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.

EXPRESSION OF E8

Human papillomavirus (HPV) E1 and E2 proteins activate genome replication. E2 also modulates viral gene expression and is involved in the segregation of viral genomes. In addition to full length E2, almost all PV share the ability to encode an E8^E2 protein, that is a fusion of E8 with the C-terminal half of E2 which mediates specific DNA-binding and dimerization. HPV E8^E2 acts as a repressor of viral gene expression and genome replication. To analyze the function of E8^E2 in vivo, we used the Mus musculus PV1 (MmuPV1)-mouse model system. Characterization of the MmuPV1 E8^E2 protein revealed that it inhibits transcription from viral promoters in the absence and presence of E1 and E2 proteins and that this is partially dependent upon the E8 domain. MmuPV1 genomes, in which the E8 ATG start codon was disrupted (E8-), displayed a 10- to 25-fold increase in viral gene expression compared to wt genomes in cultured normal mouse tail keratinocytes in short-term experiments. This suggests that the function and mechanism of E8^E2 is conserved between MmuPV1 and HPVs. Surprisingly, challenge of athymic nude Foxn1nu/nu mice with MmuPV1 E8- genomes did not induce warts on the tail in contrast to wt MmuPV1. Furthermore, viral gene expression was completely absent at E8- MmuPV1 sites 20 - 22 weeks after DNA challenge on the tail or quasivirus challenge in the vaginal vault. This reveals that expression of E8^E2 is necessary to form tumors in vivo and that this is independent from the presence of T-cells.IMPORTANCE HPV encode an E8^E2 protein which acts as repressors of viral gene expression and genome replication. In cultured normal keratinocytes, E8^E2 is essential for long-term episomal maintenance of HPV31 genomes, but not for HPV16. To understand E8^E2's role in vivo, the Mus musculus PV1 (MmuPV1)-mouse model system was used. This revealed that E8^E2's function as a repressor of viral gene expression is conserved. Surprisingly, MmuPV1 E8^E2 knock out genomes did not induce warts in T-cell deficient mice. This shows for the first time that expression of E8^E2 is necessary for tumor formation in vivo independently of T cell immunity. This indicates that E8^E2 could be an interesting target for anti-viral therapy in vivo.

PD-1 blockade synergizes with intratumoral vaccination of a therapeutic HPV protein vaccine and elicits regression of tumor in a preclinical model.

INTRODUCTION: The human papillomavirus (HPV) encoded oncoproteins E6 and E7 are constitutively expressed in HPV-associated cancers, making them logical therapeutic targets. Intramuscular immunization of patients with HPV16 L2E7E6 fusion protein vaccine (TA-CIN) is well tolerated and induces HPV-specific cellular immune responses. Efficacy of PD-1 immune checkpoint blockade correlates with the level of tumor-infiltrating CD8 + T cells, yet most patients lack significant tumor infiltration of immune cells making immune checkpoint blockade suboptimal. We hypothesized that intratumoral vaccination with TA-CIN could increase the number of tumor-infiltrating CD8 + T cells, synergize with PD-1 blockade and result in better control of tumors compared with either PD-1 blockade or vaccination alone. METHODS: We examined the immunogenicity and antitumor effects of intratumoral vaccination with TA-CIN alone or in combination with PD-1 blockade in the TC-1 syngeneic murine tumor model expressing HPV16 E6/E7. RESULTS: Intratumoral vaccination with TA-CIN induced stronger antigen-specific CD8 + T cell responses and antitumor effects. Intratumoral TA-CIN vaccination generated a systemic immune response that was able to control distal TC-1 tumors. Furthermore, intratumoral TA-CIN vaccination induced tumor infiltration of antigen-specific CD8 + T cells. Knockout of Batf3 abolished antigen-specific CD8 + T cell responses and antitumor effects of intratumoral TA-CIN vaccination. Finally, PD-1 blockade synergizes with intratumoral TA-CIN vaccination resulting in significantly enhanced antigen-specific CD8 + T cell responses and complete regression of tumors, whereas either alone failed to control established TC-1 tumor. CONCLUSIONS: Our results provide rationale for future clinical testing of intratumoral TA-CIN vaccination in combination with PD-1 blockade for the control of HPV16-associated tumors.

