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Zika virus (ZIKV) is a mosquito-borne flavivirus that replicates in both vertebrate and insect cells, whereas insect-specific flaviviruses (ISF) replicate only in insect cells. We sought to convert ZIKV, from a dual-tropic flavivirus, into an insect-specific virus for the eventual development of a safe ZIKV vaccine. Reverse genetics was used to introduce specific mutations into the furin cleavage motif within the ZIKV pre-membrane protein (prM). Mutant clones were selected, which replicated well in C6/36 insect cells but exhibited reduced replication in non-human primate (Vero) cells. Further characterization of the furin cleavage site mutants indicated they replicated poorly in both human (HeLa, U251), and baby hamster kidney (BHK-21) cells. One clone with the induced mutation in the prM protein and at positions 291and 452 within the NS3 protein was totally and stably replication-defective in vertebrate cells (VSRD-ZIKV). Preliminary studies in ZIKV sensitive, immunodeficient mice demonstrated that VSRD-ZIKV-infected mice survived and were virus-negative. Our study indicates that a reverse genetic approach targeting the furin cleavage site in prM can be used to select an insect-specific ZIKV with the potential utility as a vaccine strain.

Aims Hereditary leiomyomatosis and renal cell cancer (HLRCC) syndrome is caused by germline mutations in the Fumarate hydratase (FH) gene. In young women, the syndrome often presents with symptomatic uterine leiomyomas, leading to myomectomy or hysterectomy. In this study, we aimed to investigate the incidence and mutational profiles of FH-negative leiomyomas from young patients, thus allowing for early identification and triage of syndromic patients for surveillance. Methods and results We evaluated 153 cases of uterine leiomyomas from women aged up to 30 years for loss of FH expression by tissue microarray (TMA)-based immunohistochemical staining. Mutational analysis of tumours with loss of FH was carried out by polymerase chain reaction (PCR) amplification of 10 exons within the FH gene and subsequent Sanger sequencing. The status of promoter methylation was assessed by bisulphite sequencing. Loss of FH protein expression was detected in seven (4.6%) of 153 tested uterine leiomyomas from young patients. All FH-negative leiomyomas displayed staghorn vasculature and fibrillary/neurophil-like cytoplasm. We found that six (86%) of seven FH-negative tumours detected by immunohistochemistry harboured FH mutations, 50% of which contained germline mutations. In particular, the germline mutational rate in FH gene was 2.0% (three of 153 cases). Bisulphite sequencing analysis failed to detect promoter methylation in any of the seven tumours. Conclusion Our study showed a relatively high rate of FH germline mutation in FH-negative uterine leiomyomas from patients aged up to 30 years. While genetic mutations confer protein expression loss, epigenetic regulation of the FH gene appears to be unrelated to this phenotype.

As severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spreads, variants with enhanced virulence and transmissibility have emerged. Although in vitro systems allow rapid characterization, they do not fully recapitulate the dynamic interaction of virions and neutralizing antibodies in the airway. Here, we demonstrate that the N501Y variant permits respiratory infection in unmodified mice. We utilize N501Y to survey in vivo pseudovirus infection dynamics and susceptibility to reinfection with the L452R (Los Angeles), K417N + E484K (South Africa), and L452R + K417N + E484Q (India) variants. Human coronavirus disease 2019 (COVID-19)+ or vaccinated antibody isotypes, titers, variant receptor binding domain (RBD) binding, and neutralization potential are studied, revealing numerous significant correlations. Immune escape of the K417N + E484K variant is observed because infection can be appreciated in the nasopharynx, but not lungs, of mice transferred with low-antibody-tier plasma. Conversely, near-complete protection is observed in animals receiving high-antibody-tier plasma, a phenomenon that can only be appreciated in vivo., Graphical abstract, Lam et al. develop a model of SARS-CoV-2 infection in laboratory mice. This allows researchers to study the threat of emerging variants in a more physiological context than by using cell culture systems. Interactions between SARS-CoV-2 variants and immunity are explored in the airway of mice.

