Licensed human papillomavirus (HPV) vaccines do not target cutaneous ßHPV types implicated in skin cancer development in immunosuppressed individuals. The conserved N-terminus of minor capsid protein L2 contains cross-neutralization epitopes, offering the opportunity to develop broad-spectrum vaccine candidates. A sequential immunization strategy using N-terminal L2 fragments of five ßHPVs induced type-common humoral immunity. Four monoclonal antibodies (mAb) were generated that cross-neutralized multiple ßHPV types and conferred in vivo protection against potentially oncogenic HPV5/38/24. MAb epitopes were inserted individually into the DE-surface loop of HPV16 L1 virus-like particles (VLP). Immunizations with chimeric VLPs induced cross-neutralizing antibodies against multiple ßHPV types and conferred in vivo protection against HPV5 infection. Chimeric VLPs displaying ßHPV-L2 cross-neutralizing epitopes are promising broad-spectrum vaccine candidates against the plethora of ßHPVs. Immunization of patients prior to immunosuppression may reduce the burden of ßHPV infection and thus lower their highly increased risk to develop keratinocytic skin cancer.

Zusammenfassung Infektionen mit >12 sexuell übertragbaren genitalen „high-risk“ (hr) humanen Papillomviren (HPV) sind hauptverantwortlich für anogenitale Karzinome, insbesondere Zervix- und Analkarzinome sowie oropharyngeale Karzinome, insgesamt für 5 % der Karzinome weltweit. Genitale „low-risk“ (lr) HPV und kutane HPV verursachen Anogenitalwarzen (Kondylome) bzw. Hautwarzen, kutane Genus β‑HPV sind ein potenzieller Kofaktor für die Entwicklung nichtmelanozytärer Hautkarzinome in Immunsupprimierten. Die zugelassenen HPV-Vakzinen sind Spaltimpfstoffe bestehend aus leeren Hauptkapsidproteinhüllen (L1-virus-like particles, VLP). Die prophylaktische Impfung mit dem modernen nonavalenten Impfstoff Gardasil‑9 (HPV6/11/16/18/31/33/45/52/58) verhindert persistierende Infektionen mit Typen, die bis zu 90 % der Zervixkarzinome und Kondylome verursachen. Der Impfschutz ist vorwiegend typenspezifisch, daher besteht kein Schutz gegen Infektionen mit den übrigen genitalen hrHPV oder Hauttypen. RG1-VLP ist ein experimenteller „next generation“-Impfstoff, bestehend aus HPV16L1-VLP, welche ein Kreuzneutralisierungs-Epitop des HPV16 Nebenkapsidproteins L2 („RG1“; Aminosäuren 17–36) repetitiv (360×) an der Oberfläche tragen. Eine Vakzinierung mit RG1-VLP schützt im Tierversuch gegen experimentelle Infektionen mit allen relevanten genitalen hrHPV (~96 % aller Zervixkarzinome), lrHPV (~90 % der Kondylome) sowie gegen einige kutane und β‑HPV. Präklinische Daten zeigen langanhaltende Protektion ohne Boosterimmunisierung ein Jahr nach der Impfung sowie Wirksamkeit nach nur 2 Dosen. Auch in lyophilisierter, thermostabiler Form bleibt die Immunogenität der RG1-VLP erhalten. Eine Phase-I-Studie ist mit Unterstützung des US NCI/NIH in Vorbereitung. Der vorliegende Artikel diskutiert Fragestellungen zur HPV-Impfstoffoptimierung und präsentiert den pan-HPV-Impfstoffkandidat RG1-VLP.