Insights into the Role of Innate Immunity in Cervicovaginal Papillomavirus Infection from Studies Using Gene-Deficient Mice.

We demonstrate that female C57BL/6J mice are susceptible to a transient lower genital tract infection with MmuPV1 mouse papillomavirus and display focal histopathological abnormalities resembling those of human papillomavirus (HPV) infection. We took advantage of strains of genetically deficient mice to study in vivo the role of innate immune signaling in the control of papillomavirus. At 4 months, we sacrificed MmuPV1-infected mice and measured viral 757/3139 spliced transcripts by TaqMan reverse transcription-PCR (RT-PCR), localization of infection by RNAscope in situ hybridization, and histopathological abnormities by hematoxylin and eosin (H&E) staining. Among mice deficient in receptors for pathogen-associated molecular patterns, MyD88-/- and STING-/- mice had 1,350 and 80 copies of spliced transcripts/µg RNA, respectively, while no viral expression was detected in MAVS-/- and Ripk2-/- mice. Mice deficient in an adaptor molecule, STAT1-/-, for interferon signaling had 46,000 copies/µg RNA. Among mice with targeted deficiencies in the inflammatory response, interleukin-1 receptor knockout (IL-1R-/-) and caspase-1-/- mice had 350 and 30 copies/µg RNA, respectively. Among mice deficient in chemokine receptors, CCR6-/- mice had 120 copies/µg RNA, while CXCR2-/- and CXCR3-/- mice were negative. RNAscope confirmed focal infection in MyD88-/-, STAT1-/-, and CCR6-/- mice but was negative for other gene-deficient mice. Histological abnormalities were seen only in the latter mice. Our findings and the literature support a working model of innate immunity to papillomaviruses involving the activation of a MyD88-dependent pathway and IL-1 receptor signaling, control of viral replication by interferon-stimulated genes, and clearance of virus-transformed dysplastic cells by the action of the CCR6/CCL20 axis.IMPORTANCE Papillomaviruses infect stratified squamous epithelia, and the viral life cycle is linked to epithelial differentiation. Additionally, changes occur in viral and host gene expression, and immune cells are activated to modulate the infectious process. In vitro studies with keratinocytes cannot fully model the complex viral and host responses and do not reflect the contribution of local and migrating immune cells. We show that female C57BL/6J mice are susceptible to a transient papillomavirus cervicovaginal infection, and mice deficient in select genes involved in innate immune responses are susceptible to persistent infection with variable manifestations of histopathological abnormalities. The results of our studies support a working model of innate immunity to papillomaviruses, and the model provides a framework for more in-depth studies. A better understanding of mechanisms of early viral clearance and the development of approaches to induce clearance will be important for cancer prevention and the treatment of HPV-related diseases.

A novel pseudovirus-based mouse model of SARS-CoV-2 infection to test COVID-19 interventions.

BACKGROUND: The spread of SARS-CoV-2, the virus that causes Coronavirus Disease 2019 (COVID-19), has been characterized as a worldwide pandemic. Currently, there are few preclinical animal models that suitably represent infection, as the main point of entry to human cells is via human angiotensin-converting enzyme 2 (ACE2) which is not present in typical preclinical mouse strains. Additionally, SARS-CoV-2 is highly virulent and unsafe for use in many research facilities. Here we describe the development of a preclinical animal model using intranasal administration of ACE2 followed by non-infectious SARS-CoV-2 pseudovirus (PsV) challenge. METHODS: To specifically generate our SARS-CoV-2 PsV, we used a lentivirus system. Following co-transfection with a packaging plasmid containing HIV Gag and Pol, luciferase-expressing lentiviruses, and a plasmid carrying the SARS-CoV-2 spike protein, SARS-CoV-2 PsVs can be isolated and purified. To better understand and maximize the infectivity of SARS-CoV-2 PsV, we generated PsV carrying spike protein variants known to have varying human ACE2 binding properties, including 19 deletion (19del) and 19del + D614G. RESULTS: Our system demonstrated the ability of PsVs to infect the respiratory passage of mice following intranasal hACE2 transduction. Additionally, we demonstrate in vitro and in vivo manipulability of our system using recombinant receptor-binding domain protein to prevent PsV infection. CONCLUSIONS: Our PsV system is able to model SARS-CoV-2 infections in a preclinical mouse model and can be used to test interventions or preventative treatments. We believe that this method can be extended to work in various mouse strains or to model infection with different coronaviruses. A simple in vivo system such as our model is crucial for rapidly and effectively responding to the current COVID-19 pandemic in addition to preparing for future potential coronavirus outbreaks.

