Christina Schellenbacher

Chimeric L1-L2 Virus-Like Particles as Potential Broad-Spectrum Human Papillomavirus Vaccines

ABSTRACT The amino (N) terminus of the human papillomavirus (HPV) minor capsid protein L2 can induce low-titer, cross-neutralizing antibodies. The aim of this study was to improve immunogenicity of L2 peptides by surface display on highly ordered, self-assembled virus-like particles (VLP) of major capsid protein L1, and to more completely characterize neutralization epitopes of L2. Overlapping peptides comprising amino acids (aa) 2 to 22 (hereafter, chimera or peptide 2-22), 13 to 107, 18 to 31, 17 to 36, 35 to 75, 75 to 112, 115 to 154, 149 to 175, and 172 to 200 of HPV type 16 (HPV16) L2 were genetically engineered into the DE surface loop of bovine papillomavirus type 1 L1 VLP. Except for chimeras 35-75 and 13-107, recombinant fusion proteins assembled into VLP. Vaccination of rabbits with Freunds adjuvanted native VLP induced higher L2-specific antibody titers than vaccination with corresponding sodium dodecyl sulfate-denatured proteins. Immune sera to epitopes within residues 13 to 154 neutralized HPV16 in pseudovirion neutralization assays, whereas chimera 17-36 induced additional cross-neutralization to divergent high-risk HPV18, -31, -45, -52, and -58; low-risk HPV11; and beta-type HPV5 (titers of 50 to 10,000). Aluminum hydroxide-monophosphoryl lipid A (Alum-MPL)-adjuvanted VLP induced similar patterns of neutralization in both rabbits and mice, albeit with 100-fold-lower titers than Freunds adjuvant. Importantly, Alum-MPL-adjuvanted immunization with chimeric HPV16L1-HPV16L2 (peptide 17-36) VLP induced neutralization or cross-neutralization of HPV16, -18, -31, -45, -52, and -58; HPV6 and -11; and HPV5 (titers of 50 to 100,000). Immunization with HPV16 L1-HPV16 L2 (chimera 17-36) VLP in adjuvant applicable for human use induces broad-spectrum neutralizing antibodies against HPV types evolutionarily divergent to HPV16 and thus may protect against infection with mucosal high-risk, low-risk, and beta HPV types and associated disease.

Efficacy of RG1-VLP Vaccination against Infections with Genital and Cutaneous Human Papillomaviruses

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.

Diseases caused by human papillomaviruses (HPV)

Human papillomaviruses (HPV) are non‐enveloped tumor viruses with a double stranded DNA approximately 8 kilobases in length. The viral genome is enclosed by a spherical capsid with icosahedral symmetry and a diameter of about 55 nm. More than 100 HPV types have been identified. They infect the squamous epithelia of skin and mucosa and usually cause benign papillomas or warts. Persistent infection with high‐risk oncogenic HPV causes all cervical cancers, most anal cancers, and a subset of vulvar, vaginal, penile and oropha‐ryngeal cancers. In recent years cutaneous beta‐HPV types have been associated with the pathogenesis of non‐melanoma skin cancers. Two prophylactic HPV vaccines based on virus‐like particles (VLP) are licensed. These are up to 100% effective in preventing HPV 16 and HPV 18 infections and associated genital lesions in women, who have not been previously infected with these types. One vaccine also prevents genital warts caused by HPV 6 and HPV 11.

A quadrivalent HPV vaccine induces humoral and cellular immune responses in WHIM immunodeficiency syndrome.

WHIM-syndrome is an inherited immunodeficiency disorder with abnormal susceptibility to human papillomavirus (HPV) infection and diseases. We determined safety and immunogenicity to a quadrivalent HPV vaccine in WHIM-syndrome by detection of HPV-specific antibodies and lymphoproliferation. In virus-like-particle (VLP)-ELISA, a WHIM patient showed antibody titers up to 400 for HPV-6/11/16/18, whereas immuno-competent controls developed titers of 6400-25,600. In pseudovirion assays, the patients neutralization titers ranged from 20 to 400 to the four HPV vaccine types, while titers of 1600-25,600 were detected in healthy vaccinees. Specific proliferation of PBMC of the WHIM patient to the HPV vaccine was demonstrated. This first report on response to HPV vaccination in WHIM-immunodeficiency highlights that patients with WHIM-syndrome, and probably other immunodeficiencies, may benefit from HPV immunoprophylaxis.

