RG2-VLP: a Vaccine Designed to Broadly Protect against Anogenital and Skin Human Papillomaviruses Causing Human Cancer.

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 ßHPVs 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 ßHPV. To comprehensively target both α- and ßHPVs, 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 ßHPV 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 (Tmc6Δ/Δ or Tmc8Δ/Δ) with RG2-VLP induced robust L2-specific antibody titers and protected against ß-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 ßHPVs tested. Taken together, these observations suggest RG2-VLP's potential as a broad-spectrum vaccine to prevent αHPV-driven anogenital, oropharyngeal, and ßHPV-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 ßHPV 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 ßHPVs by incorporation into HPV16 and HPV18 L1 VLP of 20-amino-acid conserved protective epitopes derived from minor capsid protein L2.

Roles of Fc Domain and Exudation in L2 Antibody-Mediated Protection against Human Papillomavirus.

To address how L2-specific antibodies prevent human papillomavirus (HPV) infection of the genital tract, we generated neutralizing monoclonal antibodies (MAbs) WW1, a rat IgG2a that binds L2 residues 17 to 36 (like mouse MAb RG1), and JWW3, a mouse IgG2b derivative of Mab24 specific for L2 residues 58 to 64. By Western blotting, WW1 recognized L2 of 29/34 HPV genotypes tested, compared to only 13/34 for RG1 and 25/34 for JWW3. WW1 IgG and F(ab')2 bound HPV16 pseudovirions similarly; however, whole IgG provided better protection against HPV vaginal challenge. Passive transfer of WW1 IgG was similarly protective in wild-type and neonatal Fc receptor (FcRn)-deficient mice, suggesting that protection by WW1 IgG is not mediated by FcRn-dependent transcytosis. Rather, local epithelial disruption, required for genital infection and induced by either brushing or nonoxynol-9 treatment, released serum IgG in the genital tract, suggesting Fc-independent exudation. Depletion of neutrophils and macrophages reduced protection of mice upon passive transfer of whole WW1 or JWW3 IgGs. Similarly, IgG-mediated protection by L2 MAbs WW1, JWW3, and RG1 was reduced in Fc receptor knockout compared to wild-type mice. However, levels of in vitro neutralization by WW1 IgG were similar in TRIM21 knockout and wild-type cells, indicating that Fc does not contribute to antibody-dependent intracellular neutralization (ADIN). In conclusion, the Fc domain of L2-specific IgGs is not active for ADIN, but it opsonizes bound extracellular pseudovirions for phagocytes in protecting mice from intravaginal HPV challenge. Systemically administered neutralizing IgG can access the site of infection in an abrasion via exudation without the need for FcRn-mediated transcytosis.IMPORTANCE At least 15 alpha HPV types are causative agents for 5% of all cancers worldwide, and beta types have been implicated in nonmelanoma skin cancer, whereas others produce benign papillomas, such as genital warts, associated with considerable morbidity and health systems costs. Vaccines targeting the minor capsid protein L2 have the potential to provide broad-spectrum immunity against medically relevant HPVs of divergent genera via the induction of broadly cross-neutralizing serum IgG. Here we examine the mechanisms by which L2-specific serum IgG reaches the viral inoculum in the genital tract to effect protection. Abrasion of the vaginal epithelium allows the virus to access and infect basal keratinocytes, and our findings suggest that this also permits the local exudation of neutralizing IgG and vaccine-induced sterilizing immunity. We also demonstrate the importance of Fc-mediated phagocytosis of L2 antibody-virion complexes for humoral immunity, a protective mechanism that is not detected by current in vitro neutralization assays.

Spontaneous and Vaccine-Induced Clearance of Mus Musculus Papillomavirus 1 Infection.

