Development of prophylactic human papillomavirus vaccines

The demonstration of the etiologic role of oncogenic type HPV infection in cervical cancer has led to the development of the prophylactic vaccines Cervarix and Gardasil. Despite their licensure, current vaccines do not elicit complete protection against all 15 oncogenic HPV genotypes, and the high cost of these vaccines is a major hurdle in access to vaccination. During my thesis, I have compared several promising technologies as candidate preventive vaccines with the potential to target a broad swath of medically significant HPV types using approaches that are simple and inexpensive to manufacture, and thus represent potential alternatives to highly multivalent L1 VLP vaccines. An initial critical step was the development of pseudovirions derived from medically significant HPV types, and examining their assembly and infectious pathway for commonalities using inhibitors and antibodies. The HPV pseudovirions were utilized for measuring the neutralizing antibody responses to candidate vaccines and for vaginal challenge studies in mice. In passive transfer studies neutralizing antibodies by all vaccines tested were sufficient to mediate protection against experimental vaginal challenge, implying their central role in effecting immunity and their relevance as a correlate of protection. We compared four L2-based HPV vaccine candidates: 1. in vivo electroporation of a DNA vector expressing a codon-optimized L1 gene, 2. HPV16 L1 capsomers linked with amino acids 13-47 of HPV18, HPV31, and HPV45 L2 in tandem (GST-HPV16L1Δ-L2x3), 3. A chimeric VLP presenting HPV16 L2 amino acids 17-36 in an immunodominant loop of HPV16 L1 (RG1-VLP), and 4. L2 multimer fusions comprising L2 protective domains (amino acids 11-88) derived from 8 or 5 different HPV subtypes (11-88×8 and 11-88×5). Electroporation of L1 DNA vector elicited type-restricted immunity but when different HPV type vectors were co-administered heterotypic L1 interactions produced interference. GST-HPV16L1Δ-L2x3 and RG1-VLP induced high titers of HPV16 L1-specific neutralizing antibody but moderate titers of L2-specific antibody, although sufficient for cross-protection against other HPV types. L2 multimers induced substantially broader protection against vaginal challenge with medically significant HPV types than either Gardasil or Cervarix in the mouse model. Our data suggest the potential of L2 multimer and RG1-VLP as next generation prophylactic HPV vaccines

The high risk HPV16 L2 minor capsid protein has multiple transport signals that mediate its nucleocytoplasmic traffic

AbstractIn this study we examined the transport signals contributing to HPV16 L2 nucleocytoplasmic traffic using confocal microscopy analysis of enhanced green fluorescent protein—L2 (EGFP-L2) fusions expressed in HeLa cells. We confirmed that both nuclear localization signals (NLSs), the nNLS (1MRHKRSAKRTKR12) and cNLS (456RKRRKR461), previously characterized in vitro (Darshan et al., 2004), function independently in vivo. We discovered that a middle region rich in arginine residues (296SRRTGIRYSRIGNKQTLRTRS316) functions as a nuclear retention sequence (NRS), as mutagenesis of critical arginine residues within this NRS reduced the fraction of L2 in the nucleus despite the presence of both NLSs. Significantly, the infectivity of HPV16 pseudoviruses containing either RR297AA or RR297EE within the L2 NRS was strongly reduced both in HaCaT cells and in a murine challenge model. Experiments using Ratjadone A nuclear export inhibitor and mutation-localization analysis lead to the discovery of a leucine-rich nuclear export signal (462LPYFFSDVSL) mediating 16L2 nuclear export. These data indicate that HPV16 L2 nucleocytoplasmic traffic is dependent on multiple functional transport signals

Capsomer Vaccines Protect Mice from Vaginal Challenge with Human Papillomavirus

Capsomers were produced in bacteria as glutathione-S-transferase (GST) fusion proteins with human papillomavirus type 16 L1 lacking the first nine and final 29 residues (GST-HPV16L1Δ) alone or linked with residues 13–47 of HPV18, HPV31 and HPV45 L2 in tandem (GST-HPV16L1Δ-L2x3). Subcutaneous immunization of mice with GST-HPV16L1Δ or GST-HPV16L1Δ-L2x3 in alum and monophosphoryl lipid A induced similarly high titers of HPV16 neutralizing antibodies. GST-HPV16L1Δ-L2x3 also elicited moderate L2-specific antibody titers. Intravaginal challenge studies showed that immunization of mice with GST-HPV16 L1Δ or GST-HPV16L1Δ-L2x3 capsomers, like Cervarix®, provided complete protection against HPV16. Conversely, vaccination with GST-HPV16 L1Δ capsomers failed to protect against HPV18 challenge, whereas mice immunized with either GST-HPV16L1Δ-L2x3 capsomers or Cervarix® were each completely protected. Thus, while the L2-specific response was moderate, it did not interfere with immunity to L1 in the context of GST-HPV16L1Δ-L2x3 and is sufficient to mediate L2-dependent protection against an experimental vaginal challenge with HPV18

