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Bortezomib and the other licensed 20S proteasome inhibitors show robust activity against liquid tumors like multiple myeloma, but have disappointed against solid tumors including ovarian cancer. Consequently, interest is mounting in alternative non-peptide based drugs targeting the proteasome’s 19S regulatory particle subunit, including its ubiquitin receptor RPN13. RA183 and RA375 are more potent analogs of the prototypic inhibitor of RPN13 (iRPN13) called RA190, and they show promise for the treatment of ovarian cancer. Here we demonstrate that rendering these candidate RPN13 inhibitors chiral and asymmetric through the addition of a single methyl to the core piperidone moiety increases their potency against cancer cell lines, with the S-isomer being more active than the R-isomer. The enhanced cancer cell cytotoxicities of these compounds are associated with improved binding to RPN13 in cell lysates, ATP depletion by inhibition of glycolysis and mitochondrial electron chain transport, mitochondrial depolarization and perinuclear clustering, oxidative stress and glutathione depletion, and rapid accumulation of high molecular weight polyubiquitinated proteins with a consequent unresolved ubiquitin proteasome system (UPS) stress response. Cytotoxicity was associated with an early biomarker of apoptosis, increased surface annexin V binding. As for cisplatin, BRCA2 and ATM deficiency conferred increased sensitivity to these iRPN13s. Ubiquitination plays an important role in coordinating DNA damage repair and the iRPN13s may compromise this process by depletion of monomeric ubiquitin following its sequestration in high molecular weight polyubiquitinated protein aggregates. Indeed, a synergistic cytotoxic response was evident upon treatment of several ovarian cancer cell lines with either cisplatin or doxorubicin and our new candidate iRPN13s, suggesting that such a combination approach warrants further exploration for the treatment of ovarian cancer.

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., Author Summary While most patients clear human papillomavirus (HPV) infection, some develop persistent papillomas, especially if immunocompromised. Likewise, we find a fraction of outbred SKH-1 mice challenged with Mus musculus papillomavirus type 1 (MusPV1/MmuPV1) develop persistent papillomas, whereas most SKH-1 mice, as seen for the inbred C57BL/6 and BALB/c strains, clear the infection. Viral clearance requires both CD4+ and CD8+ T cells, and depletion of either subset permits persistent but subclinical infection. In C57BL/6 mice, CD8+ T cell epitopes were mapped to MusPV1 E6 and E7; however the CD8+ T cell response to E6 dominated and correlated with spontaneous regression. A MusPV1 E6-specific CD8+ T cell line was developed by vaccination and culture in vitro, and its systemic administration once was sufficient to effect papilloma clearance in an immunodeficient mouse. Our observations in inbred and outbred mice challenged with MusPV1 suggest promise for immunotherapy to treat HPV-associated disease.

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.

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.

The origin recognition complex (ORC) coordinates a series of events that lead to initiation of DNA strand duplication. As a nuclear double stranded DNA plasmid, the papillomavirus (PV) genome resembles a mini-chromosome in infected cells. To initiate its replication, the viral E2 protein binds to and recruits the E1 DNA helicase at the viral origin. PV genome replication program exhibits three stages: initial amplification from a single genome upon infection to a few copies per cell, a cell cycle linked maintenance phase, and a differentiation dependent late stage where the genome is amplified to thousands of copies. Involvement of ORC or other pre-replication complex (pre-RC) factors has not been described. We report that human PV (HPV) and bovine PV (BPV-1) E2 proteins bind to ORC2, however, ORC2 was not detected at the viral origin. Depletion of ORC2 enhanced PV replication in a transient replication model and in keratinocytes stably maintaining viral episomes, while there was no effect on copy number in a cell line with integrated HPV genomes. Consistent with this, occupancy of E1 and E2 at the viral origin increased following ORC2 silencing. These data imply that ORC2 is not necessary for activation of the PV origin by E1 and E2 but instead suppresses E2 replicative function. Furthermore, we observed that over-expression of HPV E2 decreased ORC2 occupation at two known mammalian origins of replication, suggesting that E2 restricts pre-ORC assembly that could otherwise compete for host replication complexes necessary for viral genome amplification. We infer that the ORC2 complex with E2 restricts viral replication in the maintenance phase of the viral replication program and that elevated levels of E2 that occur during the differentiation dependent amplification stage subvert ORC loading and hence DNA synthesis at cellular origins., Author Summary Papillomavirus genome replication occurs during three distinct stages that are linked to the differentiation state of the infected epithelium. The viral proteins E1 and E2 recognize the viral origin and initiate a process that attracts host DNA replication factors. The origin recognition complex (ORC) coordinates initiation of chromosome duplication. While ORC2 binds to the E2 protein, its depletion does not impair PV genome replication. Instead, depletion of ORC2 stimulates viral replication, while over-expression of E2 protein decreases ORC2 occupancy at mammalian origins. We propose that the relative abundance of E2 and ORC2 in complex regulates viral and cellular origin licensing.

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.

The human papillomavirus (HPV) minor capsid protein L2 is a promising candidate for a broadly protective HPV vaccine yet the titers obtained in most experimental systems are rather low. Here we examine the potential of empty AAV2 particles (AAVLPs), assembled from VP3 alone, for display of L2 epitopes to enhance their immunogenicity. Insertion of a neutralizing epitope (amino acids 17–36) from L2 of HPV16 and HPV31 into VP3 at positions 587 and 453, respectively, permitted assembly into empty AAV particles (AAVLP(HPV16/31L2)). Intramuscularly vaccination of mice and rabbits with AAVLP(HPV16/31L2)s in montanide adjuvant, induced high titers of HPV16 L2 antibodies as measured by ELISA. Sera obtained from animals vaccinated with the AAVLP(HPV16/31L2)s neutralized infections with several HPV types in a pseudovirion infection assay. Lyophilized AAVLP(HPV16/31L2) particles retained their immunogenicity upon reconstitution. Interestingly, vaccination of animals that were pre-immunized with AAV2 - simulating the high prevalence of AAV2 antibodies in the population - even increased cross neutralization against HPV31, 45 and 58 types. Finally, passive transfer of rabbit antisera directed against AAVLP(HPV16/31L2)s protected naïve mice from vaginal challenge with HPV16 pseudovirions. In conclusion, AAVLP(HPV16/31L2) particles have the potential as a broadly protective vaccine candidate regardless of prior exposure to AAV.

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.