Mus musculus papillomavirus 1 (MmuPV1/MusPV1) induces persistent papillomas in immunodeficient mice but not in common laboratory strains. To facilitate the study of immune control, we sought an outbred and immunocompetent laboratory mouse strain in which persistent papillomas could be established. We found that challenge of SKH1 mice (Crl:SKH1-Hrhr) with MmuPV1 by scarification on their tail resulted in three clinical outcomes: (i) persistent (>2-month) papillomas (∼20%); (ii) transient papillomas that spontaneously regress, typically within 2 months (∼15%); and (iii) no visible papillomas and viral clearance (∼65%). SKH1 mice with persistent papillomas were treated by using a candidate preventive/therapeutic naked-DNA vaccine that expresses human calreticulin (hCRT) fused in frame to MmuPV1 E6 (mE6) and mE7 early proteins and residues 11 to 200 of the late protein L2 (hCRTmE6/mE7/mL2). Three intramuscular DNA vaccinations were delivered biweekly via in vivo electroporation, and both humoral and CD8 T cell responses were mapped and measured. Previously persistent papillomas disappeared within 2 months after the final vaccination. Coincident virologic clearance was confirmed by in situ hybridization and a failure of disease to recur after CD3 T cell depletion. Vaccination induced strong mE6 and mE7 CD8+ T cell responses in all mice, although they were significantly weaker in mice that initially presented with persistent warts than in those that spontaneously cleared their infection. A human papillomavirus 16 (HPV16)-targeted version of the DNA vaccine also induced L2 antibodies and protected mice from vaginal challenge with an HPV16 pseudovirus. Thus, MmuPV1 challenge of SKH1 mice is a promising model of spontaneous and immunotherapy-directed clearances of HPV-related disease.IMPORTANCE High-risk-type human papillomaviruses (hrHPVs) cause 5% of all cancer cases worldwide, notably cervical, anogenital, and oropharyngeal cancers. Since preventative HPV vaccines have not been widely used in many countries and do not impact existing infections, there is considerable interest in the development of therapeutic vaccines to address existing disease and infections. The strict tropism of HPV requires the use of animal papillomavirus models for therapeutic vaccine development. However, MmuPV1 failed to grow in common laboratory strains of mice with an intact immune system. We show that MmuPV1 challenge of the outbred immunocompetent SKH1 strain produces both transient and persistent papillomas and that vaccination of the mice with a DNA expressing an MmuPV1 E6E7L2 fusion with calreticulin can rapidly clear persistent papillomas. Furthermore, an HPV16-targeted version of the DNA can protect against vaginal challenge with HPV16, suggesting the promise of this approach to both prevent and treat papillomavirus-related disease.

Optimization of heterologous DNA-prime, protein boost regimens and site of vaccination to enhance therapeutic immunity against human papillomavirus-associated disease.

BACKGROUND: Human papillomavirus (HPV) has been identified as the primary etiologic factor of cervical cancer as well as subsets of anogenital and oropharyngeal cancers. The two HPV viral oncoproteins, E6 and E7, are uniquely and consistently expressed in all HPV infected cells and are therefore promising targets for therapeutic vaccination. Both recombinant naked DNA and protein-based HPV vaccines have been demonstrated to elicit HPV-specific CD8+ T cell responses that provide therapeutic effects against HPV-associated tumor models. Here we examine the immunogenicity in a preclinical model of priming with HPV DNA vaccine followed by boosting with filterable aggregates of HPV 16 L2E6E7 fusion protein (TA-CIN). RESULTS: We observed that priming twice with an HPV DNA vaccine followed by a single TA-CIN booster immunization generated the strongest antigen-specific CD8+ T cell response compared to other prime-boost combinations tested in C57BL/6 mice, whether naïve or bearing the HPV16 E6/E7 transformed syngeneic tumor model, TC-1. We showed that the magnitude of antigen-specific CD8+ T cell response generated by the DNA vaccine prime, TA-CIN protein vaccine boost combinatorial strategy is dependent on the dose of TA-CIN protein vaccine. In addition, we found that a single booster immunization comprising intradermal or intramuscular administration of TA-CIN after priming twice with an HPV DNA vaccine generated a comparable boost to E7-specific CD8+ T cell responses. We also demonstrated that the immune responses elicited by the DNA vaccine prime, TA-CIN protein vaccine boost strategy translate into potent prophylactic and therapeutic antitumor effects. Finally, as seen for repeat TA-CIN protein vaccination, we showed that the heterologous DNA prime and protein boost vaccination strategy is well tolerated by mice. CONCLUSIONS: Our results provide rationale for future clinical testing of HPV DNA vaccine prime, TA-CIN protein vaccine boost immunization regimen for the control of HPV-associated diseases.