A batch-effect adjusted Simon's two-stage design for cancer vaccine clinical studies.

In the development of cancer treatment vaccines, phase II clinical studies are conducted to examine the efficacy of a vaccine in order to screen out vaccines with minimal activity. Immune responses are commonly used as the primary endpoint for assessing vaccine efficacy. With respect to study design, Simon's two-stage design is a popular format for phase II cancer clinical studies because of its simplicity and ethical considerations. Nonetheless, it is not straightforward to apply Simon's two-stage design to cancer vaccine studies when performing immune assays in batches, as outcomes from multiple patients may be correlated with each other in the presence of batch effects. This violates the independence assumption of Simon's two-stage design. In this paper, we numerically explore the impact of batch effects on Simon's two-stage design, propose a batch-effect adjusted Simon's two-stage design, demonstrate the proposed design by both a simulation study and a therapeutic human papillomavirus vaccine trial, and briefly introduce a software that implements the proposed design.

Next generation polyphosphazene immunoadjuvant: Synthesis, self-assembly and in vivo potency with human papillomavirus VLPs-based vaccine.

Poly[di(carboxylatomethylphenoxy)phosphazene] (PCMP), a new member of polyphosphazene immunoadjuvant family, is synthesized. In vitro assessment of a new macromolecule revealed hydrolytic degradation profile and immunostimulatory activity comparable to its clinical stage homologue PCPP; however, PCMP was characterized by a beneficial reduced sensitivity to the ionic environment. In vivo evaluation of PCMP potency was conducted with human papillomavirus (HPV) virus-like particles (VLPs) based RG1-VLPs vaccine. In contrast with previously reported self-assembly of polyphosphazene adjuvants with proteins, which typically results in the formation of complexes with multimeric display of antigens, PCMP surface modified VLPs in a composition dependent pattern, which at a high polymer-to VLPs ratio led to stabilization of antigenic particles. Immunization experiments in mice demonstrated that PCMP adjuvanted RG1-VLPs vaccine induced potent humoral immune responses, in particular, on the level of highly desirable protective cross-neutralizing antibodies, and outperformed PCPP and Alhydrogel adjuvanted formulations.

Bis-benzylidine Piperidone RA190 treatment of hepatocellular carcinoma via binding RPN13 and inhibiting NF-κB signaling.

BACKGROUND: According to GLOBOSCAN, hepatocellular carcinoma (HCC) claimed 782,000 lives in 2018. The tyrosine kinase inhibitor sofafenib is used to treat HCC, but new anticancer agents targeting different pathways are urgently needed to improve outcomes for patients with advanced disease. The aberrant metabolism and aggressive growth of cancer cells can render them particularly susceptible to proteasome inhibition, as demonstrated by bortezomib treatment of multiple myeloma. However, resistance does emerge, and this 20S proteasome inhibitor has not proven active against HCC. The bis-benzylidine piperidone RA190 represents a novel class of proteasome inhibitor that covalently binds to cysteine 88 of RPN13, an ubiquitin receptor subunit of the proteasome's 19S regulatory particle. RA190 treatment inhibits proteasome function, causing rapid accumulation of polyubiquitinated proteins. Considerable evidence suggests that nuclear factor κB (NF-κB) signaling, which is dependent upon the proteasome, is a major driver of inflammation-associated cancers, including HCC. METHODS: Human HCC cell lines were treated with titrations of RA190. The time course of endoplasmic reticulum stress and NF-κB-related mechanisms by which RA190 may trigger apoptosis were assessed. The therapeutic activity of RA190 was also determined in an orthotopic HCC xenograft mouse model. RESULTS: RA190 is toxic to HCC cells and synergizes with sofafenib. RA190 triggers rapid accumulation of polyubiquitinated proteins, unresolved endoplasmic reticulum stress, and cell death via apoptosis. RA190 blocks proteasomal degradation of IκBα and consequent release of NF-κB into the nuclei of HCC cells. Treatment of mice bearing an orthotopic HCC model with RA190 significantly reduced tumor growth. CONCLUSIONS: RA190 has therapeutic activity in a xenograft model, and with sorafenib exhibited synergetic killing of HCC cells in vitro, suggesting further exploration of such a combination treatment of HCC is warranted.

