Quasi-targeted metabolomics revealed isoliquiritigenin and lauric acid associated with resistance to tobacco black shank.

Tobacco black shank (TBS), caused by Phytophthora nicotianae, is a severe disease. Plant root exudates play a crucial role in mediating plant-pathogen interactions in the rhizosphere. However, the specific interaction between key secondary metabolites present in root exudates and the mechanisms of disease resistance remains poorly understood. This study conducted a comprehensive comparison via quasi-targeted metabolomic analysis on the root exudate metabolites from the tobacco cultivar Yunyan87 and K326, both before and after inoculation with P. nicotianae. The results showed that the root exudate metabolites changed after P. nicotianae inoculation, and the root exudate metabolites of different tobacco cultivar was significantly different. Furthermore, homovanillic acid, lauric acid, and isoliquiritigenin were identified as potential key compounds for TBS resistance based on their impact on the mycelium growth of the pathogens. The pot experiment showed that isoliquiritigenin reduced the incidence by 55.2%, while lauric acid reduced it by 45.8%. This suggests that isoliquiritigenin and lauric acid have potential applications in the management of TBS. In summary, this study revealed the possible resistance mechanisms of differential metabolites in resistance of commercial tobacco cultivar, and for the first time discovered the inhibitory effects of isoliquiritigenin and homovanillic acid on P. nictianae, and attempt to use plants secondary metabolites of for plant protection.

Immune responses, therapeutic anti-tumor effects, and tolerability upon therapeutic HPV16/18 E6/E7 DNA vaccination via needle-free biojector.

IMPORTANCE: Respectively, HPV16 and HPV18 cause 50% and 20% of cervical cancer cases globally. Viral proteins E6 and E7 are obligate drivers of oncogenic transformation. We recently developed a candidate therapeutic DNA vaccine, pBI-11, that targets HPV16 and HPV18 E6 and E7. Single-site intramuscular delivery of pBI-11 via a needle elicited therapeutic anti-tumor effects in mice and is now being tested in high-risk human papillomavirus+ head and neck cancer patients (NCT05799144). Needle-free biojectors such as the Tropis device show promise due to ease of administration, high patient acceptability, and the possibility of improved delivery. For example, vaccination of patients with the ZyCoV-D DNA vaccine using the Tropis device is effective against COVID19, well tolerated, and licensed. Here we show that split-dose, multi-site administration and intradermal delivery via the Tropis biojector increase the delivery of pBI-11 DNA vaccine, enhance HPV antigen-specific CD8+ T-cell responses, and improve anti-tumor therapeutic effects, suggesting its translational potential to treat HPV16/18 infection and disease.

Identification of human MHC-I HPV18 E6/E7-specific CD8 + T cell epitopes and generation of an HPV18 E6/E7-expressing adenosquamous carcinoma in HLA-A2 transgenic mice.

BACKGROUND: Human Papillomavirus type 18 (HPV18) is a high-risk HPV that is commonly associated with cervical cancer. HPV18 oncogenes E6 and E7 are associated with the malignant transformation of cells, thus the identification of human leukocyte antigen (HLA)-restricted E6/E7 peptide-specific CD8 + T cell epitopes and the creation of a HPV18 E6/E7 expressing cervicovaginal tumor in HLA-A2 transgenic mice will be significant for vaccine development. METHODS: In the below study, we characterized various human HLA class I-restricted HPV18 E6 and E7-specific CD8 + T cells mediated immune responses in HLA class I transgenic mice using DNA vaccines encoding HPV18E6 and HPV18E7. We then confirmed HLA-restricted E6/E7 specific CD8 + T cell epitopes using splenocytes from vaccinated mice stimulated with HPV18E6/E7 peptides. Furthermore, we used oncogenic DNA plasmids encoding HPV18E7E6(delD70), luciferase, cMyc, and AKT to create a spontaneous cervicovaginal carcinoma model in HLA-A2 transgenic mice. RESULTS: Therapeutic HPV18 E7 DNA vaccination did not elicit any significant CD8 + T cell response in HLA-A1, HLA-24, HLA-B7, HLA-B44 transgenic or wild type C57BL/6 mice, but it did generate a strong HLA-A2 and HLA-A11 restricted HPV18E7-specific CD8 + T cell immune response. We found that a single deletion of aspartic acid (D) at location 70 in HPV18E6 DNA abolishes the presentation of HPV18 E6 peptide (aa67-75) by murine MHC class I. We found that the DNA vaccine with this mutant HPV18 E6 generated E6-specific CD8 + T cells in HLA-A2. HLA-A11, HLA-A24 and HLA-b40 transgenic mice. Of note, HLA-A2 restricted, HPV18 E7 peptide (aa7-15)- and HPV18 E6 peptide (aa97-105)-specific epitopes are endogenously processed by HPV18 positive Hela-AAD (HLA-A*0201/Dd) cells. Finally, we found that injection of DNA plasmids encoding HPV18E7E6(delD70), AKT, cMyc, and SB100 can result in the development of adenosquamous carcinoma in the cervicovaginal tract of HLA-A2 transgenic mice. CONCLUSIONS: We characterized various human HLA class I-restricted HPV18 E6/E7 peptide specific CD8 + T cell epitopes in human HLA class I transgenic mice. We demonstrated that HPV18 positive Hela cells expressing chimeric HLA-A2 (AAD) do present both HLA-A2-restricted HPV18 E7 (aa7-15)- and HPV18 E6 (aa97-105)-specific CD8 + T cell epitopes. A mutant HPV18E6 that had a single deletion at location 70 obliterates the E6 presentation by murine MHC class I and remains oncogenic. The identification of these human MHC restricted HPV antigen specific epitopes as well as the HPV18E6/E7 expressing adenosquamous cell carcinoma model may have significant future translational potential.