Immunologic Control of Mus musculus Papillomavirus Type 1.

Persistent papillomas developed in ~10% of out-bred immune-competent SKH-1 mice following MusPV1 challenge of their tail, and in a similar fraction the papillomas were transient, suggesting potential as a model. However, papillomas only occurred in BALB/c or C57BL/6 mice depleted of T cells with anti-CD3 antibody, and they completely regressed within 8 weeks after depletion was stopped. Neither CD4+ nor CD8+ T cell depletion alone in BALB/c or C57BL/6 mice was sufficient to permit visible papilloma formation. However, low levels of MusPV1 were sporadically detected by either genomic DNA-specific PCR analysis of local skin swabs or in situ hybridization of the challenge site with an E6/E7 probe. After switching to CD3+ T cell depletion, papillomas appeared upon 14/15 of mice that had been CD4+ T cell depleted throughout the challenge phase, 1/15 of CD8+ T cell depleted mice, and none in mice without any prior T cell depletion. Both control animals and those depleted with CD8-specific antibody generated MusPV1 L1 capsid-specific antibodies, but not those depleted with CD4-specific antibody prior to T cell depletion with CD3 antibody. Thus, normal BALB/c or C57BL/6 mice eliminate the challenge dose, whereas infection is suppressed but not completely cleared if their CD4 or CD8 T cells are depleted, and recrudescence of MusPV1 is much greater in the former following treatment with CD3 antibody, possibly reflecting their failure to generate capsid antibody. Systemic vaccination of C57BL/6 mice with DNA vectors expressing MusPV1 E6 or E7 fused to calreticulin elicits potent CD8 T cell responses and these immunodominant CD8 T cell epitopes were mapped. Adoptive transfer of a MusPV1 E6-specific CD8+ T cell line controlled established MusPV1 infection and papilloma in RAG1-knockout mice. These findings suggest the potential of immunotherapy for HPV-related disease and the importance of host immunogenetics in the outcome of infection.

Durable immunity to oncogenic human papillomaviruses elicited by adjuvanted recombinant Adeno-associated virus-like particle immunogen displaying L2 17-36 epitopes.

Vaccination with the minor capsid protein L2, notably the 17-36 neutralizing epitope, induces broadly protective antibodies, although the neutralizing titers attained in serum are substantially lower than for the licensed L1 VLP vaccines. Here we examine the impact of other less reactogenic adjuvants upon the induction of durable neutralizing serum antibody responses and protective immunity after vaccination with HPV16 and HPV31 L2 amino acids 17-36 inserted at positions 587 and 453 of VP3, respectively, for surface display on Adeno-Associated Virus 2-like particles [AAVLP (HPV16/31L2)]. Mice were vaccinated three times subcutaneously with AAVLP (HPV16/31L2) at two week intervals at several doses either alone or formulated with alum, alum and MPL, RIBI adjuvant or Cervarix. The use of adjuvant with AAVLP (HPV16/31L2) was necessary in mice for the induction of L2-specific neutralizing antibody and protection against vaginal challenge with HPV16. While use of alum was sufficient to elicit durable protection (>3 months after the final immunization), antibody titers were increased by addition of MPL and RIBI adjuvants. To determine the breadth of immunity, rabbits were immunized three times with AAVLP (HPV16/31L2) either alone, formulated with alum±MPL, or RIBI adjuvants, and after serum collection, the animals were concurrently challenged with HPV16/31/35/39/45/58/59 quasivirions or cottontail rabbit papillomavirus (CRPV) at 6 or 12 months post-immunization. Strong protection against all HPV types was observed at both 6 and 12 months post-immunization, including robust protection in rabbits receiving the vaccine without adjuvant. In summary, vaccination with AAVLP presenting HPV L2 17-36 epitopes at two sites on their surface induced cross-neutralizing serum antibody, immunity against HPV16 in the genital tract, and long-term protection against skin challenge with the 7 most common oncogenic HPV types when using a clinically relevant adjuvant.

