. In this study, 2939 (27.60%, 2939/10650) positive samples were detected, and 174 (5.92%, 174/2939) were HPV68. 150 HPV68 E6-E7 were successful sequenced, 4 non-synonymous mutations were detected in E6, and E7 were 12. N133S non-synonymous mutations of HPV 68 E6 and C67G, T68 A/M of HPV68 E7 are E6, E7 positive selection sites, they all located in the key domains and major motifs of E6/E7 protein, the above amino-acid substitutions changed the protein structure, disturbed the interaction with other protein or cellular factors and make a difference in epitopes affinity, may affect the pathogenicity and adaptability of HPV68 to the environment. The enrichment of HPV68 data is of great significance for understanding the inherent geographical and biological differences of HPV68 in China.

Depletion of Human Papilloma Virus E6- and E7-Oncoprotein-Specific T-Cell Responses in Women Living With HIV.

Background: Cervical cancer - caused by persistent High Risk Human Papilloma Virus (HR HPV) infections - is the second most common cancer affecting women globally. HIV infection increases the risk for HPV persistence, associated disease progression and malignant cell transformation. We therefore hypothesized that this risk increase is directly linked to HIV infection associated dysfunction or depletion of HPV-oncoprotein-specific T-cell responses. Methods: The 2H study specifically included HIV+ and HIV- women with and without cervical lesions and cancer to analyze HPV oncogene-specific T cell responses in relation to HPV infection, cervical lesion status and HIV status. Oncoprotein E6 and E7 specific T-cell responses were quantified for the most relevant types HPV16, 18 and 45 and control antigens (CMV-pp65) and M.tb-PPD in 373 women, using fresh peripheral blood mononuclear cells in an IFN-γ release ELISpot assay. Results: Overall, systemic E6- and E7-oncoprotein-specific T-cell responses were infrequent and of low magnitude, when compared to CMV-pp65 and M.tb-PPD (p < 0.001 for all HR HPV types). Within HIV negative women infected with either HPV16, 18 or 45, HPV16 infected women had lowest frequency of autologous-type-E6/E7-specific T-cell responses (33%, 16/49), as compared to HPV18 (46% (6/13), p = 0.516) and HPV45 (69% (9/13), p = 0.026) infected women. Prevalent HPV18 and 45, but not HPV16 infections were linked to detectable oncoprotein-specific T-cell responses, and for these infections, HIV infection significantly diminished T-cell responses targeting the autologous infecting genotype. Within women living with HIV, low CD4 T-cell counts, detectable HIV viremia as well as cancerous and precancerous lesions were significantly associated with depletion of HPV oncoprotein-specific T-cell responses. Discussion: Depletion of HPV-oncoprotein-specific T-cell responses likely contributes to the increased risk for HR HPV persistence and associated cancerogenesis in women living with HIV. The low inherent immunogenicity of HPV16 oncoproteins may contribute to the exceptional potential for cancerogenesis associated with HPV16 infections.

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.

Targeting Human Papillomavirus-Associated Cancer by Oncoprotein-Specific Recombinant Antibodies.

In recent decades, recombinant antibodies against specific antigens have shown great promise for the therapy of infectious diseases and cancer. Human papillomaviruses (HPVs) are involved in the development of around 5% of all human cancers and HPV16 is the high-risk genotype with the highest prevalence worldwide, playing a dominant role in all HPV-associated cancers. Here, we describe the main biological activities of the HPV16 E6, E7, and E5 oncoproteins, which are involved in the subversion of important regulatory pathways directly associated with all known hallmarks of cancer. We then review the state of art of the recombinant antibodies targeted to HPV oncoproteins developed so far in different formats, and outline their mechanisms of action. We describe the advantages of a possible antibody-based therapy against the HPV-associated lesions and discuss the critical issue of delivery to tumour cells, which must be addressed in order to achieve the desired translation of the antibodies from the laboratory to the clinic.

Immunological responses and anti-tumor effects of HPV16/18 L1-L2-E7 multiepitope fusion construct along with curcumin and nanocurcumin in C57BL/6 mouse model.