First Report of Trichoderma parareesei Causing Seedling Blight on Eggplant in China.

Eggplant (Solanum melongena L.) is an economically important vegetable crop in subtropics and tropics. In March 2021, a serious disease on eggplant seedlings about 20 days after transplanting was found in Rong'an County (25°28' N; 109°53' E), Guangxi, China, with an incidence of diseased plants of 35%. The initial symptom was water-soaked spots on the leaves, followed by irregular black-brown spots that gradually expanded outward, causing leaf necrosis and defoliation. Even parts of eggplant seedlings died after lesions extended to the stem and the surface of the diseased tissues was covered with white to blue mold. Four diseased eggplants were randomly collected from different fields. Small pieces of the symptomatic tissues were surface sterilized and incubated on potato dextrose agar (PDA) at 28°C for 4 days. A total of 12 strains with similar morphological characteristics were isolated, and four representative strains (FW-01 to FW-04) were characterized. The colony was initially white, changing to yellow-green after 7 days. Phialides were lageniform or ampulliform, 2.9 to 9.75 µm × 1.36 to 4.3 µm (n=50). Conidia were green, ellipsoidal to oblong, smooth, 2.1 to 3.3 µm × 1.6 to 2.33 µm (n=50). Chlamydospores were not observed on PDA. These morphological characteristics are consistent with the description of the genus Trichoderma (Samuels et al. 2012). To confirm the identification, from mycelia of the four isolates and DNA was extracted using the Fungal Genomic DNA Extraction Kit (Bioer Technology [Hangzhou] Co., Ltd.). Three gene regions (ITS, tef1 and rpb2) were amplified (Sadfi-Zouaoui et al. 2009; Atanasova et al. 2010) and sequenced (GenBank Accessions: OL677389 to OL677392 for ITS, OL743178 to OL743181 for tef1 and OL743182 to OL743185 for rpb2). ITS sequences shared 100% identity with sequences of T. reesei (MW514156) and T. parareesei (HM466668), and tef1 and rpb2 sequences showed more than 99% similarity with sequences of T. parareesei (KM263190 and HM182962). The phylogenetic tree of the concatenated sequences showed that four isolates were clustered with T. parareesei. Therefore, the isolates were identified as T. parareesei. To satisfy Koch's postulates, the pathogenicity of four strains was tested on healthy eggplant seedlings planted in a sterile potting mix. Eggplants at four leaves stage were inoculated using conidial suspensions (with a concentration of 1 × 106 conidia/ml), with two leaves of each eggplant inoculated with each isolate and the test repeated three times. The control eggplants leaves were inoculated with sterile water. All plants were placed in a greenhouse at 22 ± 3°C and 85% relative humidity, with a photoperiod of 12 h. The water-soaked spots appeared 48 h after inoculation. All inoculated leaves showed symptoms 3 days post-inoculation. The diseased leaves became brittle and abcissed, while the control leaves remained symptomless. Only T. parareesei was successfully re-isolated from the lesions. Atanasova et al. (2010) found that T. parareesei inhibited the growth of Lepidium sativum seedlings under in vitro conditions (Atanasova et al. 2010). To our knowledge, this is the first report of T. parareesei causing eggplant seedling blight in China. The pathogen can cause substantial economic losses in eggplant production. Therefore, the identification of the pathogen is of great significance for the diagnosis and control of the disease. The results of this study deepen the understanding of the pathogenicity of Trichoderma.

