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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 βHPVs 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 βHPV. To comprehensively target both α- and βHPVs, 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. Unlike vaccination with Gardasil 9, vaccination of wild-type and EV model mice (Tmc6(Δ/Δ) or Tmc8(Δ/Δ)) with RG2-VLP induced robust L2-specific antibody titers and protected against β-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 βHPVs tested. Taken together, these observations suggest RG2-VLP’s potential as a broad-spectrum vaccine to prevent αHPV-driven anogenital, oropharyngeal, and βHPV-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 βHPV 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 βHPVs by incorporation into HPV16 and HPV18 L1 VLP of 20-amino-acid conserved protective epitopes derived from minor capsid protein L2.

As severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spreads, variants with enhanced virulence and transmissibility have emerged. Although in vitro systems allow rapid characterization, they do not fully recapitulate the dynamic interaction of virions and neutralizing antibodies in the airway. Here, we demonstrate that the N501Y variant permits respiratory infection in unmodified mice. We utilize N501Y to survey in vivo pseudovirus infection dynamics and susceptibility to reinfection with the L452R (Los Angeles), K417N + E484K (South Africa), and L452R + K417N + E484Q (India) variants. Human coronavirus disease 2019 (COVID-19)+ or vaccinated antibody isotypes, titers, variant receptor binding domain (RBD) binding, and neutralization potential are studied, revealing numerous significant correlations. Immune escape of the K417N + E484K variant is observed because infection can be appreciated in the nasopharynx, but not lungs, of mice transferred with low-antibody-tier plasma. Conversely, near-complete protection is observed in animals receiving high-antibody-tier plasma, a phenomenon that can only be appreciated in vivo., Graphical abstract, Lam et al. develop a model of SARS-CoV-2 infection in laboratory mice. This allows researchers to study the threat of emerging variants in a more physiological context than by using cell culture systems. Interactions between SARS-CoV-2 variants and immunity are explored in the airway of mice.

We demonstrate that female C57BL/6J mice are susceptible to a transient lower genital tract infection with MmuPV1 mouse papillomavirus and display focal histopathological abnormalities resembling those of human papillomavirus (HPV) infection. We took advantage of strains of genetically deficient mice to study in vivo the role of innate immune signaling in the control of papillomavirus. At 4 months, we sacrificed MmuPV1-infected mice and measured viral 757/3139 spliced transcripts by TaqMan reverse transcription-PCR (RT-PCR), localization of infection by RNAscope in situ hybridization, and histopathological abnormities by hematoxylin and eosin (H&E) staining. Among mice deficient in receptors for pathogen-associated molecular patterns, MyD88(−/−) and STING(−/−) mice had 1,350 and 80 copies of spliced transcripts/μg RNA, respectively, while no viral expression was detected in MAVS(−/−) and Ripk2(−/−) mice. Mice deficient in an adaptor molecule, STAT1(−/−), for interferon signaling had 46,000 copies/μg RNA. Among mice with targeted deficiencies in the inflammatory response, interleukin-1 receptor knockout (IL-1R(−/−)) and caspase-1(−/−) mice had 350 and 30 copies/μg RNA, respectively. Among mice deficient in chemokine receptors, CCR6(−/−) mice had 120 copies/μg RNA, while CXCR2(−/−) and CXCR3(−/−) mice were negative. RNAscope confirmed focal infection in MyD88(−/−), STAT1(−/−), and CCR6(−/−) mice but was negative for other gene-deficient mice. Histological abnormalities were seen only in the latter mice. Our findings and the literature support a working model of innate immunity to papillomaviruses involving the activation of a MyD88-dependent pathway and IL-1 receptor signaling, control of viral replication by interferon-stimulated genes, and clearance of virus-transformed dysplastic cells by the action of the CCR6/CCL20 axis. IMPORTANCE Papillomaviruses infect stratified squamous epithelia, and the viral life cycle is linked to epithelial differentiation. Additionally, changes occur in viral and host gene expression, and immune cells are activated to modulate the infectious process. In vitro studies with keratinocytes cannot fully model the complex viral and host responses and do not reflect the contribution of local and migrating immune cells. We show that female C57BL/6J mice are susceptible to a transient papillomavirus cervicovaginal infection, and mice deficient in select genes involved in innate immune responses are susceptible to persistent infection with variable manifestations of histopathological abnormalities. The results of our studies support a working model of innate immunity to papillomaviruses, and the model provides a framework for more in-depth studies. A better understanding of mechanisms of early viral clearance and the development of approaches to induce clearance will be important for cancer prevention and the treatment of HPV-related diseases.