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.

A Bayesian design for phase I cancer therapeutic vaccine trials.

Phase I clinical trials are the first step in drug development to test a new drug or drug combination on humans. Typical designs of Phase I trials use toxicity as the primary endpoint and aim to find the maximum tolerable dosage. However, these designs are poorly applicable for the development of cancer therapeutic vaccines because the expected safety concerns for these vaccines are not as much as cytotoxic agents. The primary objectives of a cancer therapeutic vaccine phase I trial thus often include determining whether the vaccine shows biologic activity and the minimum dose necessary to achieve a full immune or even clinical response. In this paper, we propose a new Bayesian phase I trial design that allows simultaneous evaluation of safety and immunogenicity outcomes. We demonstrate the proposed clinical trial design by both a numeric study and a therapeutic human papillomavirus vaccine trial.

Evaluation of osteopenia and osteoporosis in younger breast cancer survivors compared with cancer-free women: a prospective cohort study.

BACKGROUND: Osteoporosis, an indicator of significant bone loss, has been consistently reported among older breast cancer survivors. Data are limited on the incidence of osteopenia, an earlier indicator of bone loss, and osteoporosis in younger breast cancer survivors compared with cancer-free women. METHODS: We prospectively examined bone loss in 211 breast cancer survivors (mean age at breast cancer diagnosis = 47 years) compared with 567 cancer-free women in the same cohort with familial risk for breast cancer. Multivariable-adjusted Cox proportional hazards models were used to estimate HRs and 95% CIs of osteopenia and/or osteoporosis incidence based on physician diagnosis. RESULTS: During a mean follow-up of 5.8 years, 66% of breast cancer survivors and 53% of cancer-free women reported having a bone density examination, and 112 incident cases of osteopenia and/or osteoporosis were identified. Breast cancer survivors had a 68% higher risk of osteopenia and osteoporosis compared to cancer-free women (HR = 1.68, 95% CI = 1.12-2.50). The association was stronger among recent survivors after only 2 years of follow-up (HR = 2.74, 95% CI = 1.37-5.47). A higher risk of osteopenia and osteoporosis was also observed among survivors aged ≤ 50 years, estrogen receptor-positive tumors, and those treated with aromatase inhibitors alone or chemotherapy plus any hormone therapy relative to cancer-free women. CONCLUSIONS: Younger breast cancer survivors are at higher risk for osteopenia and osteoporosis compared to cancer-free women. Studies are needed to determine effective approaches to minimize bone loss in this population.

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.

The Proteasome Ubiquitin Receptor hRpn13 and Its Interacting Deubiquitinating Enzyme Uch37 Are Required for Proper Cell Cycle Progression.

Recently, we reported that bisbenzylidine piperidone RA190 adducts to Cys-88 of the proteasome ubiquitin receptor hRpn13, triggering accumulation of ubiquitinated proteins and endoplasmic reticulum stress-related apoptosis in various cancer cell lines. hRpn13 contains an N-terminal pleckstrin-like receptor for ubiquitin domain that binds ubiquitin and docks it into the proteasome as well as a C-terminal deubiquitinase adaptor (DEUBAD) domain that binds the deubiquitinating enzyme Uch37. Here we report that hRpn13 and Uch37 are required for proper cell cycle progression and that their protein knockdown leads to stalling at G0/G1 Moreover, serum-starved cells display reduced hRpn13 and Uch37 protein levels with hallmarks of G0/G1 stalling and recovery to their steady-state protein levels following release from nutrient deprivation. Interestingly, loss of hRpn13 correlates with a small but statistically significant reduction in Uch37 protein levels, suggesting that hRpn13 interaction may stabilize this deubiquitinating enzyme in human cells. We also find that RA190 treatment leads to a loss of S phase, suggesting a block of DNA replication, and G2 arrest by using fluorescence-activated cell sorting. Uch37 deprivation further indicated a reduction of DNA replication and G0/G1 stalling. Overall, this work implicates hRpn13 and Uch37 in cell cycle progression, providing a rationale for their function in cellular proliferation and for the apoptotic effect of the hRpn13-targeting molecule RA190.