Development of a Spontaneous HPV16 E6/E7-Expressing Head and Neck Squamous Cell Carcinoma in HLA-A2 Transgenic Mice.

Human papillomavirus (HPV)-associated head and neck squamous cell carcinoma (HNSCC) is a growing global health problem. HPV16 has been attributed to a majority of HPV-associated HNSCCs. In order to test candidate immunotherapies, we developed a spontaneous HPV16-driven HNSCC model in HLA-A2 (AAD) transgenic mice. We sought to eliminate the confounding effects of dominant HPV antigen presentation through murine major histocompatibility complex class I (MHC-I) via epitope mutagenesis (without compromising tumorigenicity). We generated HPV16 E6(R55K)(delK75) and E7(N53S) expression constructs with mutations in known dominant H-2Db epitopes and characterized their presentation through murine and human MHC-I molecules using in vitro and in vivo activation of HPV16 E6/E7 antigen-specific CD8+ T cells. In addition, we tested the ability of E6(R55K)(delK75) and E7(N53S) for oncogenicity. The mutated E7(N53S) abolished the presentation of murine H-2Db-restricted HPV16 E7 peptide (i.e., amino acids [aa] 49 to 57) cytotoxic T lymphocyte (CTL) epitope and resulted in HLA-A2-restricted presentation of the HPV16 E7 (aa 11 to 20)-specific CTL epitope. The mutated E6(R55K)(delK75) abolished the activation of murine MHC-I-restricted E6-specific CD8+ T cell-mediated immune responses in C57BL/6 mice. In addition, the vaccination led to the activation of human HLA-A2-restricted E6-specific CD8+ T cell-mediated immune responses in HLA-A2 (AAD) transgenic mice. Injection of DNA plasmids encoding LucE7(N53S)E6(R55K)(delK75), AKT, c-Myc, and SB100 followed by electroporation results in development of squamous cell carcinoma in the oral/pharyngeal cavity of all of the HLA-A2 (AAD) transgenic mice (5/5), with 2/5 tumor-bearing mice developing metastatic carcinoma in the neck lymph nodes. IMPORTANCE Our data indicate that mutated HPV16 E6(R55K)(delK75) and mutated HPV16 E7(N53S) DNA abolishes the presentation of HPV16 E6 and E7 through murine MHC-I and results in their presentation through human HLA-A2 molecules. Additionally, the mutated HPV16 E6 and E7 remain oncogenic. Our approach is potentially applicable to different human MHC-I transgenic mice for the identification of human MHC-I restricted HPV16 E6/E7-specific CTL epitopes as well as the generation of spontaneous HPV E6/E7-expressing oral/pharyngeal carcinoma.

Control of Spontaneous HPV16 E6/E7 Expressing Oral Cancer in HLA-A2 (AAD) Transgenic Mice with Therapeutic HPV DNA Vaccine.