Recurrent respiratory papillomatosis (RRP) is a benign disease caused by human papillomavirus (HPV) types 6 and 11. Although a prophylactic vaccine against RRP is available, a therapeutic vaccine is needed to treat those already infected. The objective of our study was to design and test a DNA vaccine targeting HPV11 proteins.Preclinical scientific investigation.A DNA vaccine encoding the HPV11 E6 and E7 genes linked to calreticulin (CRT) was generated. Immunologic response to the HPV11 CRT/E6E7 vaccine was measured by vaccinating C57BL/6 mice via electroporation and measuring CD8 + T cell responses from harvested splenocytes. A tumor cell line containing HPV11-E6E7 was created, and the ability of novel DNA vaccine to control tumor growth was measured in vivo.Our vaccine generated a significant and specific CD8 + T-cell response against the HPV11-E6aa41-70 peptide. The CD8 + T-cell responses did not recognize E7 epitopes, indicating E6 immunodominance. CD8 + responses were augmented in the CRT-linked vaccine compared to a control non-CRT vaccine. The HPV11 CRT/E6E7 vaccine was used to treat mice inoculated with a HPV11 E6E7 expressing tumor cell line after temporary CD3 depletion to facilitate tumor growth. Vaccinated mice had a significantly lower tumor growth rate (P = .029) and smaller tumor volumes compared to control mice, indicating an augmented immunologic response in vaccinated mice.A DNA vaccine targeting HPV11 E6E7 generates a specific HPV11 CD-8 + T-cell response capable of reducing the growth of HPV11-expressing tumors. DNA vaccines are a promising immunologic strategy for treating RRP.NA. Laryngoscope, 127:2713-2720, 2017.

Although it has been shown that prophylactic vaccination can induce genital immunity, there is inadequate information on human papillomavirus (HPV) vaccine-induced oral immunity, which is of particular interest due to HPV-associated oropharyngeal malignancies and recurrent respiratory papillomatosis. Therefore, we assessed the efficacy of various HPV vaccines against oral HPV pseudovirus (PsV) infection in mice.Preclinical scientific investigation.C57BL/6 mice were vaccinated three times at 2-week intervals with either Gardasil (Merck, Kenilworth, NJ) (50 µL intramuscular injection) or a candidate pan-HPV L2 vaccine with alum adjuvant (25 µg subcutaneous injection). Additional mice were immunized with passive transfer of either Gardasil (Merck) human antisera or nonimmunized sera (100 µL intraperitoneal injection). All vaccinated and naïve control mice were then challenged with HPV16 E6E7 luciferase PsV in the oral mucosa. Visualization of HPV PsV infection was monitored through in vivo luciferase imaging.Oral luciferase-expressing HPV16 PsV infection was not detected in Gardasil (Merck), L2 vaccine, and Gardasil (Merck) antisera-immunized mice, whereas robust luciferase expression was observed in all control mice. An in vitro neutralization assay from sera of Gardasil-vaccinated (Merck) mice confirmed that vaccine efficacy was due to neutralizing antibodies.Oral HPV16 PsV infection in mice was completely prevented with all methods of prophylactic HPV immunization. These findings provide preliminary evidence that human vaccines induce protection against oral HPV infection, which has significant public health implications for HPV-associated oropharyngeal malignancies.NA. Laryngoscope, 128:E16-E20, 2018.