Purpose: Choriocarcinoma is most commonly gestational (androgenetic or biparental) but can be of germ cell origin or can develop as a component of a somatic neoplasm (genetically related to the patient). The latter type are aggressive neoplasms for which the underlying genetic alterations are not well characterized. Experimental Design: To investigate the relationship between the different components of somatic neoplasms with choriocarcinomatous elements, the genetic differences between gestational and nongestational tumors, and identify potential targetable alterations, we analyzed 23 samples from 11 tumors, including five gynecologic-type somatic neoplasms with choriocarcinomatous differentiation (two to three different components each) and six pure choriocarcinomas, for somatic mutations, single-nucleotide polymorphisms, and PD-L1 expression. Results: In mixed tumors, gynecologic-type carcinoma components demonstrated lineage-characteristic and lineage-specific alterations, with choriocarcinomatous components sharing some of these as well as demonstrating novel alterations, supporting a clonal relationship with divergent differentiation of the choriocarcinoma from the somatic carcinoma. TP53 mutation only occurred in nongestational tumors. Diffuse PD-L1 expression was characteristic of choriocarcinoma in both pure and mixed tumors but not seen in the gynecologic-type carcinoma components. Conclusions: Given that the somatic carcinomatous and choriocarcinomatous components of mixed tumors have distinct genetic alterations and biomarker expression, separate analysis of these components is required to guide targeted therapy. High PD-L1 expression suggests a role for checkpoint inhibitor–based immunotherapy in tumors with a choriocarcinoma component. The underlying mechanisms by which cancer stem cells reprogram and initiate trophoblastic retrodifferentiation in some somatic tumors warrant further investigation.

The flavivirus Zika (ZIKV) has emerged as a global threat, making the development of a ZIKV vaccine a priority. While live-attenuated vaccines are known to induce long-term immunity but reduced safety, inactivated vaccines exhibit a weaker immune response as a trade-off for increased safety margins. To overcome the trade-off between immunogenicity and safety, the concept of a third-generation flavivirus vaccine based on single-cycle flaviviruses has been developed. These third-generation flavivirus vaccines have demonstrated extreme potency with a high level of safety in animal models. However, the production of these single-cycle, encapsidation-defective flaviviruses requires a complicated virion packaging system. Here, we investigated a new single-cycle flavivirus vaccine, a vertebrate-specific replication-defective ZIKV (VSRD-ZIKV), in a mouse model. VSRD-ZIKV replicates to high titers in insect cells but can only initiate a single-round infection in vertebrate cells. During a single round of infection, VSRD-ZIKV can express all the authentic viral antigens in vertebrate hosts. VSRD-ZIKV immunization elicited a robust cellular and humoral immune response that protected against a lethal ZIKV challenge in AG129 mice. Additionally, VSRD-ZIKV-immunized pregnant mice were protected against vertically transferring a lethal ZIKV infection to their offspring. Immunized male mice were protected and prevented viral accumulation in the testes after being challenged with lethal ZIKV. Overall, our results indicate that VSRD-ZIKV induces a potent protective immunity against ZIKV in a mouse model and represents a promising approach to develop novel single-cycle arbovirus vaccines.

Interleukin 2 (IL-2) plays a fundamental role in both immune activation and tolerance and has revolutionized the field of cancer immunotherapy since its discovery. The ability of IL-2 to mediate tumor regression in preclinical and clinical settings led to FDA approval for its use in the treatment of metastatic renal cell carcinoma and metastatic melanoma in the 1990s. Although modest success is observed in the clinic, cancer patients receiving IL-2 therapy experience a wide array of side effects ranging from flu-like symptoms to life-threatening conditions such as vascular leak syndrome. Over the past three decades, efforts have focused on circumventing IL-2-related toxicities by engineering methods to localize IL-2 to the tumor or secondary lymphoid tissue, preferentially activate CD8+ T cells and NK cells, and alter pharmacokinetic properties to increase bioavailability. This review summarizes the various IL-2-based strategies that have emerged, with a focus on chimeric fusion methods.

A 44-yr-old woman with menorrhagia and uterine fibroids underwent total laparoscopic hysterectomy, revealing several submucosal, intramural, and subserosal tan-white nodules in the uterus. Microscopic examination revealed tumors displaying 3 distinct morphologies: 1 tumor with features of conventional leiomyoma; 1 tumor with increased cellularity, staghorn/hemangiopericytoma-like vasculature, and occasional atypical cells with prominent red nucleoli and some perinucleolar halos suggesting a fumarate hydratase (FH)-deficient atypical leiomyoma; and 1 tumor with an admixture of epithelioid and spindled cells with the former arranged around blood vessels suggesting a perivascular epithelioid cell tumor (PEComa). Immunohistochemical studies confirmed these diagnoses by demonstrating loss of FH expression in the atypical leiomyoma and diffuse expression of HMB45 and cathepsin K in the tumor with epithelioid features. Sanger sequencing analysis revealed that the FH-deficient atypical leiomyoma harbored a c.181A>G (p.Lys61Glu) mutation in exon 2 of the FH gene. As this mutation was not present in either the other tumors or peripheral blood, the mutation is somatic and hereditary leiomyomatosis and renal cell cancer syndrome is excluded. This case highlights the importance of thorough examination of uterine mesenchymal tumors with atypical and epithelioid features so that tumors with some potential for recurrence (PEComas) and those that might indicate a hereditary cancer syndrome (FH-deficient atypical leiomyoma) are identified and can trigger appropriate clinical investigation and follow-up.