BACKGROUND: Human Papillomavirus type 16 (HPV16) has been associated with a subset of head and neck cancers. Two HPV encoded oncogenic proteins, E6 and E7, are important for the malignant progression of HPV-associated cancers. A spontaneous HPV16 E6/E7-expressing oral tumor model in human HLA-A2 (AAD) transgenic mice will be important for the development of therapeutic HPV vaccines for the control of HPV-associated head and neck cancers. METHODS: In the current studies, we characterized the HLA-A2 restricted HPV16 E7-specific CD8 + T cell mediated immune responses in the HLA-A2 (AAD) transgenic mice using a therapeutic naked DNA vaccine encoding calreticulin (CRT) linked to a mutated E7(N53S). We also employed oncogenic DNA plasmids that encoded HPV16E6/E7/Luc, NRasG12V, and sleeping beauty transposase for the transfection into the submucosal of oral cavity of the transgenic mice with electroporation to create a spontaneous oral tumor. Furthermore, we characterized the therapeutic antitumor effects of CRT/E7(N53S) DNA vaccine using the spontaneous HPV16 E6/E7-expressing oral tumor model in HLA-A2 (AAD) transgenic mice. RESULTS: We found that CRT/E7(N53S) DNA vaccine primarily generated human HPV16 E7 peptide (aa11-20) specific CD8 + T cells, as compared to the wild-type CRT/E7 vaccine, which primarily generated murine H-2Db restricted E7 peptide (aa49-57) specific CD8 + T cell responses. We also observed transfection of the oncogenic DNA plasmids with electroporation generated spontaneous oral tumor in all of the injected mice. Additionally, treatment with CRT/E7(N53S) DNA vaccine intramuscularly followed by electroporation resulted in significant antitumor effects against the spontaneous HPV16 E6/E7-expressing oral tumors in HLA-A2 (AAD) transgenic mice. CONCLUSIONS: Taken together, the data indicated that the combination of HPV16 E6/E7-expressing DNA, NRasG12V DNA and DNA encoding sleeping beauty transposase is able to generate spontaneous oral tumor in HLA-A2 (AAD) transgenic mice, which can be successfully controlled by treatment with CRT/E7(N53S) DNA vaccine. The translational potential of our studies are discussed.

Development of DNA Vaccine Targeting E6 and E7 Proteins of Human Papillomavirus 16 (HPV16) and HPV18 for Immunotherapy in Combination with Recombinant Vaccinia Boost and PD-1 Antibody.

Immunotherapy for cervical cancer should target high-risk human papillomavirus types 16 and 18, which cause 50% and 20% of cervical cancers, respectively. Here, we describe the construction and characterization of the pBI-11 DNA vaccine via the addition of codon-optimized human papillomavirus 18 (HPV18) E7 and HPV16 and 18 E6 genes to the HPV16 E7-targeted DNA vaccine pNGVL4a-SigE7(detox)HSP70 (DNA vaccine pBI-1). Codon optimization of the HPV16/18 E6/E7 genes in pBI-11 improved fusion protein expression compared to that in DNA vaccine pBI-10.1 that utilized the native viral sequences fused 3' to a signal sequence and 5' to the HSP70 gene of Mycobacterium tuberculosis Intramuscular vaccination of mice with pBI-11 DNA better induced HPV antigen-specific CD8+ T cell immune responses than pBI-10.1 DNA. Furthermore, intramuscular vaccination with pBI-11 DNA generated stronger therapeutic responses for C57BL/6 mice bearing HPV16 E6/E7-expressing TC-1 tumors. The HPV16/18 antigen-specific T cell-mediated immune responses generated by pBI-11 DNA vaccination were further enhanced by boosting with tissue-antigen HPV vaccine (TA-HPV). Combination of the pBI-11 DNA and TA-HPV boost vaccination with PD-1 antibody blockade significantly improved the control of TC-1 tumors and extended the survival of the mice. Finally, repeat vaccination with clinical-grade pBI-11 with or without clinical-grade TA-HPV was well tolerated in vaccinated mice. These preclinical studies suggest that the pBI-11 DNA vaccine may be used with TA-HPV in a heterologous prime-boost strategy to enhance HPV 16/18 E6/E7-specific CD8+ T cell responses, either alone or in combination with immune checkpoint blockade, to control HPV16/18-associated tumors. Our data serve as an important foundation for future clinical translation.IMPORTANCE Persistent expression of high-risk human papillomavirus (HPV) E6 and E7 is an obligate driver for several human malignancies, including cervical cancer, wherein HPV16 and HPV18 are the most common types. PD-1 antibody immunotherapy helps a subset of cervical cancer patients, and its efficacy might be improved by combination with active vaccination against E6 and/or E7. For patients with HPV16+ cervical intraepithelial neoplasia grade 2/3 (CIN2/3), the precursor of cervical cancer, intramuscular vaccination with a DNA vaccine targeting HPV16 E7 and then a recombinant vaccinia virus expressing HPV16/18 E6-E7 fusion proteins (TA-HPV) was safe, and half of the patients cleared their lesions in a small study (NCT00788164). Here, we sought to improve upon this therapeutic approach by developing a new DNA vaccine that targets E6 and E7 of HPV16 and HPV18 for administration prior to a TA-HPV booster vaccination and for application against cervical cancer in combination with a PD-1-blocking antibody.