Cathelicidins have been reported to inhibit human papillomavirus infection in vitro; however, nothing is known about their activity in vivo. In this study, experimental skin infection with Mus musculus papillomavirus 1 resulted in robust development of cutaneous papillomas in cyclosporine A-treated C57BL/6J mice deficient for the murine cathelicidin-related antimicrobial peptide (CRAMP), in contrast to wild-type controls. Analysis of the underlying mechanisms revealed moderate disruption of virion integrity and lack of interference with viral entry and intracellular trafficking by a synthetic CRAMP peptide. Differences in the immune response to Mus musculus papillomavirus 1 infection were observed between CRAMP-deficient and wildtype mice. These included a stronger reduction in CD4(+) and CD8(+) T-cell numbers in infected skin, and lack of Mus musculus papillomavirus 1-specific neutralizing antibodies in response to cyclosporine A in the absence of endogenous CRAMP. CRAMP has modest direct anti-papillomaviral effects in vitro, but exerts protective functions against Mus musculus papillomavirus 1 skin infection and disease development in vivo, primarily by modulation of cellular and humoral immunity.

The candidate pan-Human Papillomavirus (HPV) vaccine RG1-VLP are HPV16 major capsid protein L1 virus-like-particles (VLP) comprising a type-common epitope of HPV16 minor capsid protein L2 (RG1; aa17-36). Vaccinations have previously demonstrated efficacy against genital high-risk (hr), low-risk (1r) and cutaneous HPV. To compare RG1-VLP to licensed vaccines, rabbits (n = 3) were immunized thrice with 1 mu g, 5 mu g, 25 mu g, or 125 mu g of RG1-VLP or a 1/4 dose of Cervarix (R). 5 mu g of RG1-VLP or 16L1-VLP (Cervarix) induced comparable HPV16 capsid-reactive and neutralizing antibodies titers (62,500/12,500-62,500 or 1000/10,000). 25 mu g RG1-VLP induced robust cross-neutralization titers (50-1000) against hrHPV18/31/33/45/52/58/26/70. To mimic reduced immunization schedules in adolescents, mice (n = 10) were immunized twice with RG1-VLP (5 mu g) plus 18L1-VLP (5 mu g). HPV16 neutralization (titers of 10,000) similar to Cervarix and Gardasil and cross-protection against hrHPV58 vaginal challenge was observed. RG1-VLP vaccination induces hrHPV16 neutralization comparable to similar doses of licensed vaccines, plus cross-neutralization to heterologous hrHPV even when combined with HPV18L1-VLP. (C) 2019 Published by Elsevier Ltd.

We have previously shown that immunization of horses with bovine papillomavirus type 1 (BPV1) L1 virus-like particles (VLPs) is safe and highly immunogenic and that BPV1 and bovine papillomavirus type 2 (BPV2) are closely related serotypes. Here we evaluated the protective potential of a BPV1 L1 VLP vaccine against experimental BPV1 and BPV2 challenge and studied the safety and immunogenicity of a bivalent equine papillomavirus type 2 (EcPV2)/BPV1 L1 VLP vaccine. Fourteen healthy horses were immunized with BPV1 L1 VLPs (100 µg per injection) plus adjuvant on days 0 and 28, while seven remained unvaccinated. On day 42, all 21 horses were challenged intradermally at 10 sites of the neck with 107 BPV1 virions per injection. In analogy, 14 horses immunized twice with EcPV2 plus BPV1 L1 VLPs (50 µg each) and seven control animals were challenged with 107 BPV2 virions per injection. Immunization with BPV1 L1 VLPs alone induced a robust antibody response (day 42 median titre: 12 800), and BPV1-inoculated skin remained unchanged in 13/14 vaccinated horses. Immunization with the bivalent vaccine was safe, resulted in lower median day 42 antibody titres of 400 for BPV1 and 1600 for EcPV2 and conferred significant yet incomplete cross-protection from BPV2-induced tumour formation, with 11/14 horses developing small, short-lived papules. Control horses developed pseudo-sarcoids at all inoculation sites. The monovalent BPV1 L1 VLP vaccine proved highly effective in protecting horses from BPV1-induced pseudo-sarcoid formation. Incomplete protection from BPV2-induced tumour development conferred by the bivalent vaccine is due to the poorer immune response by immune interference or lower cross-neutralization titres to heterologous BPV2 virions.