Immunization with human papillomavirus (HPV) L1 virus-like particles (VLPs) prevents infection with HPV. However, the expense and logistical demands of current VLP vaccines will limit their widespread use in resource-limited settings, where most HPV-induced cervical cancer occurs. Live oral adenovirus vaccines have properties that are well-suited for use in such settings. We have described a live recombinant adenovirus vaccine prototype that produces abundant HPV16 L1 protein from the adenovirus major late transcriptional unit and directs the assembly of HPV16 VLPs in tissue culture. Recombinant-derived VLPs potently elicit neutralizing antibodies in mice. Here, we characterize the immune response to the recombinant after dual oral and intranasal immunization of pigtail macaques, in which the virus replicates as it would in immunized humans. The immunization of macaques induced vigorous humoral responses to adenovirus capsid and nonstructural proteins, although, surprisingly, not against HPV L1. In contrast, immunization elicited strong T-cell responses to HPV VLPs as well as adenovirus virions. T-cell responses arose immediately after the primary immunization and were boosted by a second immunization with recombinant virus. T-cell immunity contributes to protection against a wide variety of pathogens, including many viruses. The induction of a strong cellular response by the recombinant indicates that live adenovirus recombinants have potential as vaccines for those agents. These studies encourage and will inform the continued development of viable recombinant adenovirus vaccines.

Background Infection by any one of 15 high risk human papillomavirus (hrHPV) types causes most invasive cervical cancers. Their oncogenic genome is encapsidated by L1 (major) and L2 (minor) coat proteins. Current HPV prophylactic vaccines are composed of L1 virus-like particles (VLP) that elicit type restricted immunity. An N-terminal region of L2 protein identified by neutralizing monoclonal antibodies comprises a protective epitope conserved among HPV types, but it is weakly immunogenic compared to L1 VLP. The major antigenic capsid protein of adenovirus type 5 (Ad5) is hexon which contains 9 hypervariable regions (HVRs) that form the immunodominant neutralizing epitopes. Insertion of weakly antigenic foreign B cell epitopes into these HVRs has shown promise in eliciting robust neutralizing antibody responses. Thus here we sought to generate a broadly protective prophylactic HPV vaccine candidate by inserting a conserved protective L2 epitope into the Ad5 hexon protein for VLP-like display. Methods Four recombinant adenoviruses were generated without significant compromise of viral replication by introduction of HPV16 amino acids L2 12–41 into Ad5 hexon, either by insertion into, or substitution of, either hexon HVR1 or HVR5. Results Vaccination of mice three times with each of these L2-recombinant adenoviruses induced similarly robust adenovirus-specific serum antibody but weak titers against L2. These L2-specific responses were enhanced by vaccination in the presence of alum and monophoryl lipid A adjuvant. Sera obtained after the third immunization exhibited low neutralizing antibody titers against HPV16 and HPV73. L2-recombinant adenovirus vaccination without adjuvant provided partial protection of mice against HPV16 challenge to either the vagina or skin. In contrast, vaccination with each L2-recombinant adenovirus formulated in adjuvant provided robust protection against vaginal challenge with HPV16, but not against HPV56. Conclusion We conclude that introduction of HPV16 L2 12–41 epitope into Ad5 hexon HVR1 or HVR5 is a feasible method of generating a protective HPV vaccine, but further optimization is required to strengthen the L2-specific response and broaden protection to the more diverse hrHPV.

The identification of human papillomavirus (HPV) as a cause of cervical cancer and its precursor lesions indicates that HPV vaccines can potentially be used to prevent or treat cervical cancer and other HPV-associated malignancies. Prophylactic HPV vaccines aim to prevent infection by producing neutralizing antibodies against HPV capsid proteins L1 and L2. However, because HPV-infected basal keratinocytes and HPV-transformed cells generally do not express L1 or L2, therapeutic HPV vaccines are being developed to treat established HPV infections and HPV-associated malignancies by targeting non-structural early viral antigens of HPV, such as E6 and E7 (two viral oncogenic proteins required for the induction and maintenance of malignant cancer). Results from preclinical HPV vaccine studies have led to several HPV vaccine clinical trials. If these prophylactic and therapeutic HPV vaccines prove as successful in patients as they have in animal models, HPV vaccines may have a role in the control of HPV infection and HPV-associated disease.