AIMS: Human papillomavirus (HPV) L1, L2 and E7 proteins were used as target antigens for development of preventive and therapeutic vaccines. Moreover, linkage of antigens to heat shock proteins (HSPs) could enhance the potency of vaccines. Curcumin and nanocurcumin compounds were suggested as the chemopreventive and chemotherapeutic agents against cancer. In this study, two multiepitope DNA and peptide-based vaccine constructs (L1-L2-E7 and HSP70-L1-L2-E7) were used along with curcumin and nanocurcumin to evaluate immune responses, and protective/therapeutic effects in tumor mouse model. MAIN METHODS: At first, the multiepitope L1-L2-E7 and HSP70-L1-L2-E7 fusion genes were subcloned in eukaryotic and prokaryotic expression vectors. The recombinant multiepitope peptides were generated in E. coli strain. Then, the cytotoxic effects of curcumin and nanocurcumin were evaluated on HEK-293 T non-cancerous and C3 cancerous cells. Finally, mice vaccination was performed using different regimens. Curcumin and nanocurcumin compounds were administered alone or along with different vaccine constructs. KEY FINDINGS: Our data indicated that the use of nanocurcumin along with the multiepitope HSP70-L1-L2-E7 vaccine construct could completely protect mice against HPV-related C3 tumor cells, and eradicate tumors in a therapeutic test. Furthermore, nanocurcumin showed higher protection than curcumin alone. Generally, curcumin and nanocurcumin compounds could reduce tumor growth synergistically with the multiepitope vaccine constructs, but they did not influence the immune responses in different regimens. SIGNIFICANCE: These data demonstrated that the designed multiepitope vaccine constructs along with curcumin and nanocurcumin can be used as a promising method for HPV vaccine development.

Functional HPV-specific PD-1+ stem-like CD8 T cells in head and neck cancer.

T cells are important in tumour immunity but a better understanding is needed of the differentiation of antigen-specific T cells in human cancer1,2. Here we studied CD8 T cells in patients with human papillomavirus (HPV)-positive head and neck cancer and identified several epitopes derived from HPV E2, E5 and E6 proteins that allowed us to analyse virus-specific CD8 T cells using major histocompatibility complex (MHC) class I tetramers. HPV-specific CD8 T cells expressed PD-1 and were detectable in the tumour at levels that ranged from 0.1% to 10% of tumour-infiltrating CD8 T lymphocytes (TILs) for a given epitope. Single-cell RNA-sequencing analyses of tetramer-sorted HPV-specific PD-1+ CD8 TILs revealed three transcriptionally distinct subsets. One subset expressed TCF7 and other genes associated with PD-1+ stem-like CD8 T cells that are critical for maintaining T cell responses in conditions of antigen persistence. The second subset expressed more effector molecules, representing a transitory cell population, and the third subset was characterized by a terminally differentiated gene signature. T cell receptor clonotypes were shared between the three subsets and pseudotime analysis suggested a hypothetical differentiation trajectory from stem-like to transitory to terminally differentiated cells. More notably, HPV-specific PD-1+TCF-1+ stem-like TILs proliferated and differentiated into more effector-like cells after in vitro stimulation with the cognate HPV peptide, whereas the more terminally differentiated cells did not proliferate. The presence of functional HPV-specific PD-1+TCF-1+CD45RO+ stem-like CD8 T cells with proliferative capacity shows that the cellular machinery to respond to PD-1 blockade exists in HPV-positive head and neck cancer, supporting the further investigation of PD-1 targeted therapies in this malignancy. Furthermore, HPV therapeutic vaccination efforts have focused on E6 and E7 proteins; our results suggest that E2 and E5 should also be considered for inclusion as vaccine antigens to elicit tumour-reactive CD8 T cell responses of maximal breadth.

Potent, Selective CARs as Potential T-Cell Therapeutics for HPV-positive Cancers.

Next-generation T-cell therapies will likely continue to utilize T-cell receptors (TCRs) and chimeric antigen receptors (CARs) because each receptor type has advantages. TCRs often possess exceptional properties even when tested unmodified from patients' T cells. CARs are generally less sensitive, possibly because their ligand-binding domains are grafted from antibodies selected for binding affinity or avidity and not broadly optimized for a functional response. Because of the disconnect between binding and function among these receptor types, the ultimate potential of CARs optimized for sensitivity and selectivity is not clear. Here, we focus on a thoroughly studied immuno-oncology target, the HLA-A*02/HPV-E629-38 complex, and show that CARs can be optimized by a combination of high-throughput binding screens and low-throughput functional assays to have comparable activity to clinical TCRs in acute assays in vitro. These results provide a case study for the challenges and opportunities of optimizing high-performing CARs, especially in the context of targets utilized naturally by TCRs.

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.

Design of a multi-epitope vaccine against cervical cancer using immunoinformatics approaches.