Common cutaneous human papillomavirus (HPV) types induce skin warts, whereas species beta HPV are implicated, together with UV-radiation, in the development of non-melanoma skin cancer (NMSC) in immunosuppressed patients. Licensed HPV vaccines contain virus-like particles (VLP) self-assembled from L1 major capsid proteins that provide type-restricted protection against mucosal HPV infections causing cervical and other ano-genital and oro-pharyngeal carcinomas and warts (condylomas), but do not target heterologous HPV. Experimental papillomavirus vaccines have been designed based on L2 minor capsid proteins that contain type-common neutralization epitopes, to broaden protection to heterologous mucosal and cutaneous HPV types. Repetitive display of the HPV16 L2 cross-neutralization epitope RG1 (amino acids (aa) 17-36) on the surface of HPV16 L1 VLP has greatly enhanced immunogenicity of the L2 peptide. To more directly target cutaneous HPV, L1 fusion proteins were designed that incorporate the RG1 homolog of beta HPV17, the beta HPV5 L2 peptide aa53-72, or the common cutaneous HPV4 RG1 homolog, inserted into DE surface loops of HPV1, 5, 16 or 18 L1 VLP scaffolds. Baculovirus expressed chimeric proteins self-assembled into VLP and VLP-raised NZW rabbit immune sera were evaluated by ELISA and L1- and L2-based pseudovirion (PsV) neutralizing assays, including 12 novel beta PsV types. Chimeric VLP displaying the HPV17 RG1 epitope, but not the HPV5L2 aa53-72 epitope, induced cross-neutralizing humoral immune responses to beta HPV. In vivo cross-protection was evaluated by passive serum transfer in a murine PsV challenge model. Immune sera to HPV16L1-17RG1 VLP (cross-) protected against beta HPV5/20/24/38/96/16 (but not type 76), while antisera to HPV5L1-17RG1 VLP cross-protected against HPV20/24/96 only, and sera to HPV1L1-4RG1 VLP cross-protected against HPV4 challenge. In conclusion, RG1-based VLP are promising next generation vaccine candidates to target cutaneous HPV infections.

Equine sarcoids are common therapy-resistant skin tumours induced by bovine papillomavirus type 1 or 2 (BPV1, BPV2) infection. We have previously shown that prophylactic vaccination with BPV1 L1 virus-like particles (VLPs) efficiently protects horses from experimental BPV1-induced pseudo-sarcoid development. Here, we assessed BPV1 L1 VLP vaccine-mediated long-term protection from experimental tumour formation in seven horses 5 years after immunization with three different doses of BPV1 L1 VLPs, and three unvaccinated control animals. Horses were challenged by intradermal inoculation with infectious BPV1 virions at 10 sites on the neck (106 virions per injection). In vaccinated horses, BPV1 challenge did not result in any apparent lesions irrespective of vaccine dosage and BPV1-neutralizing antibody titres that had dropped considerably over time and below the detection limit in one individual. Control horses developed pseudo-sarcoids at all inoculation sites. We conclude that immunization of horses with BPV1 L1 VLPs induces long-lasting protection against experimental BPV1 virion-induced disease.