Development of prophylactic human papillomavirus vaccines

The demonstration of the etiologic role of oncogenic type HPV infection in cervical cancer has led to the development of the prophylactic vaccines Cervarix and Gardasil. Despite their licensure, current vaccines do not elicit complete protection against all 15 oncogenic HPV genotypes, and the high cost of these vaccines is a major hurdle in access to vaccination. During my thesis, I have compared several promising technologies as candidate preventive vaccines with the potential to target a broad swath of medically significant HPV types using approaches that are simple and inexpensive to manufacture, and thus represent potential alternatives to highly multivalent L1 VLP vaccines. An initial critical step was the development of pseudovirions derived from medically significant HPV types, and examining their assembly and infectious pathway for commonalities using inhibitors and antibodies. The HPV pseudovirions were utilized for measuring the neutralizing antibody responses to candidate vaccines and for vaginal challenge studies in mice. In passive transfer studies neutralizing antibodies by all vaccines tested were sufficient to mediate protection against experimental vaginal challenge, implying their central role in effecting immunity and their relevance as a correlate of protection. We compared four L2-based HPV vaccine candidates: 1. in vivo electroporation of a DNA vector expressing a codon-optimized L1 gene, 2. HPV16 L1 capsomers linked with amino acids 13-47 of HPV18, HPV31, and HPV45 L2 in tandem (GST-HPV16L1Δ-L2x3), 3. A chimeric VLP presenting HPV16 L2 amino acids 17-36 in an immunodominant loop of HPV16 L1 (RG1-VLP), and 4. L2 multimer fusions comprising L2 protective domains (amino acids 11-88) derived from 8 or 5 different HPV subtypes (11-88×8 and 11-88×5). Electroporation of L1 DNA vector elicited type-restricted immunity but when different HPV type vectors were co-administered heterotypic L1 interactions produced interference. GST-HPV16L1Δ-L2x3 and RG1-VLP induced high titers of HPV16 L1-specific neutralizing antibody but moderate titers of L2-specific antibody, although sufficient for cross-protection against other HPV types. L2 multimers induced substantially broader protection against vaginal challenge with medically significant HPV types than either Gardasil or Cervarix in the mouse model. Our data suggest the potential of L2 multimer and RG1-VLP as next generation prophylactic HPV vaccines

Impact of inhibitors and L2 antibodies upon the infectivity of diverse alpha and beta human papillomavirus types.

The licensed human papillomavirus (HPV) vaccines elicit type-restricted immunity but do not target cutaneous HPV types of the beta genus that are associated with non-melanoma skin cancer in immune-compromised patients, and it is unclear if these diverse types share a common mechanism of infection. Residues 11-88 of minor capsid protein L2 contain cross-protective epitopes, and vaccination with concatamers of this region derived from as many as eight alpha HPV (L2 α11-88x8) is being developed as an alternative prophylactic vaccine with potentially broader efficacy. There is also interest in developing broadly protective topical microbicides, such as carrageenan or heparin that block HPV receptor interactions, or small molecule inhibitors of infection. Here we have examined several inhibitors of HPV infection and antisera to L2 α11-88x8 for their breadth of activity against infection by 34 HPV types from within both the alpha and beta families using pseudovirions (PsV) carrying a luciferase reporter as surrogates for native virus. We observed that both heparin and carrageenan prevented infection by mucosatropic HPV types, but surprisingly PsV of several epidermotropic alpha4 and beta HPV types exhibited increased infectivity especially at low inhibitor concentrations. Furin and γ-secretase inhibitors and L2 α11-88x8 antiserum blocked infection by all HPV PsV types tested. These findings suggest that the distinct tropism of mucosal and cutaneous HPV may reflect distinct cell surface receptor interactions, but a common uptake mechanism dependent upon furin and γ-secretase proteolytic activities. Carrageenan, which is being tested as a vaginal microbicide, broadly inhibited infection by the high-risk mucosatropic HPV PsV, but not most skin tropic alpha and beta HPV. Vaccination with an L2 multimer derived exclusively from alpha papillomavirus sequences induced antibodies that broadly neutralized PsV of all 34 HPVs from within both the alpha and beta families, suggesting each displays conserved L2 neutralizing epitopes

Optimization of multimeric human papillomavirus L2 vaccines.