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.

Structure-function analyses of candidate small molecule RPN13 inhibitors with antitumor properties.

We sought to design ubiquitin-proteasome system inhibitors active against solid cancers by targeting ubiquitin receptor RPN13 within the proteasome's 19S regulatory particle. The prototypic bis-benzylidine piperidone-based inhibitor RA190 is a michael acceptor that adducts Cysteine 88 of RPN13. In probing the pharmacophore, we showed the benefit of the central nitrogen-bearing piperidone ring moiety compared to a cyclohexanone, the importance of the span of the aromatic wings from the central enone-piperidone ring, the contribution of both wings, and that substituents with stronger electron withdrawing groups were more cytotoxic. Potency was further enhanced by coupling of a second warhead to the central nitrogen-bearing piperidone as RA375 exhibited ten-fold greater activity against cancer lines than RA190, reflecting its nitro ring substituents and the addition of a chloroacetamide warhead. Treatment with RA375 caused a rapid and profound accumulation of high molecular weight polyubiquitinated proteins and reduced intracellular glutathione levels, which produce endoplasmic reticulum and oxidative stress, and trigger apoptosis. RA375 was highly active against cell lines of multiple myeloma and diverse solid cancers, and demonstrated a wide therapeutic window against normal cells. For cervical and head and neck cancer cell lines, those associated with human papillomavirus were significantly more sensitive to RA375. While ARID1A-deficiency also enhanced sensitivity 4-fold, RA375 was active against all ovarian cancer cell lines tested. RA375 inhibited proteasome function in muscle for >72h after single i.p. administration to mice, and treatment reduced tumor burden and extended survival in mice carrying an orthotopic human xenograft derived from a clear cell ovarian carcinoma.

Epithelial boost enhances antigen expression by vaccinia virus for the generation of potent CD8+ T cell-mediated antitumor immunity following DNA priming vaccination.

While both pNGVL4a-Sig/E7(detox)/HSP70 DNA vaccine and TA-HPV recombinant vaccinia viral vector-based vaccines have elicited HPV-specific CD8+ T cell responses in HPV16/E7-expressing tumor models, and been used as prime-boost regimen to enhance HPV-specific immune responses in humans (NCT00788164), the optimal route of administration for TA-HPV remains unclear. In a preclinical model, we examined the immunogenicity of priming with intramuscular pNGVL4a-Sig/E7(detox)/HSP70 followed by TA-HPV boost through different administration routes. We observed that priming twice with a pNGVL4a-Sig/E7(detox)/HSP70 followed by a single TA-HPV immunization boost through skin scarification generated the strongest antigen-specific CD8+ T cell response in C57BL/6 mice. These data translate to tumor control and prolonged survival of treated mice. Our results provide rationale for future clinical testing of intramuscular pNGVL4a-Sig/E7(detox)/HSP70 DNA vaccine prime, TA-HPV vaccine skin scarification boost immunization regimen for the control of HPV-associated diseases.

Covalent Rpn13-Binding Inhibitors for the Treatment of Ovarian Cancer.

Substitution of the m,p-chloro groups of bis-benzylidinepiperidone RA190 for p-nitro, generating RA183, enhanced covalent drug binding to Cys88 of RPN13. Treatment of cancer cell lines with RA183 inhibited ubiquitin-mediated protein degradation, resulting in rapid accumulation of high-molecular-weight polyubiquitinated proteins, blockade of NFκB signaling, endoplasmic reticulum stress, an unfolded protein response, production of reactive oxygen species, and apoptotic cell death. High-grade ovarian cancer, triple-negative breast cancer, and multiple myeloma cell lines were particularly vulnerable to RA183. RA183 stabilized a tetraubiquitin-linked firefly luciferase reporter protein in cancer cell lines and mice, demonstrating in vitro and in vivo proteasomal inhibition, respectively. However, RA183 was rapidly cleared from plasma, likely reflecting its rapid degradation to the active compound RA9, as seen in human liver microsomes. Intraperitoneal administration of RA183 inhibited proteasome function and orthotopic tumor growth in mice bearing human ovarian cancer model ES2-luc ascites or syngeneic ID8-luc tumor.

Intramuscular vaccination targeting mucosal tumor draining lymph node enhances integrins-mediated CD8+ T cell infiltration to control mucosal tumor growth.