Production of Furin-Cleaved Papillomavirus Pseudovirions and Their Use for In Vitro Neutralization Assays of L1- or L2-Specific Antibodies.

Immunization with Human Papillomavirus (HPV) L1 virus-like particles or L2 capsid protein elicits neutralizing antibodies that mediate protection. A high-throughput and sensitive in vitro neutralization assay is therefore valuable for prophylactic HPV vaccine studies. Over several hours during infection of the genital tract, virions take on a distinct intermediate conformation, including a required furin cleavage of L2 at its N-terminus. This intermediate is an important target for neutralization by L2-specific antibody, but it is very transiently exposed during in vitro infection of most cell lines resulting in insensitive measurement for L2, but not L1-specific neutralizing antibodies. To model this intermediate, we describe a protocol to generate furin-cleaved HPV pseudovirions (fc-PsV), which deliver an encapsidated reporter plasmid to facilitate infectivity measurements. We also describe a protocol for use of fc-PsV in a high-throughput in vitro neutralization assay for the sensitive measurement of both L1 and L2-specific neutralizing antibodies.

Seroepidemiology of Human Papillomavirus 16 (HPV16) L2 and Generation of L2-Specific Human Chimeric Monoclonal Antibodies.

Presently, the seroprevalence of human papillomavirus (HPV) minor capsid antigen L2-reactive antibody is not well understood, and no serologic standard exists for L2-specific neutralizing antibodies. Therefore, we screened a total of 1,078 serum samples for HPV16 L2 reactivity, and these were obtained from four prior clinical studies: a population-based (n = 880) surveillance study with a high-risk HPV DNA prevalence of 10.8%, a cohort study of women (n = 160) with high-grade cervical intraepithelial neoplasia (CIN), and two phase II trials in women with high-grade vulvar intraepithelial neoplasia (VIN) receiving imiquimod therapy combined with either photodynamic therapy (PDT) (n = 19) or vaccination with a fusion protein comprising HPV16 L2, E7, and E6 (TA-CIN) (n = 19). Sera were screened sequentially by HPV16 L2 enzyme-linked immunosorbent assay (ELISA) and then Western blot. Seven of the 1,078 serum samples tested had L2-specific antibodies, but none were detectably neutralizing for HPV16. To develop a standard, we substituted human IgG1 sequences into conserved regions of two rodent monoclonal antibodies (MAbs) specific for neutralizing epitopes at HPV16 L2 residues 17 to 36 and 58 to 64, creating JWW-1 and JWW-2, respectively. These chimeric MAbs retained neutralizing activity and together reacted with 33/34 clinically relevant HPV types tested. In conclusion, our inability to identify an HPV16 L2-specific neutralizing antibody response even in the sera of patients with active genital HPV disease suggests the subdominance of L2 protective epitopes and the value of the chimeric MAbs JWW-1 and JWW-2 as standards for immunoassays to measure L2-specific human antibodies.

Sequential cisplatin therapy and vaccination with HPV16 E6E7L2 fusion protein in saponin adjuvant GPI-0100 for the treatment of a model HPV16+ cancer.