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.

An integrated proteomic and glycoproteomic approach uncovers differences in glycosylation occupancy from benign and malignant epithelial ovarian tumors.

BACKGROUND: Epithelial ovarian carcinomas encompass a heterogeneous group of diseases with a poor 5-year survival rate. Serous carcinoma is the most common type. Most FDA-approved serum tumor markers are glycoproteins. These glycoproteins on cell surface or shed into the bloodstream could serve as therapeutic targets as well as surrogates of tumor. In addition to glycoprotein expressions, the analysis of protein glycosylation occupancy could be important for the understanding of cancer biology as well as the identification of potential glycoprotein changes in cancer. In this study, we used an integrated proteomics and glycoproteomics approach to analyze global glycoprotein abundance and glycosylation occupancy for proteins from high-grade ovarian serous carcinoma (HGSC) and serous cystadenoma, a benign epithelial ovarian tumor, by using LC-MS/MS-based technique. METHODS: Fresh-frozen ovarian HGSC tissues and benign serous cystadenoma cases were quantitatively analyzed using isobaric tags for relative and absolute quantitation for both global and glycoproteomic analyses by two dimensional fractionation followed by LC-MS/MS analysis using a Orbitrap Velos mass spectrometer. RESULTS: Proteins and N-linked glycosite-containing peptides were identified and quantified using the integrated global proteomic and glycoproteomic approach. Among the identified N-linked glycosite-containing peptides, the relative abundances of glycosite-containing peptide and the glycoprotein levels were compared using glycoproteomic and proteomic data. The glycosite-containing peptides with unique changes in glycosylation occupancies rather than the protein expression levels were identified. CONCLUSION: In this study, we presented an integrated proteomics and glycoproteomics approach to identify changes of glycoproteins in protein expression and glycosylation occupancy in HGSC and serous cystadenoma and determined the changes of glycosylation occupancy that are associated with malignant and benign tumor tissues. Specific changes in glycoprotein expression or glycosylation occupancy have the potential to be used in the discrimination between benign and malignant epithelial ovarian tumors and to improve our understanding of ovarian cancer biology.

Evaluation of cancer-associated myositis and scleroderma autoantibodies in breast cancer patients without rheumatic disease.

OBJECTIVES: Systemic sclerosis (scleroderma) and dermatomyositis are two prototypic autoimmune diseases that are strongly associated with malignancy. While specific autoantibodies in these diseases are markers of an increased risk of cancer at scleroderma and dermatomyositis onset, it is not known whether these autoantibodies are biomarkers of cancer risk in patients without rheumatic disease. METHODS: In a matched case-control study of women without rheumatic disease, identified from a familial breast cancer cohort, 50 breast cancer cases and 50 controls were assayed for 3 autoantibodies that are known markers of cancer-associated scleroderma and dermatomyositis: anti-RNA polymerase III, anti-NXP2, and anti-TIF1γ. RESULTS: No subject had moderate or strong autoantibody positivity. Eleven women were borderline positive for at least one autoantibody. The prevalence of borderline autoantibody positivity did not differ between cases and controls. CONCLUSIONS: Our results suggest that scleroderma and dermatomyositis autoantibodies are cancer biomarkers only in patients with clinical manifestations of specific rheumatic diseases and are unlikely to improve risk stratification for cancer in the general population. However, prospective studies are needed to examine whether scleroderma and dermatomyositis autoantibodies are markers of malignancy in other cancer types.