Purpose: Mucosal immunization is suggested to be crucial for controlling tumors in the mucosal region; however, therapeutic DNA vaccination with electroporation in various mucosal sites has yet to become clinically adaptable. Since tumor-draining lymph nodes (tdLNs) have been suggested as immune-educated sites that can be utilized to mount a potent antitumor immune response, we examined whether intramuscular DNA vaccination with electroporation at sites that target the mucosal tdLNs could elicit mucosal immune response to restrict tumor growth. Experimental Design: The efficacy and mechanism of intramuscular administration of a therapeutic DNA vaccine with electroporation at different sites was examined by lymphocyte analysis, tumor growth, mouse survival, as well as integrin expression, in mice bearing orthotopic HPV16 E6/E7+ syngeneic TC-1 tumors in various mucosal areas. Results: While provoking comparable systemic CD8+ T cell responses, intramuscular hind leg vaccination generated stronger responses in cervicovaginal-draining LNs to control cervicovaginal tumors, whereas intramuscular front leg vaccination generated stronger responses in oral-draining LNs to control buccal tumors. Surgical removal of tdLNs abolished the antitumor effects of therapeutic vaccination. Mucosal-tdLN-targeted intramuscular vaccination induced the expression of mucosal-homing integrins LPAM-1 and CD49a by tumor-specific CD8+ T cells in the tdLNs. Inhibition of these integrins abolished the therapeutic effects of vaccination and the infiltration of tumor-specific CD8+ T cells into mucosal tumors. Conclusions: Our findings demonstrate that tumor draining lymph nodes targeted intramuscular immunization can effectively control mucosal tumors, which represents a readily adaptable strategy for treating mucosal cancers in humans.

Programmed self-assembly of peptide-major histocompatibility complex for antigen-specific immune modulation.

A technology to prime desired populations of T cells in the body-particularly those that possess low avidity against target antigen-would pave the way for the design of new types of vaccination for intractable infectious diseases or cancer. Here, we report such a technology based on positive feedback-driven, programmed self-assembly of peptide-major histocompatibility complex (pMHC) directly on the membrane of cognate T cells. Our design capitalizes on the unique features of the protein annexin V (ANXA5), which-in a concerted and synergistic manner-couples the early onset of TCR signaling by cognate pMHC with a surge in pMHC-TCR affinity, with repeated pMHC encounters, and with widespread TCR cross-linking. In our system, ANXA5 is linked to pMHC and firmly engages the plasma membrane of cognate T cells upon (and only upon) the early onset of TCR signaling. ANXA5, in turn, exerts a mechanical force that stabilizes interactions at the TCR-pMHC interface and facilitates repeated, serial pMHC encounters. Furthermore, ANXA5 quickly arranges into uniform 2D matrices, thereby prompting TCR cross-linking. Fusion of ANXA5 to pMHC augments lymphocyte activation by several orders of magnitude (>1,000-fold), bypasses the need for costimulation, and breaks tolerance against a model self-antigen in vivo. Our study opens the door to the application of synthetic, feedback-driven self-assembly platforms in immune modulation.

Integration of Oncogenes via Sleeping Beauty as a Mouse Model of HPV16+ Oral Tumors and Immunologic Control.

Human papillomavirus type 16 (HPV16) is the etiologic factor for cervical cancer and a subset of oropharyngeal cancers. Although several prophylactic HPV vaccines are available, no effective therapeutic strategies to control active HPV diseases exist. Tumor implantation models are traditionally used to study HPV-associated buccal tumors. However, they fail to address precancerous phases of disease progression and display tumor microenvironments distinct from those observed in patients. Previously, K14-E6/E7 transgenic mouse models have been used to generate spontaneous tumors. However, the rate of tumor formation is inconsistent, and the host often develops immune tolerance to the viral oncoproteins. We developed a preclinical, spontaneous, HPV16+ buccal tumor model using submucosal injection of oncogenic plasmids expressing HPV16-E6/E7, NRas G12V , luciferase, and sleeping beauty (SB) transposase, followed by electroporation in the buccal mucosa. We evaluated responses to immunization with a pNGVL4a-CRT/E7(detox) therapeutic HPV DNA vaccine and tumor cell migration to distant locations. Mice transfected with plasmids encoding HPV16-E6/E7, NRas G12V , luciferase, and SB transposase developed tumors within 3 weeks. We also found transient anti-CD3 administration is required to generate tumors in immunocompetent mice. Bioluminescence signals from luciferase correlated strongly with tumor growth, and tumors expressed HPV16-associated markers. We showed that pNGVL4a-CRT/E7(detox) administration resulted in antitumor immunity in tumor-bearing mice. Lastly, we demonstrated that the generated tumor could migrate to tumor-draining lymph nodes. Our model provides an efficient method to induce spontaneous HPV+ tumor formation, which can be used to identify effective therapeutic interventions, analyze tumor migration, and conduct tumor biology research. Cancer Immunol Res; 6(3); 305-19. ©2018 AACR.