Clinical studies suggest that responses to HPV16 E6E7L2 fusion protein (TA-CIN) vaccination alone are modest, and GPI-0100 is a well-tolerated, potent adjuvant. Here we sought to optimize both the immunogenicity of TA-CIN via formulation with GPI-0100 and treatment of HPV16+ cancer by vaccination after cisplatin chemotherapy. HPV16 neutralizing serum antibody titers, CD4+ T cell proliferative and E6/E7-specific CD8+ T cell responses were significantly enhanced when mice were vaccinated subcutaneously (s.c.) or intramuscularly (i.m.) with TA-CIN formulated with GPI-0100. Vaccination was tested for therapy of mice bearing syngeneic HPV16 E6/E7+ tumors (TC-1) either in the lung or subcutaneously. Mice treated with TA-CIN/GPI-0100 vaccination exhibited robust E7-specific CD8+ T cell responses, which were associated with reduced tumor burden in the lung, whereas mice receiving either component alone were similar to controls. Since vaccination alone was not sufficient for cure, mice bearing s.c. TC-1 tumor were first treated with two doses of cisplatin and then vaccinated. Vaccination with TA-CIN/GPI-0100 i.m. substantially retarded tumor growth and extended survival after cisplatin therapy. Injection of TA-CIN alone, but not GPI-0100, into the tumor (i.t.) was similarly efficacious after cisplatin therapy, but the mice eventually succumbed. However, tumor regression and extended remission was observed in 80% of the mice treated with cisplatin and then intra-tumoral TA-CIN/GPI-0100 vaccination. These mice also exhibited robust E7-specific CD8+ T cell and HPV16 neutralizing antibody responses. Thus formulation of TA-CIN with GPI-0100 and intra-tumoral delivery after cisplatin treatment elicits potent therapeutic responses in a murine model of HPV16+ cancer.

Immunoprevention of human papillomavirus-associated malignancies.

Persistent infection by one of 15 high-risk human papillomavirus (hrHPV) types is a necessary but not sufficient cause of 5% of all human cancers. This provides a remarkable opportunity for cancer prevention via immunization. Since Harald zur Hausen's pioneering identification of hrHPV types 16 and 18, found in approximately 50% and 20% of cervical cancers, respectively, two prophylactic HPV vaccines containing virus-like particles (VLP) of each genotype have been widely licensed. These vaccines are beginning to affect infection and HPV-associated neoplasia rates after immunization campaigns in adolescents. Here, we review recent progress and opportunities to better prevent HPV-associated cancers, including broadening immune protection to cover all hrHPV types, reducing the cost of HPV vaccines especially for developing countries that have the highest rates of cervical cancer, and immune-based treatment of established HPV infections. Screening based upon George Papanicolaou's cervical cytology testing, and more recently detection of hrHPV DNA/RNA, followed by ablative treatment of high-grade cervical intraepithelial neoplasia (CIN2/3) have substantially reduced cervical cancer rates, and we examine their interplay with immune-based modalities for the prevention and eventual elimination of cervical cancer and other HPV-related malignancies.

Measurement of neutralizing serum antibodies of patients vaccinated with human papillomavirus L1 or L2-based immunogens using furin-cleaved HPV Pseudovirions.

Antibodies specific for neutralizing epitopes in either Human papillomavirus (HPV) capsid protein L1 or L2 can mediate protection from viral challenge and thus their accurate and sensitive measurement at high throughput is likely informative for monitoring response to prophylactic vaccination. Here we compare measurement of L1 and L2-specific neutralizing antibodies in human sera using the standard Pseudovirion-Based Neutralization Assay (L1-PBNA) with the newer Furin-Cleaved Pseudovirion-Based Neutralization Assay (FC-PBNA), a modification of the L1-PBNA intended to improve sensitivity towards L2-specific neutralizing antibodies without compromising assay of L1-specific responses. For detection of L1-specific neutralizing antibodies in human sera, the FC- PBNA and L1-PBNA assays showed similar sensitivity and a high level of correlation using WHO standard sera (n = 2), and sera from patients vaccinated with Gardasil (n = 30) or an experimental human papillomavirus type 16 (HPV16) L1 VLP vaccine (n = 70). The detection of L1-specific cross-neutralizing antibodies in these sera using pseudovirions of types phylogenetically-related to those targeted by the L1 virus-like particle (VLP) vaccines was also consistent between the two assays. However, for sera from patients (n = 17) vaccinated with an L2-based immunogen (TA-CIN), the FC-PBNA was more sensitive than the L1-PBNA in detecting L2-specific neutralizing antibodies. Further, the neutralizing antibody titers measured with the FC-PBNA correlated with those determined with the L2-PBNA, another modification of the L1-PBNA that spacio-temporally separates primary and secondary receptor engagement, as well as the protective titers measured using passive transfer studies in the murine genital-challenge model. In sum, the FC-PBNA provided sensitive measurement for both L1 VLP and L2-specific neutralizing antibody in human sera. Vaccination with TA-CIN elicits weak cross-protective antibody in a subset of patients, suggesting the need for an adjuvant.