Cervical cancer, caused by human papillomavirus (HPV), is the fourth most common type of cancer among women worldwide. While HPV prophylactic vaccines are available, they have no therapeutic effects and do not clear up existing infections. This study aims to design a therapeutic vaccine against cervical cancer using reverse vaccinology. In this study, the E6 and E7 oncoproteins from HPV16 were chosen as the target antigens for epitope prediction. Cytotoxic T lymphocytes (CTL) and helper T lymphocytes (HTL) epitopes were predicted, and the best epitopes were selected based on antigenicity, allergenicity, and toxicity. The final vaccine construct was composed of the selected epitopes, along with the appropriate adjuvant and linkers. The multi-epitope vaccine was evaluated in terms of physicochemical properties, antigenicity, and allergenicity. The tertiary structure of the vaccine construct was predicted. Furthermore, several analyses were also carried out, including molecular docking, molecular dynamics (MD) simulation, and in silico cloning of the vaccine construct. The results showed that the final proposed vaccine could be considered an effective therapeutic vaccine for HPV; however, in vitro and in vivo experiments are required to validate the efficacy of this vaccine candidate.

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.

Worldwide genetic variations in high-risk human papillomaviruses capsid L1 gene and their impact on vaccine efficiency.

BACKGROUND: Human papillomavirus is the most common sexually transmitted infection. It is associated with different cancers, mainly cervical cancer, which remains the fourth most frequent cancer among women worldwide; it is also related to anogenital (anus, vulvar, vagina, and penis) and oropharyngeal cancers. Vaccination against HPV infection is the major way of prevention, and it has demonstrated impressive efficacy in reducing cervical cancer incidence. Nowadays, all the licensed HPV recombinant vaccines were designed based on HPV major capsid L1 protein. However, some variations in the HPV L1 gene sequence may induce structural changes within the L1 protein, which may alter the affinity and interaction of monoclonal antibodies (MAbs) with L1 protein epitopes, and influence host immune response and recognition. Hence, the importance of accuracy in delineating epitopes relevant to vaccine design and defining genetic variations within antigenic regions in the L1 gene to predict its impact on prophylactic vaccine efficiency. The present review reports the sequence variations in HR-HPV L1 gene isolates from different countries around the world, which may help to understand the effect of HPV L1 gene variations on vaccine efficiency. METHODS: Research studies were retrieved from PubMed, Google Scholar, Science direct, and the National Center for Biotechnology Information (NCBI) database. A total of 31 articles describing genetic variations within the major capsid L1 gene and conducted in Africa, Europe, America and Asia were found. Only 26 studies conducted on HPV16, 18, 31, 33, 58, 45 and 52 which are the targets of HPV prophylactic vaccines, and which reported genetic variations within the L1 gene, were selected and evaluated in this review. FINDINGS: We found a total of 87, 49, 11, 7, 22, 3, and 17 non-synonymous single nucleotide polymorphisms (SNPs) within HPV16, HPV18, HPV31, HPV58, HPV45, and HPV52 L1 gene, respectively. Four mutations were frequently observed in HPV16 L1 sequences: T353P in the HI loop, H228D in the EF loop, T266A in the FG loop, and T292A in the FG loop. Two mutations in HPV58 L1 sequences: T375N in the HI loop and L150F in the DE loop. Three mutations in HPV33 L1 sequences: T56N in the BC loop, G133S in the DE loop, T266K in the FG loop. Other mutations were found in HPV18, HPV45, and HPV52 L1 sequences. Some were found in different countries, and others were specific to a given population. Furthermore, some variations were located on peptide binding epitopes and lead to a modification of epitopes, which may influence MAbs interactions. Others need further investigations due to the lack of studies. CONCLUSION: This study investigated the major capsid L1 genetic diversity of HPV16, 18, 31, 33, 58, 45, and 52 circulating in different populations around the world. Further investigations should be conducted to confirm their effect on immunogenicity and prophylactic vaccine efficiency.

A novel intracellular nanobody against HPV16 E6 oncoprotein.

Cervical cancer occurs as a result of the persistent infection of high-risk human papillomavirus (HPV). HPV16 oncoproteins E6 and E7 exert different and concerted pro-tumor actions in cell transformation and malignance maintenance in various m echanisms. Nanobody expressed as "intracellular antibodies" (intrabodies) can target intracellular antigens to hamper their function efficaciously and specifically. In this work, phage-display approach was employed to select the high affinity HPV16 E6-specific nanobody, nanobody Nb9 against HPV16 E6 was selected. Nb9 has high affinity (Kaff =6.3 × 108 M-) and can specifically bind endogenous HPV16 E6 protein in HPV16 positive CaSki and SiHa cells. In Nb9 overexpressed SiHa and CaSki cells, nucleus localization of HPV16 E6 was inhibited, p53 inactivation was prevented and increased apoptosis was observed. Moreover, tumor growth was inhibited in mouse xenograft model. Taken together, our results suggested that nanobody Nb9 could be a useful inhibitor for HPV16 E6 function and particularly appropriate for the treatment of HPV-associated disease.