Persistent infection with oncogenic human papillomaviruses (HPV) types causes all cervical and a subset of other anogenital and oropharyngeal carcinomas. Four high-risk (hr) mucosal types HPV16, 18, 45, or 59 cause almost all cervical adenocarcinomas (AC), a subset of cervical cancer (CxC). Although the incidence of cervical squamous cell carcinoma (SCC) has dramatically decreased following introduction of Papanicolaou (PAP) screening, the proportion of AC has relatively increased. Cervical SCC arise mainly from the ectocervix, whereas AC originate primarily from the endocervical canal, which is less accessible to obtain viable PAP smears. Licensed (bivalent and quadrivalent) HPV vaccines comprise virus-like particles (VLP) of the most important hr HPV16 and 18, self-assembled from the major capsid protein L1. Due to mainly type-restricted efficacy, both vaccines do not target 13 additional hr mucosal types causing 30% of CxC. The papillomavirus genus alpha species 7 (α7) includes a group of hr types of which HPV18, 45, 59 are proportionally overrepresented in cervical AC and only partially (HPV18) targeted by current vaccines. To target these types, we generated a chimeric vaccine antigen that consists of a cross-neutralizing epitope (homologue of HPV16 RG1) of the L2 minor capsid protein of HPV45 genetically inserted into a surface loop of HPV18 L1 VLP (18L1-45RG1). Vaccination of NZW rabbits with 18L1-45RG1 VLP plus alum-MPL adjuvant induced high-titer neutralizing antibodies against homologous HPV18, that cross-neutralized non-cognate hr α7 types HPV39, 45, 68, but not HPV59, and low risk HPV70 in vitro, and induced a robust L1-specific cellular immune response. Passive immunization protected mice against experimental vaginal challenge with pseudovirions of HPV18, 39, 45 and 68, but not HPV59 or the distantly related α9 type HPV16. 18L1-45RG1 VLP might be combined with our previously described 16L1-16RG1 VLP to develop a second generation bivalent vaccine with extended spectrum against hr HPV.

The consistent and specific presence of Equus caballus papillomavirus type 2 (EcPV2) DNA and mRNA in equine genital squamous cell carcinoma (gSCC) is suggestive of an etiological role in tumor development. To further validate this concept, EcPV2-neutralizing serum antibody titers were determined by an EcPV2 pseudovirion (PsV) neutralization assay. Furthermore, an EcPV2 L1 virus-like particle (VLP)-based vaccine was generated and its prophylactic efficacy evaluated in vivo. All 6/6 gSCC-affected, but only 3/20 tumor-free age-matched animals revealed EcPV2-neutralizing serum antibody titers by PsV assay. Vaccination of NZW rabbits and BalbC mice with EcPV2 L1 VLP using Freund׳s or alum respectively as adjuvant induced high-titer neutralizing serum antibodies (1600-12,800). Passive transfer with rabbit EcPV2-VLP immune sera completely protected mice from experimental vaginal EcPV2 PsV infection. These findings support the impact of EcPV2 in equine gSCC development and recommend EcPV2 L1 VLP as prophylactic vaccine against EcPV2 infection and associated disease in equids.

Persistent infection with high-risk human papillomavirus (HPV) types, most often HPV16 and HPV18, causes all cervical and most anal cancers, and a subset of vulvar, vaginal, penile and oropharyngeal carcinomas. Two prophylactic virus-like particle (VLPs)-based vaccines, are available that protect against vaccine type-associated persistent infection and associated disease, yet have no therapeutic effect on existing lesions or infections. We have generated recombinant live-attenuated influenza A viruses expressing the HPV16 oncogenes E6 and E7 as experimental immunotherapeutic vaccine candidates. The influenza A virus life cycle lacks DNA intermediates as important safety feature. Different serotypes were generated to ensure efficient prime and boost immunizations. The immune response to vaccination in C57BL/6 mice was characterized by peptide ELISA and IFN-γ ELISpot, demonstrating induction of cell-mediated immunity to HPV16 E6 and E7 oncoproteins. Prophylactic and therapeutic vaccine efficacy was analyzed in the murine HPV16-positive TC-1 tumor challenge model. Subcutaneous (s.c.) prime and boost vaccinations of mice with recombinant influenza A serotypes H1N1 and H3N2, followed by challenge with TC-1 cells resulted in complete protection or significantly reduced tumor growth as compared to control animals. In a therapeutic setting, s.c. vaccination of mice with established TC-1 tumors decelerated tumor growth and significantly prolonged survival. Importantly, intralesional vaccine administration induced complete tumor regression in 25% of animals, and significantly reduced tumor growth in 50% of mice. These results suggest recombinant E6E7 influenza viruses as a promising new approach for the development of a therapeutic vaccine against HPV-induced disease.