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.

Control of HPV-associated tumors by innovative therapeutic HPV DNA vaccine in the absence of CD4+ T cells.

Human papillomavirus (HPV) infections are particularly problematic for HIV + and solid organ transplant patients with compromised CD4+ T cell-dependent immunity as they produce more severe and progressive disease compared to healthy individuals. There are no specific treatments for chronic HPV infection, resulting in an urgent unmet need for a modality that is safe and effective for both immunocompromised and otherwise normal patients with recalcitrant disease. DNA vaccination is attractive because it avoids the risks of administration of live vectors to immunocompromised patients, and can induce potent HPV-specific cytotoxic T cell responses. We have developed a DNA vaccine (pNGVL4a-hCRTE6E7L2) encoding calreticulin (CRT) fused to E6, E7 and L2 proteins of HPV-16, the genotype associated with approximately 90% vaginal, vulvar, anal, penile and oropharyngeal HPV-associated cancers and the majority of cervical cancers. Administration of the DNA vaccine by intramuscular (IM) injection followed by electroporation induced significantly greater HPV-specific immune responses compared to IM injection alone or mixed with alum. Furthermore, pNGVL4a-hCRTE6E7L2 DNA vaccination via electroporation of mice carrying an intravaginal HPV-16 E6/E7-expressing syngeneic tumor demonstrated more potent therapeutic effects than IM vaccination alone. Of note, administration of the DNA vaccine by IM injection followed by electroporation elicited potent E6 and E7-specific CD8+ T cell responses and antitumor effects despite CD4+ T cell-depletion, although no antibody response was detected. While CD4+ T cell-depletion did reduce the E6 and E7-specific CD8+ T cell response, it remained sufficient to prevent subcutaneous tumor growth and to eliminate circulating tumor cells in a model of metastatic HPV-16+ cancer. Thus, the antibody response was CD4-dependent, whereas CD4+ T cell help enhanced the E6/E7-specific CD8+ T cell immunity, but was not required. Taken together, our data suggest that pNGVL4a-hCRTE6E7L2 DNA vaccination via electroporation warrants testing in otherwise healthy patients and those with compromised CD4+ T cell immunity to treat HPV-16-associated anogenital disease and cancer.

Preparation and properties of a papillomavirus infectious intermediate and its utility for neutralization studies.

We show that minor capsid protein L2 is full length in clinical virion isolates and prepare furin-cleaved pseudovirus (fcPsV) as a model of the infectious intermediate for multiple human papillomavirus (HPV) types. These fcPsV do not require furin for in vitro infection, and are fully infectious in vivo. Both the γ-secretase inhibitor XXI and carrageenan block fcPsV infection in vitro and in vivo implying that they act after furin-cleavage of L2. Despite their enhanced exposure of L2 epitopes, vaccination with fcPsV particles fails to induce L2 antibody, although L1-specific responses are similar to PsV with intact L2. FcPsV can be applied in a simple, high-throughput neutralization assay that detects L2-specific neutralizing antibodies with >10-fold enhanced sensitivity compared with the PsV-based assay. The PsV and fcPsV-based assays exhibit similar sensitivity for type-specific antibodies elicited by L1 virus-like particles (VLP), but the latter improves detection of L1-specific cross-type neutralizing antibodies.