Intrabodies targeting human papillomavirus 16 E6 and E7 oncoproteins for therapy of established HPV-associated tumors.

BACKGROUND: The oncogenic activity of the high risk human papillomavirus type 16 (HPV16) is fully dependent on the E6 and E7 viral oncoproteins produced during viral infection. The oncoproteins interfere with cellular homeostasis by promoting proliferation, inhibiting apoptosis and blocking epithelial differentiation, driving the infected cells towards neoplastic progression. The causal relationship between expression of E6/E7 and cellular transformation allows inhibiting the oncogenic process by hindering the activity of the two oncoproteins. We previously developed and characterized some antibodies in single-chain format (scFvs) against the HPV16 E6 and E7 proteins, and demonstrated both in vitro and in vivo their antitumor activity consisting of protective efficacy against tumor progression of HPV16-positive cells. METHODS: Envisioning clinical application of the best characterized anti-HPV16 E6 and -HPV16 E7 scFvs, we verified their activity in the therapeutic setting, on already implanted tumors. Recombinant plasmids expressing the anti-HPV16 E6 scFvI7 with nuclear targeting sequence, or the anti-HPV16 E7 scFv43M2 with endoplasmic reticulum targeting sequence were delivered by injection followed by electroporation to three different preclinical models using C57/BL6 mice, and their effect on tumor growth was investigated. In the first model, the HPV16+ TC-1 Luc cells were used to implant tumors in mice, and tumor growth was measured by luciferase activity; in the second model, a fourfold number of TC-1 cells was used to obtain more aggressively growing tumors; in the third model, the HPV16+ C3 cells where used to rise tumors in mice. To highlight the scFv possible mechanism of action, H&E and caspase-3 staining of tumor section were performed. RESULTS: We showed that both the anti-HPV16 E6 and HPV16 E7 scFvs tested were efficacious in delaying tumor progression in the three experimental models and that their antitumor activity seems to rely on driving tumor cells towards the apoptotic pathway. CONCLUSION: Based on our study, two scFvs have been identified that could represent a safe and effective treatment for the therapy of HPV16-associated lesions. The mechanism underlying the scFv effectiveness appears to be leading cells towards death by apoptosis. Furthermore, the validity of electroporation, a methodology allowed for human treatment, to deliver scFvs to tumors was confirmed.

Development of DNA Vaccine Targeting E6 and E7 Proteins of Human Papillomavirus 16 (HPV16) and HPV18 for Immunotherapy in Combination with Recombinant Vaccinia Boost and PD-1 Antibody.

Immunotherapy for cervical cancer should target high-risk human papillomavirus types 16 and 18, which cause 50% and 20% of cervical cancers, respectively. Here, we describe the construction and characterization of the pBI-11 DNA vaccine via the addition of codon-optimized human papillomavirus 18 (HPV18) E7 and HPV16 and 18 E6 genes to the HPV16 E7-targeted DNA vaccine pNGVL4a-SigE7(detox)HSP70 (DNA vaccine pBI-1). Codon optimization of the HPV16/18 E6/E7 genes in pBI-11 improved fusion protein expression compared to that in DNA vaccine pBI-10.1 that utilized the native viral sequences fused 3' to a signal sequence and 5' to the HSP70 gene of Mycobacterium tuberculosis Intramuscular vaccination of mice with pBI-11 DNA better induced HPV antigen-specific CD8+ T cell immune responses than pBI-10.1 DNA. Furthermore, intramuscular vaccination with pBI-11 DNA generated stronger therapeutic responses for C57BL/6 mice bearing HPV16 E6/E7-expressing TC-1 tumors. The HPV16/18 antigen-specific T cell-mediated immune responses generated by pBI-11 DNA vaccination were further enhanced by boosting with tissue-antigen HPV vaccine (TA-HPV). Combination of the pBI-11 DNA and TA-HPV boost vaccination with PD-1 antibody blockade significantly improved the control of TC-1 tumors and extended the survival of the mice. Finally, repeat vaccination with clinical-grade pBI-11 with or without clinical-grade TA-HPV was well tolerated in vaccinated mice. These preclinical studies suggest that the pBI-11 DNA vaccine may be used with TA-HPV in a heterologous prime-boost strategy to enhance HPV 16/18 E6/E7-specific CD8+ T cell responses, either alone or in combination with immune checkpoint blockade, to control HPV16/18-associated tumors. Our data serve as an important foundation for future clinical translation.IMPORTANCE Persistent expression of high-risk human papillomavirus (HPV) E6 and E7 is an obligate driver for several human malignancies, including cervical cancer, wherein HPV16 and HPV18 are the most common types. PD-1 antibody immunotherapy helps a subset of cervical cancer patients, and its efficacy might be improved by combination with active vaccination against E6 and/or E7. For patients with HPV16+ cervical intraepithelial neoplasia grade 2/3 (CIN2/3), the precursor of cervical cancer, intramuscular vaccination with a DNA vaccine targeting HPV16 E7 and then a recombinant vaccinia virus expressing HPV16/18 E6-E7 fusion proteins (TA-HPV) was safe, and half of the patients cleared their lesions in a small study (NCT00788164). Here, we sought to improve upon this therapeutic approach by developing a new DNA vaccine that targets E6 and E7 of HPV16 and HPV18 for administration prior to a TA-HPV booster vaccination and for application against cervical cancer in combination with a PD-1-blocking antibody.