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, 2017

Using a comprehensive next-generation sequencing pipeline (143 genes), Oncomine Comprehensive v.2, we analyzed genetic alterations on a set of vulvar squamous cell carcinomas (SCCs) with emphasis on the primary and metastatic samples from the same patient, to identify amenable therapeutic targets. Clinicopathologic features were reported and genomic DNA was extracted from 42 paraffin-embedded tumor tissues of 32 cases. PD-L1 expression was evaluated in 20 tumor tissues (10 cases with paired primary and metastatic tumors). Fifteen (88%) of 17 successfully analyzed HPV-unrelated SCCs harbored TP53 mutations. 2 different TP53 mutations had been detected in the same tumor in 4 of 15 cases. Other recurrent genetic alterations in this group of tumors included CDKN2a mutations (41%), HRAS mutations (12%), NOTCH1 mutations (12%) and BIRC3 (11q22.1-22.2) amplification (12%). Six HPV-related tumors harbored PIK3CA, BAP1, PTEN, KDR, CTNNB1, and BRCA2 mutations, of which, one case also contained TP53 mutation. Six cases showed identical mutations in paired primary site and distant metastatic location and four cases displayed different mutational profiles. PD-L1 expression was seen in 6 of 10 primary tumors and all 6 paired cases showed discordant PD-L1 expression in the primary and metastatic sites. Our results further confirmed the genetic alterations that are amenable to targeted therapy, offering the potential for individualized management strategies for the treatment of these aggressive tumors with different etiology. Discordant PD-L1 expression in the primary and metastatic vulvar SCCs highlights the importance of evaluation of PD-L1 expression in different locations to avoid false negative information provided for immunotherapy.

Background Endoplasmic reticulum stress has a profound effect on cancer cell proliferation and survival, and also has the capacity to activate cells of the adaptive immune system. Multimodal treatment methods that utilize and combine conventional cancer therapies with antigen-specific immunotherapies have emerged as promising approaches for the treatment and control of cancer. However, it is not well known whether endoplasmic reticulum stress-inducing agents can influence the efficacy of tumor antigen-targeting vaccines. Methods In the past, we developed a therapeutic human papillomavirus (HPV) DNA vaccine that encodes for calreticulin (CRT) linked to the HPV16 E7 antigen (CRT/E7). In this study, we utilize the CRT/E7 and further encode for an endoplasmic reticulum (ER) stress-inducing agent, 3-bromopyruvate (3-BrPA), in a preclinical model, by harnessing its potential to enhance HPV16 E7-specific CD8+ T cell immune responses as well as antitumor effects against E7-expressing tumors (TC-1 cells). E7-specific CD8+ T cells were added to evaluate the cytotoxicity of luciferase-expressing TC-1 tumor cells treated with 3-BrPA in vitro, as measured with an IVIS Luminescence Imaging System. We also determined the levels of ER stress markers in 3-BrPA-treated TC-1 cells. TC-1 tumor-bearing mice were treated with either 3-BrPA (10 mg/kg, intraperitoneal injection) and/or CRT/E7 DNA vaccine (30 μg/mouse). Results Treatment of E7-expressing TC-1 tumor cells with 3-BrPA induced significantly higher in vitro cytotoxicity and resulted in upregulation of endoplasmic reticulum stress markers (CHOP and GRP78). More importantly, combination treatment of 3-BrPA and the CRT/E7 DNA vaccine led to improved antigen-specific CD8+ T cell immune responses as well as therapeutic antitumor effects in TC-1 tumor-bearing mice. Conclusions Our data indicate that 3-BrPA can enhance therapeutic HPV vaccine potency in generating improved antigen-specific immune responses and antitumor effects. These findings have important implications for future clinical translation and provide novel strategies for the treatment of HPV-associated diseases.