Reasons for performing study We have already demonstrated that vaccination with empty BPV 1 capsids termed virus‐like particles ( VLP ) protects horses from experimental infection with BPV 1 virion. Long‐term monitoring of antibody titres in experimental horses and data from other species suggest that this protection is long lasting. Objectives To test protection against experimental infection with BPV ‐1 virion in horses that were vaccinated with BPV ‐1 L 1 VLP approximately 5 years earlier. Study design Controlled experiment. Methods Seven horses, vaccinated 3 times (boosters after 4 weeks and 6 months) in 2007/2008 with doses of BPV 1 L 1 VLP ranging from 50 μg to 150 μg/dose and 3 unvaccinated control horses were challenged by intra‐dermal inoculation with cow wart derived BPV 1 virion (5 × 10 7 BPV ‐1 virions per wheal, 10 wheals per horse). Inoculation sites were monitored for 10 weeks. Blood for serum antibody titre determination by pseudovirion neutralisation assay was taken on the day of challenge and after 6 months. Results Six of 7 vaccinated horses had measurable serum antibody titres (1:50 to 1:400). These titres were boosted by inoculation (about one step of dilution). Two of 3 unvaccinated controls remained sero‐negative. One control horse showed sero‐conversion. All control horses developed tumours at all 10 inoculation sites. Tumours appeared approximately 2 weeks after inoculation and reached maximum sizes of up to 8 mm. Regression was complete by 8 weeks after their first appearance in all horses. All vaccinated horses remained completely free from tumours. No influence of dose rate or antibody titre on the level of protection could be determined. Conclusions BPV ‐1 L 1 VLP vaccination proves to be fully effective in protecting horses from experimental infection and tumour formation 5 years post immunisation. The protection was complete even in horses with low or unmeasurable antibody titres. This is another step towards establishing a vaccination against equine sarcoids. Ethical animal research: This experiment was approved by the institutional ethics committee and the national authority under license N o: GZ : 68.205/0144‐ II /3b/2012. Sources of funding: Veterinary University Vienna, A ustrian Research Fund ( FWF ). Competing interests: None.

Licensed human papillomavirus (HPV) vaccines, based on virus-like particles (VLPs) self-assembled from major capsid protein L1, afford type-restricted protection against HPV types 16/18/6/11 (or 16/18 for the bivalent vaccine), which cause 70% of cervical cancers (CxCas) and 90% of genital warts. However, they do not protect against less prevalent high-risk (HR) types causing 30% of CxCa, or cutaneous HPV. In contrast, vaccination with the minor capsid protein L2 induces low-level immunity to type-common epitopes. Chimeric RG1-VLP presenting HPV16 L2 amino acids 17-36 (RG1 epitope) within the DE-surface loop of HPV16 L1 induced cross-neutralizing antisera. We hypothesized that RG1-VLP vaccination protects against a large spectrum of mucosal and cutaneous HPV infections in vivo. Immunization with RG1-VLP adjuvanted with human-applicable alum-MPL (aluminum hydroxide plus 3-O-desacyl-4'-monophosphoryl lipid A) induced robust L2 antibodies (ELISA titers 2,500-12,500), which (cross-)neutralized mucosal HR HPV16/18/45/37/33/52/58/35/39/51/59/68/73/26/69/34/70, low-risk HPV6/11/32/40, and cutaneous HPV2/27/3/76 (titers 25-1,000) using native virion-or pseudovirion (PsV)-based assays, and a vigorous cytotoxic T lymphocyte response by enzyme-linked immunospot. In vivo, mice were efficiently protected against experimental vaginal challenge with mucosal HR PsV types HPV16/18/45/31/33/52/58/35/39/51/59/68/56/73/26/53/66/34 and low-risk HPV6/43/44. Enduring protection was demonstrated 1 year after vaccination. RG1-VLP is a promising next-generation vaccine with broad efficacy against all relevant mucosal and also cutaneous HPV types.