A bis-benzylidine piperidone targeting proteasome ubiquitin receptor RPN13/ADRM1 as a therapy for cancer.

The bis-benzylidine piperidone RA190 covalently binds to cysteine 88 of ubiquitin receptor RPN13 in the 19S regulatory particle and inhibits proteasome function, triggering rapid accumulation of polyubiquitinated proteins. Multiple myeloma (MM) lines, even those resistant to bortezomib, were sensitive to RA190 via endoplasmic reticulum stress-related apoptosis. RA190 stabilized targets of human papillomavirus (HPV) E6 oncoprotein, and preferentially killed HPV-transformed cells. After oral or intraperitoneal dosing of mice, RA190 distributed to plasma and major organs except the brain and inhibited proteasome function in skin and muscle. RA190 administration profoundly reduced growth of MM and ovarian cancer xenografts, and oral RA190 treatment retarded HPV16(+) syngeneic mouse tumor growth, without affecting spontaneous HPV-specific CD8(+) T cell responses, suggesting its therapeutic potential.

L2, the minor capsid protein of papillomavirus.

The capsid protein L2 plays major roles in both papillomavirus assembly and the infectious process. While L1 forms the majority of the capsid and can self-assemble into empty virus-like particles (VLPs), L2 is a minor capsid component and lacks the capacity to form VLPs. However, L2 co-assembles with L1 into VLPs, enhancing their assembly. L2 also facilitates encapsidation of the ∼8 kbp circular and nucleosome-bound viral genome during assembly of the non-enveloped T=7d virions in the nucleus of terminally differentiated epithelial cells, although, like L1, L2 is not detectably expressed in infected basal cells. With respect to infection, L2 is not required for particles to bind to and enter cells. However L2 must be cleaved by furin for endosome escape. L2 then travels with the viral genome to the nucleus, wherein it accumulates at ND-10 domains. Here, we provide an overview of the biology of L2.

Multivalent human papillomavirus l1 DNA vaccination utilizing electroporation.

OBJECTIVES: 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. METHODS: 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. RESULTS: 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. DISCUSSION: Multivalent vaccination via in vivo electroporation requires spatial separation of individual type L1 DNA vaccines.

Virus-like particles for the prevention of human papillomavirus-associated malignancies.

As compared with peptide- or protein-based vaccines, naked DNA vectors and even traditional attenuated or inactivated virus vaccines, virus-like particles (VLPs) are an attractive vaccine platform, as they offer a combination of safety, ease of production and both high-density B-cell epitope display and intracellular presentation of T-cell epitopes that induce potent humoral and cellular immune responses, respectively. Indeed, HPV vaccines based on VLP production by recombinant expression of major capsid antigen L1 in yeast (Gardasil(®), Merck & Co., NJ, USA) or insect cells (Cervarix(®), GlaxoSmithKline, London, UK) have been licensed for the prevention of cervical and anogenital infection and disease associated with the genotypes targeted by each vaccine. However, these HPV vaccines have not been demonstrated as effective to treat existing infections, and efforts to develop a therapeutic HPV vaccine continue. Furthermore, current HPV L1-VLP vaccines provide type-restricted protection, requiring highly multivalent formulations to broaden coverage to the dozen or more oncogenic HPV genotypes. This raises the complexity and cost of vaccine production. The lack of access to screening and high disease burden in developing countries has spurred efforts to develop second-generation HPV vaccines that are more affordable, induce wider protective coverage and offer therapeutic coverage against HPV-associated malignancies. Given the previous success with L1-VLP-based vaccines against HPV, VLPs have been also adopted as platforms for many second-generation HPV and non-HPV vaccine candidates with both prophylactic and therapeutic intent. In this article, the authors examine the progress and challenges of these efforts, with a focus on how they inform VLP vaccine design.