Capsid display of a conserved human papillomavirus L2 peptide in the adenovirus 5 hexon protein: a candidate prophylactic hpv vaccine approach.

BACKGROUND: Infection by any one of 15 high risk human papillomavirus (hrHPV) types causes most invasive cervical cancers. Their oncogenic genome is encapsidated by L1 (major) and L2 (minor) coat proteins. Current HPV prophylactic vaccines are composed of L1 virus-like particles (VLP) that elicit type restricted immunity. An N-terminal region of L2 protein identified by neutralizing monoclonal antibodies comprises a protective epitope conserved among HPV types, but it is weakly immunogenic compared to L1 VLP. The major antigenic capsid protein of adenovirus type 5 (Ad5) is hexon which contains 9 hypervariable regions (HVRs) that form the immunodominant neutralizing epitopes. Insertion of weakly antigenic foreign B cell epitopes into these HVRs has shown promise in eliciting robust neutralizing antibody responses. Thus here we sought to generate a broadly protective prophylactic HPV vaccine candidate by inserting a conserved protective L2 epitope into the Ad5 hexon protein for VLP-like display. METHODS: Four recombinant adenoviruses were generated without significant compromise of viral replication by introduction of HPV16 amino acids L2 12-41 into Ad5 hexon, either by insertion into, or substitution of, either hexon HVR1 or HVR5. RESULTS: Vaccination of mice three times with each of these L2-recombinant adenoviruses induced similarly robust adenovirus-specific serum antibody but weak titers against L2. These L2-specific responses were enhanced by vaccination in the presence of alum and monophoryl lipid A adjuvant. Sera obtained after the third immunization exhibited low neutralizing antibody titers against HPV16 and HPV73. L2-recombinant adenovirus vaccination without adjuvant provided partial protection of mice against HPV16 challenge to either the vagina or skin. In contrast, vaccination with each L2-recombinant adenovirus formulated in adjuvant provided robust protection against vaginal challenge with HPV16, but not against HPV56. CONCLUSION: We conclude that introduction of HPV16 L2 12-41 epitope into Ad5 hexon HVR1 or HVR5 is a feasible method of generating a protective HPV vaccine, but further optimization is required to strengthen the L2-specific response and broaden protection to the more diverse hrHPV.

Phylogenetic considerations in designing a broadly protective multimeric L2 vaccine.

While the oncogenic human papillomavirus (HPV) types with the greatest medical impact are clustered within the α9 and α7 species, a significant fraction of cervical cancers are caused by α5, α6, and α11 viruses. Benign genital warts are caused principally by the α10 viruses HPV6 and HPV11. In an effort to achieve broad protection against both cervical cancer- and genital wart-associated types, we produced at high levels in bacteria a multimeric protein (α11-88x8) fusing eight polypeptides corresponding to a protective domain comprising L2 residues ∼11 to 88 derived from HPV6 (α10), HPV16 (α9), HPV18 (α7), HPV31 (α9), HPV39 (α7), HPV51 (α5), HPV56 (α6), and HPV73 (α11) and a truncated derivative with the last three units deleted (α11-88x5). Mice were immunized three times with α11-88x8 or α11-88x5 adjuvanted with alum or the licensed HPV vaccines and challenged intravaginally with HPV6, HPV16, HPV26, HPV31, HPV33, HPV35, HPV45, HPV51, HPV56, HPV58, or HPV59 pseudovirions. The α11-88x5 and α11-88x8 vaccines induced similarly robust protection against each HPV type tested and indistinguishable HPV16-neutralizing antibody titers. Passive transfer of α11-88x8 antisera was protective. Further, rabbit antisera to α11-88x8 and α11-88x5 similarly neutralized native HPV18 virions. These findings suggest that immunologic competition between units is not a significant issue and that it is not necessary to include a unit of L2 derived from each species to achieve broader protection against diverse medically significant HPV types than is achieved with the licensed HPV vaccines.

Profiling of VEGF Receptors and Immune Checkpoints in Recurrent Respiratory Papillomatosis.