Prophylactic immunization with human papillomavirus vaccines induces oral immunity in mice.

OBJECTIVE: Although it has been shown that prophylactic vaccination can induce genital immunity, there is inadequate information on human papillomavirus (HPV) vaccine-induced oral immunity, which is of particular interest due to HPV-associated oropharyngeal malignancies and recurrent respiratory papillomatosis. Therefore, we assessed the efficacy of various HPV vaccines against oral HPV pseudovirus (PsV) infection in mice. STUDY DESIGN: Preclinical scientific investigation. METHODS: C57BL/6 mice were vaccinated three times at 2-week intervals with either Gardasil (Merck, Kenilworth, NJ) (50 µL intramuscular injection) or a candidate pan-HPV L2 vaccine with alum adjuvant (25 µg subcutaneous injection). Additional mice were immunized with passive transfer of either Gardasil (Merck) human antisera or nonimmunized sera (100 µL intraperitoneal injection). All vaccinated and naïve control mice were then challenged with HPV16 E6E7 luciferase PsV in the oral mucosa. Visualization of HPV PsV infection was monitored through in vivo luciferase imaging. RESULTS: Oral luciferase-expressing HPV16 PsV infection was not detected in Gardasil (Merck), L2 vaccine, and Gardasil (Merck) antisera-immunized mice, whereas robust luciferase expression was observed in all control mice. An in vitro neutralization assay from sera of Gardasil-vaccinated (Merck) mice confirmed that vaccine efficacy was due to neutralizing antibodies. CONCLUSION: Oral HPV16 PsV infection in mice was completely prevented with all methods of prophylactic HPV immunization. These findings provide preliminary evidence that human vaccines induce protection against oral HPV infection, which has significant public health implications for HPV-associated oropharyngeal malignancies. LEVEL OF EVIDENCE: NA. Laryngoscope, 128:E16-E20, 2018.

Enhancing antitumor immunogenicity of HPV16-E7 DNA vaccine by fusing DNA encoding E7-antigenic peptide to DNA encoding capsid protein L1 of Bovine papillomavirus.

BACKGROUND: Human papillomavirus (HPV) has been identified as the primary etiologic factor of cervical cancer, the fourth leading cause of cancer death in females worldwide. We have previously shown that coadministration of DNA encoding L1 capsid protein of Bovine papillomavirus (BPV) can enhance the antigen-specific immune response elicited by a therapeutic HPV16-E7 DNA vaccination. In this study, we sought to generate and evaluate the immunogenicity of a therapeutic HPV16-E7 DNA vaccine that encodes the fusion construct of HPV16-E7 and BPV-L1. RESULTS: We generated a therapeutic HPV16-E7 DNA vaccine construct, pcDNA3-BPVL1-E7(49-57), encoding the fusion sequence of full-length BPVL1 protein and a murine E7 antigenic epitope, aa49-57. Transfecting 293-Db cells with pcDNA3-BPVL1-E7(49-57) demonstrated that this DNA construct can effectively lead to the presentation of E7 epitope for the activation of E7-specific CD8+ T cells in vitro. Intramuscular vaccination of pcDNA3-BPVL1-E7(49-57) with electroporation generated a stronger E7-specific CD8+ T cell-mediated immune response than coadministration of pcDNA3-BPVL1 and pcDNA3-E7(49-57) in C57BL/6 mice. Furthermore, we observed that the strong E7-specific CD8+ T cell response elicited by pcDNA3-BPVL1-E7(49-57) vaccination translated into potent protective and therapeutic antitumor effects in C57BL/6 mice against HPV16-E7 expressing TC-1 tumor cells. Finally, using antibody depletion experiment, we showed that the antitumor immune response generated by pcDNA3-BPVL1-E7(49-57) is CD8+ T cell dependent, and CD4+ T cell and NK cell independent. CONCLUSION: Treatment with fusion construct of BPV-L1 and HPV16-E7 epitope can elicit effective E7-specific antitumor immune response in mice. Due to the potential ability of the fusion DNA construct to also trigger immune responses specific to the L1 protein, the current study serves to support future design of HPV DNA vaccines encoding fusion HPVL1-E6/E7 constructs for the generation of both T cell and B cell mediated immune responses against HPV infections and associated diseases.