Bovine papillomavirus type 1 (BPV1) and BPV2 are closely related serotypes

Infection with bovine papillomavirus type 1 (BPV1) or BPV2 induces fibropapillomas in cows and skin sarcoids in horses. Prophylactic vaccination targeting BPV1 and BPV2 may reduce the incidence of these economically important diseases. The L1 major capsid proteins of BPV1 and BPV2 were expressed in Sf-9 insect cells and both self-assembled into virus-like particles (VLPs). Using conformation-dependent monoclonal antibodies (mAb) both type-specific and shared epitopes were detected. Antisera were raised against BPV1 or BPV2 VLP using alum adjuvant, and their (cross)neutralization capacity was tested by C127 neutralization assays using native BPV1 and BPV2 virions, or by BPV1 pseudovirion assay. Antisera induced by either VLP vaccine were able to robustly (cross-)neutralize heterologous as well as homologous types, indicating that BPV1 and BPV2 are closely related serotypes. These results suggest that a monovalent BPV1 (or BPV2) VLP vaccine may potentially protect against both BPV1 and BPV2 infections and associated diseases.

A chimeric 18L1-45RG1 virus-like particle vaccine cross-protects against oncogenic alpha-7 human papillomavirus types.

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.

Chimeric L2-Based Virus-Like Particle (VLP) Vaccines Targeting Cutaneous Human Papillomaviruses (HPV).

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.

Progress and prospects for L2-based human papillomavirus vaccines

ABSTRACT Human papillomavirus (HPV) is a worldwide public health problem, particularly in resource-limited countries. Fifteen high-risk genital HPV types are sexually transmitted and cause 5% of all cancers worldwide, primarily cervical, anogenital and oropharyngeal carcinomas. Skin HPV types are generally associated with benign disease, but a subset is linked to non-melanoma skin cancer. Licensed HPV vaccines based on virus-like particles (VLPs) derived from L1 major capsid antigen of key high risk HPVs are effective at preventing these infections but do not cover cutaneous types and are not therapeutic. Vaccines targeting L2 minor capsid antigen, some using capsid display, adjuvant and fusions with early HPV antigens or Toll-like receptor agonists, are in development to fill these gaps. Progress and challenges with L2-based vaccines are summarized.

Establishment of an in vitro equine papillomavirus type 2 (EcPV2) neutralization assay and a VLP-based vaccine for protection of equids against EcPV2-associated genital tumors

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.

Abstract 2367: cGMP production of a chimeric virus-like particle vaccine for prevention of HPV-associated cancers

The NCI Division of Cancer Prevention9s PREVENT Cancer Preclinical Drug Development Program supports preclinical development and clinical translation of novel cancer preventive interventions. One of the projects currently supported came from principal investigators, Drs. Reinhard Kirnbauer and Richard Roden, who requested cGMP production of a novel chimeric virus-like particle (VLP)-based human papillomavirus (HPV) vaccine, comprising HPV16 L1 VLP with HPV16 L2-RG1 epitope repetitively displayed on the capsid surface (RG1-VLP). The vaccine is designed to prevent infection by a broad range of HPV types, including mucosal and cutaneous types not targeted by current vaccines (J Invest Dermatol 2013, 133:2706), and thus has the potential to become a broadly effective next generation HPV vaccine. The cGMP process is currently being developed and validated by Paragon Bioservices in Baltimore, MD, under NCI contract with MRIGlobal. RG1-VLP is expressed in Sf9 insect cells infected with a triple-plaque-purified recombinant baculovirus. After cell culture clarification and capsid maturation steps, VLPs are purified by a series of chromatography steps to remove host cell derived impurities. The purified VLPs are analyzed by ELISA and Western blot immunoassays. Removal of host cell protein and DNA is confirmed by ELISA and qPCR, respectively. Assembly of a chimeric RG1-VLP protein into full size VLP structures (approximately 50-60 nm in diameter) is verified by transmission electron microscopy. cGMP-produced VLPs should be available during the first half of 2016. The PREVENT Program will perform the required toxicology studies and will assist with IND filing, for a first-in-human Phase I study of RG1-VLP vaccine. While the clinical protocol is still under development, it is anticipated that 15-20 healthy adult volunteers will be enrolled into each of three to four escalating dose groups. While the primary objective of the Phase I study will be to establish the safety of RG1-VLP, preliminary immunogenicity studies will be included. Immunogenicity of RG1-VLP will be evaluated by serum anti-L1 and anti-RG1 antibody levels as well as protective and cross-neutralizing antibodies against HPV16 and a broad panel of HPV types including at least one type not covered by marketed vaccines. Citation Format: George W. Buchman, Brian P. Howard, Nadia Abdallah, Bala Medicherla, Mary Anne Fisher, Jonathan M. White, Michelle L. Kennedy, Shizuko Sei, Richard B.S. Roden, Christina Schellenbacher, Reinhard Kirnbauer, Robert H. Shoemaker. cGMP production of a chimeric virus-like particle vaccine for prevention of HPV-associated cancers. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2367.