OBJECTIVES: Recurrent respiratory papillomatosis (RRP) is caused by human papilloma virus (HPV) infection of the aerodigestive tract that significantly impacts quality-of-life including the ability to communicate and breathe. Treatment was traditionally limited to serial ablative procedures in the O.R. with possible local adjuvant therapy, but new systemic therapies, such as Vascular endothelial growth factor (VEGF) inhibitors, are showing significant promise. This study aims to determine whether rationale exists for combination therapeutic approaches using VEGF inhibitors and/or immune checkpoint blockade. METHODS: Using fresh specimens from the O.R., we performed flow cytometry on papilloma, normal adjacent tissue, and blood. Papilloma and surrounding tissue were examined for expression of PD-L1, PD-L2, Galectin-9, VEGFR2, and VEGFR3. CD8+ and CD4+ T cells were assayed for expression of PD-1, TIGIT, LAG3, and TIM3. RESULTS: Our data shows that papilloma tissue exhibits significantly higher levels of PD-L1 and PD-L2 compared to adjacent tissue. Elevated levels of the VEGF receptor VEGFR3 were also observed in papilloma tissue. When examining T cells within the papilloma, elevated PD-1 and TIGIT expression was observed on CD8+ T cells, while levels of PD-1, TIGIT, and TIM3 were elevated on CD4+ T cells compared to PBMCs. Heterogenous marker expression was observed between individuals. CONCLUSIONS: Our analysis shows that RRP tissue shows elevated levels of multiple immune check point targets and VEGFR3, with varied patterns unique to each papilloma patient. Some of these immune checkpoint markers already have novel immunotherapies available or in development, providing molecular rationale to offer these systemic treatments to selected patients affected by RRP alongside VEGF inhibitors. Laryngoscope, 134:2819-2825, 2024.

Preclinical studies of RA475, a guanidine-substituted spirocyclic candidate RPN13/ADRM1 inhibitor for treatment of ovarian cancer.

There is an urgent unmet need for more targeted and effective treatments for advanced epithelial ovarian cancer (EOC). The emergence of drug resistance is a particular challenge, but small molecule covalent inhibitors have promise for difficult targets and appear less prone to resistance. Michael acceptors are covalent inhibitors that form bonds with cysteines or other nucleophilic residues in the target protein. However, many are categorized as pan-assay interference compounds (PAINS) and considered unsuitable as drugs due to their tendency to react non-specifically. Targeting RPN13/ADRM1-mediated substrate recognition and deubiquitination by the proteasome 19S Regulatory Particle (RP) is a promising treatment strategy. Early candidate RPN13 inhibitors (iRPN13) produced a toxic accumulation of very high molecular weight polyubiquitinated substrates, resulting in therapeutic activity in mice bearing liquid or solid tumor models, including ovarian cancer; however, they were not drug-like (PAINS) because of their central piperidone core. Up284 instead has a central spiro-carbon ring. We hypothesized that adding a guanidine moiety to the central ring nitrogen of Up284 would produce a compound, RA475, with improved drug-like properties and therapeutic activity in murine models of ovarian cancer. RA475 produced a rapid accumulation of high molecular polyubiquitinated proteins in cancer cell lines associated with apoptosis, similar to Up284 although it was 3-fold less cytotoxic. RA475 competed binding of biotinylated Up284 to RPN13. RA475 shows improved solubility and distinct pharmacodynamic properties compared to Up284. Specifically, tetraubiquitin firefly luciferase expressed in leg muscle was stabilized in mice more effectively upon IP treatment with RA475 than with Up284. However, pharmacologic analysis showed that RA475 was more rapidly cleared from the circulation, and less orally available than Up284. RA475 shows reduced ability to cross the blood-brain barrier and in vitro inhibition of HERG. Treatment of mice with RA475 profoundly inhibited the intraperitoneal growth of the ID8-luciferase ovarian tumor model. Likewise, RA475 treatment of immunocompetent mice inhibited the growth of spontaneous genetically-engineered peritoneal tumor, as did weekly cisplatin dosing. The combination of RA475 and cisplatin significantly extended survival compared to individual treatments, consistent with synergistic cytotoxicity in vitro. In sum, RA475 is a promising candidate covalent RPN13i with potential utility for treatment of patients with advanced EOC in combination with cisplatin.