Immunologic responses to a novel DNA vaccine targeting human papillomavirus-11 E6E7.

OBJECTIVES/HYPOTHESIS: Recurrent respiratory papillomatosis (RRP) is a benign disease caused by human papillomavirus (HPV) types 6 and 11. Although a prophylactic vaccine against RRP is available, a therapeutic vaccine is needed to treat those already infected. The objective of our study was to design and test a DNA vaccine targeting HPV11 proteins. STUDY DESIGN: Preclinical scientific investigation. METHODS: A DNA vaccine encoding the HPV11 E6 and E7 genes linked to calreticulin (CRT) was generated. Immunologic response to the HPV11 CRT/E6E7 vaccine was measured by vaccinating C57BL/6 mice via electroporation and measuring CD8 + T cell responses from harvested splenocytes. A tumor cell line containing HPV11-E6E7 was created, and the ability of novel DNA vaccine to control tumor growth was measured in vivo. RESULTS: Our vaccine generated a significant and specific CD8 + T-cell response against the HPV11-E6aa41-70 peptide. The CD8 + T-cell responses did not recognize E7 epitopes, indicating E6 immunodominance. CD8 + responses were augmented in the CRT-linked vaccine compared to a control non-CRT vaccine. The HPV11 CRT/E6E7 vaccine was used to treat mice inoculated with a HPV11 E6E7 expressing tumor cell line after temporary CD3 depletion to facilitate tumor growth. Vaccinated mice had a significantly lower tumor growth rate (P = .029) and smaller tumor volumes compared to control mice, indicating an augmented immunologic response in vaccinated mice. CONCLUSIONS: A DNA vaccine targeting HPV11 E6E7 generates a specific HPV11 CD-8 + T-cell response capable of reducing the growth of HPV11-expressing tumors. DNA vaccines are a promising immunologic strategy for treating RRP. LEVEL OF EVIDENCE: NA. Laryngoscope, 127:2713-2720, 2017.

Characterization of HPV18 E6-specific T cell responses and establishment of HPV18 E6-expressing tumor model.

Human papillomavirus (HPV) has been identified as the primary etiologic factor of cervical cancer, and subsets of anogenital and oropharyngeal cancers. HPV18 is the second most prevalent high-risk HPV type after HPV16. Furthermore, HPV18 is responsible for approximately 12% of cervical squamous cell carcinoma and 37% of cervical adenocarcinoma cases worldwide. In this study, we aimed to characterize the HPV18-E6-specific epitope and establish an HPV18 animal tumor model to evaluate the E6-specific immune response induced by our DNA vaccine. We vaccinated naïve C57BL/6 mice with a prototype DNA vaccine, pcDNA3-HPV18-E6, via intramuscular injection followed by electroporation, and analyzed the E6-specific CD8+ T cell responses by flow cytometry using a reported T cell epitope. We then characterized the MHC restriction element for the characterized HPV18-E6 epitope. Additionally, we generated an HPV18-E6-expressing tumor cell line to study the antitumor effect mediated by E6-specific immunity. We observed a robust HPV18-E6aa67-75 peptide-specific CD8+ T cell response after vaccination with pcDNA3-HPV18-E6. Further characterization demonstrated that this epitope was mainly restricted by H-2Kb, but was also weakly presented by HLA-A∗0201, as previously reported. We observed that vaccination with pcDNA3-HPV18-E6 significantly inhibited the growth of HPV18-E6-expressing tumor cells, TC-1/HPV18-E6, in mice. An antibody depletion study demonstrated that both CD4+ and CD8+ T cells are necessary for the observed antitumor immunity. The characterization of HPV18-E6-specific T cell responses and the establishment of HPV18-E6-expressing tumor cell line provide infrastructures for further development of HPV18-E6 targeted immunotherapy.