Detection of HPV16/18 E6 Oncoproteins in Head and Neck Squamous Cell Carcinoma Using a Protein Immunochromatographic Assay.

OBJECTIVES/HYPOTHESIS: The accurate diagnostic assessment of clinically relevant human papillomavirus (HPV) infections in patients with head and neck squamous cell carcinoma represents an urgent unmet medical need. The aim of this study was to determine feasibility, accuracy, and clinical significance of HPV16/18 E6 oncoprotein detection on cytological specimens from oropharyngeal squamous cell carcinoma (OPSCC) and neck lymph node metastasis of SCC from unknown primary tumor (CUP) via a protein immunochromatographic assay. STUDY DESIGN: Cross-sectional study. METHODS: Cytological specimens from primary tumor and neck metastases were collected from 34 patients with OPSCC or CUP and applied to a lateral flow format test that detects HPV16 and HPV18 E6 oncoproteins. E6 oncoprotein positivity or negativity in these specimens was compared to the specimens' "HPV-driven" reference status, defined by presence of HPV-DNA in combination with p16INK4a overexpression and/or HPV E6 seropositivity. RESULTS: Eighteen of 29 OPSCC (62%) and three of five CUP (60%) were HPV-driven according to our reference method. The E6 oncoprotein lateral flow test had a sensitivity of 94% (95% CI: 70%-100%) and a specificity of 100% (95% CI: 66%-100%) on primary tumor, and a sensitivity of 88% (95% CI: 64%-99%) and a specificity of 100% (95% CI: 74%-100%) on neck metastases. Test agreement between the E6 lateral flow test and the clinical reference method, HPV-DNA plus p16INK4a was excellent, both for primary lesion and neck metastases. CONCLUSIONS: We found the detection of HPV16/18 E6 oncoproteins to be a feasible, highly reliable, and low-invasive method to assess "HPV-driven" status in OPSCC and CUP. LEVEL OF EVIDENCE: II Laryngoscope, 131:1042-1048, 2021.

First-in-Human Phase I Clinical Trial of an SFV-Based RNA Replicon Cancer Vaccine against HPV-Induced Cancers.

A first-in-human phase I trial of Vvax001, an alphavirus-based therapeutic cancer vaccine against human papillomavirus (HPV)-induced cancers was performed assessing immunological activity, safety, and tolerability. Vvax001 consists of replication-incompetent Semliki Forest virus replicon particles encoding HPV16-derived antigens E6 and E7. Twelve participants with a history of cervical intraepithelial neoplasia were included. Four cohorts of three participants were treated per dose level, ranging from 5 × 105 to 2.5 × 108 infectious particles per immunization. The participants received three immunizations with a 3-week interval. For immune monitoring, blood was drawn before immunization and 1 week after the second and third immunization. Immunization with Vvax001 was safe and well tolerated, with only mild injection site reactions, and resulted in both CD4+ and CD8+ T cell responses against E6 and E7 antigens. Even the lowest dose of 5 × 105 infectious particles elicited E6/E7-specific interferon (IFN)-γ responses in all three participants in this cohort. Overall, immunization resulted in positive vaccine-induced immune responses in 12 of 12 participants in one or more assays performed. In conclusion, Vvax001 was safe and induced immune responses in all participants. These data strongly support further clinical evaluation of Vvax001 as a therapeutic vaccine in patients with HPV-related malignancies.