We sought to define the protective epitopes within the amino terminus of human papillomavirus (HPV) type 16 minor capsid protein L2. Passive transfer of mice with rabbit antisera to HPV16 L2 peptides 17-36, 32-51 and 65-81 provided significant protection against vaginal HPV16 challenge, whereas antisera to 47-66, 108-120 or 373-392 did not. Vaccination with L1 virus-like particles induces a high titer, but generally type-restricted neutralizing antibody response. Conversely, vaccination with L2 11-88, especially multimers thereof, induces antibodies that neutralize a broad range of papillomavirus types, albeit at lower titers than for L1 VLP. With the intent of enhancing the immunogenicity and the breadth of protection by focusing the immune response to the key protective epitopes, we designed L2 fusion proteins consisting of residues ∼11-88 of eight divergent mucosal HPV types 6, 16, 18, 31, 39, 51, 56, 73 (11-88×8) or residues ∼13-47 of fifteen HPV types (13-47×15). The 11-88×8 was significantly more immunogenic than 13-47×15 in Balb/c mice regardless of the adjuvant used, suggesting the value of including the 65-81 protective epitope in the vaccine. Since the L2 47-66 peptide antiserum failed to elicit significant protection, we generated an 11-88×8 construct deleted for this region in each subunit (11-88×8Δ). Mice were vaccinated with 11-88×8 and 11-88×8Δ to determine if deletion of this non-protective epitope enhanced the neutralizing antibody response. However, 11-88×8Δ was significantly less immunogenic than 11-88×8, and even the addition of a known T helper epitope, PADRE, to the construct (11-88×8ΔPADRE) failed to recover the immunogenicity of 11-88×8 in C57BL/6 mice, suggesting that while L2 47-66 is not a critical protective or T helper epitope, it nevertheless contributes to the immunogenicity of the L2 11-88×8 multimer vaccine

Multivalent human papillomavirus l1 DNA vaccination utilizing electroporation.

Naked DNA vaccines can be manufactured simply and are stable at ambient temperature, but require improved delivery technologies to boost immunogenicity. Here we explore in vivo electroporation for multivalent codon-optimized human papillomavirus (HPV) L1 and L2 DNA vaccination.Balb/c mice were vaccinated three times at two week intervals with a fusion protein comprising L2 residues ∼11-88 of 8 different HPV types (11-88×8) or its DNA expression vector, DNA constructs expressing L1 only or L1+L2 of a single HPV type, or as a mixture of several high-risk HPV types and administered utilizing electroporation, i.m. injection or gene gun. Serum was collected two weeks and 3 months after the last vaccination. Sera from immunized mice were tested for in-vitro neutralization titer, and protective efficacy upon passive transfer to naive mice and vaginal HPV challenge. Heterotypic interactions between L1 proteins of HPV6, HPV16 and HPV18 in 293TT cells were tested by co-precipitation using type-specific monoclonal antibodies.Electroporation with L2 multimer DNA did not elicit detectable antibody titer, whereas DNA expressing L1 or L1+L2 induced L1-specific, type-restricted neutralizing antibodies, with titers approaching those induced by Gardasil. Co-expression of L2 neither augmented L1-specific responses nor induced L2-specific antibodies. Delivery of HPV L1 DNA via in vivo electroporation produces a stronger antibody response compared to i.m. injection or i.d. ballistic delivery via gene gun. Reduced neutralizing antibody titers were observed for certain types when vaccinating with a mixture of L1 (or L1+L2) vectors of multiple HPV types, likely resulting from heterotypic L1 interactions observed in co-immunoprecipitation studies. High titers were restored by vaccinating with individual constructs at different sites, or partially recovered by co-expression of L2, such that durable protective antibody titers were achieved for each type.Multivalent vaccination via in vivo electroporation requires spatial separation of individual type L1 DNA vaccines

Impact of γ-secretase inhibitor on infection by PsV of diverse HPV.

<p>PsVs of each indicated HPV type carrying a luciferase reporter gene were transferred to 293TT cells for 72γ-secretase inhibitor XXI (n = 3). After incubation, luciferase activity was measured and percent inhibition of infectivity compared to control calculated. Red and blue bars represent mucosal and cutaneous HPV types, respectively.</p

Summary of the phylogeny, tropism, charge and in vitro neutralization by L2 11-88x8 antiserum of the 34 HPV genotypes tested.

<p>Phylogeny and tropism were taken from de Villiers et al <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097232#pone.0097232-deVilliers1" target="_blank">[58]</a>, predicted net charge of L1 at pH7.4 was calculated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097232#pone.0097232-Mistry1" target="_blank">[37]</a>. PsVs of each indicated HPV type carrying a luciferase reporter gene were mixed with titrated rabbit L2 α11-88x8 antiserum for two hours at 37°C, then the mixtures were transferred to 293TT cells and cultured for 72 hours. Cells were then lysed and luciferase activity was measured. Neutralization titer and 95% confidence interval are shown.</p

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