AimsBovine papillomavirus types 1 and 2 (BPV‐1/‐2) are chiefly involved in the pathogenesis of equine sarcoids. We have previously demonstrated that intramuscular immunisation of horses with BPV‐1 L1 virus‐like particles (VLPs) is safe and highly immunogenic, that BPV‐1 and ‐2 are closely related serotypes, and that intradermal inoculation of horses with cow wart‐derived BPV‐1 virions reliably results in the development of transient pseudo‐sarcoids. Based on these data, we next performed a virus challenge study.MethodsFourteen horses were immunised with 100 μg of BPV‐1 L1 VLP in adjuvant on Days 1 and 28; 7 horses served as nonvaccinated controls. On Day 42, all 21 horses were intradermally inoculated on their neck with 5 x 107 BPV‐1 virions per wheal (10 wheals per horse). The horses were monitored for adverse reactions and pseudo‐sarcoid formation. Antibody titres were determined for Days 0 and 42.ResultsAll 7 control horses developed pseudo‐sarcoids at every inoculation site (10/10). Tumours reached sizes of up to 16 mm in diameter and persisted for a minimum of 20 weeks. Immunisation with BPV‐1 L1 VLPs resulted in complete protection in 13 of 14 vaccinated horses, with serum antibody titres ranging between 6.400 and 25.600. One vaccinated horse developed 7 tumours, which reached a maximum size of 2 mm and completely regressed within 5 weeks, with antibody titres of 800.Conclusions and practical significanceImmunisation of 14 horses with BPV‐1 L1 VLPs conferred complete protection from experimental infection with BPV‐1 virion in 13 cases, and partial protection in one horse. The old age of this individual may account for the poor response to vaccination. Provided that the upcoming BPV‐2 challenge will yield similar results, BPV‐1 L1 VLP may be used for routine immunisation of equids.Ethical animal researchThis work was approved by the institutional ethics committee and the national authority according to Austrian Law for Animal Experiments, Tierversuchsgesetz ‐TVG. Approval Number: BMWF‐68.205/0236‐II/3b/2011. Sources of funding: This project is funded by the Veterinary University of Vienna Austrian Science Fund (FWF). Competing interests: None.

Licensed human papillomavirus (HPV) vaccines, based on virus-like particles (VLP) self-assembled from major capsid protein L1, afford type-restricted protection against types 16/18/6/11 (or 16/18 for the bivalent vaccine), which cause 70% of cervical carcinomas (Cxca) and 90% of genital warts. However, they do not protect against less prevalent high-risk types causing 30% of CxCa, or cutaneous HPV. The minor capsid protein L2 confers low-level immunity to type-common epitopes. Chimeric RG1-VLP presenting HPV16L2 amino acids 17–36 (RG1 epitope) within the DE-surface loop of HPV16L1 induce cross-neutralisation in vitro . We hypothesised, that RG1-VLP vaccination protects against a large spectrum of mucosal and cutaneous HPV infections in vivo . L2-specific antibody and CTL responses in RG1-VLP vaccinated rabbits were determined by ELISA and ELISPOT assays. Cross-neutralisation was analysed using native or pseudovirion (PsV) assays. Vaccine efficacy in vivo was determined in a mouse genital challenge model. Immunization with RG1-VLP adjuvanted with human-applicable alum-MPL induced robust L2 antibodies (ELISA titers 2,500–12,500), which cross-neutralised mucosal high-risk HPV26/33/35/39/68/59/68/73/69/53/34, low-risk HPV6/11/32/40/44/70, and cutaneous HPV2/27/3/76 (titers 25–1,000), and a vigorous CTL response. In vivo , mice were efficiently protected against experimental vaginal challenge with mucosal high-risk PsV types HPV16/18/45/31/33/52/58/35/39/51/59/68/56/73/26/53/66/34 and low-risk HPV6/43/44. Enduring protection was demonstrated 1 year after vaccination. RG1-VLP is a promising next-generation vaccine with broad efficacy against all relevant mucosal and also cutaneous HPV types.