The identification of human papillomavirus (HPV) as an etiological factor for HPV-associated malignancies creates the opportunity to control these cancers through vaccination. Currently, available preventive HPV vaccines have not yet demonstrated strong evidences for therapeutic effects against established HPV infections and lesions. Furthermore, HPV infections remain extremely common. Thus, there is urgent need for therapeutic vaccines to treat existing HPV infections and HPV-associated diseases. Therapeutic vaccines differ from preventive vaccines in that they are aimed at generating cell-mediated immunity rather than neutralizing antibodies. The HPV-encoded early proteins, especially oncoproteins E6 and E7, form ideal targets for therapeutic HPV vaccines since they are consistently expressed in HPV-associated malignancies and precancerous lesions, playing crucial roles in the generation and maintenance of HPV-associated disease. Our review will cover various therapeutic vaccines in development for the treatment of HPV-associated lesions and cancers. Furthermore, we review strategies to enhance vaccine efficacy and the latest clinical trials on therapeutic HPV vaccines.

The purpose of this study was to evaluate the safety, efficacy, and immunogenicity of a plasmid vaccine, pNGVL4a-CRT-E7(detox), administered either intradermally, intramuscularly, or directly into the cervical lesion, in patients with HPV16-associated CIN2/3.Eligible patients with HPV16(+) CIN2/3 were enrolled in treatment cohorts evaluating pNGVL4a-CRT-E7(detox), administered by either particle-mediated epidermal delivery (PMED), intramuscular injection (IM), or cervical intralesional injection, at study weeks 0, 4, and 8. Patients were monitored for local injection site and systemic toxicity. A standard therapeutic resection was performed at week 15. The primary endpoints were safety and tolerability. Secondary endpoints included histologic regression and change in cervical HPV viral load. Exploratory endpoints included immune responses in the blood and in the target tissue.Thirty-two patients with HPV16(+) CIN2/3 were enrolled onto the treatment phase of the study, and were vaccinated. Twenty-two of 32 patients (69%) experienced vaccine-specific related adverse events. The most frequent vaccine-related events were constitutional and local injection site in nature, and were grade 1 or less in severity. Histologic regression to CIN 1 or less occurred in 8 of 27 (30%) patients who received all vaccinations and underwent LEEP. In subject-matched comparisons, intraepithelial CD8+ T cell infiltrates increased after vaccination in subjects in the intralesional administration cohort.pNGVL4a-CRT-E7(detox) was well-tolerated, elicited the most robust immune response when administered intralesionally, and demonstrated preliminary evidence of potential clinical efficacy.

Recurrent respiratory papillomatosis is caused by human papillomavirus (HPV) infection, most commonly types 6 (HPV-6) and 11 (HPV-11). Due to failed host immune responses, HPV is unable to be cleared from the host, resulting in recurrent growth of HPV-related lesions that can obstruct the lumen of the airway within the upper aerodigestive tract. In our murine model, the HPV-6b and HPV-11 E7 antigens are not innately immunogenic. In order to enhance the host immune responses against the HPV E7 antigen, we linked calreticulin (CRT) to HPV-6b E7 and found that vaccinating C57BL/6 mice with the HPV-6b CRT/E7 DNA vaccine is able to induce a CD8+ T cell response that recognizes an H-2D(b)-restricted E7aa21-29 epitope. Additionally, vaccination of HLA-A*0201 transgenic mice with HPV-6b CRT/E7 DNA generated a CD8+ T cell response against the E7aa82-90 epitope that was not observed in the wild-type C57BL/6 mice, indicating this T cell response is restricted to HLA-A*0201. In vivo cytotoxic T cell killing assays demonstrated that the vaccine-induced CD8+ T cells are able to efficiently kill target cells. Interestingly, the H-2D(b)-restricted E7aa21-29 sequence and the HLA-A*0201-restricted E7aa82-90 sequence are conserved between HPV-6b and HPV-11 and may represent shared immunogenic epitopes. The identification of the HPV-6b/HPV-11 CD8+ T cell epitopes facilitates the evaluation of various immunomodulatory strategies in preclinical models. More importantly, the identified HLA-A*0201-restricted T cell epitope may serve as a peptide vaccination strategy, as well as facilitate the monitoring of vaccine-induced HPV-specific immunologic responses in future human clinical trials.

Small cell neuroendocrine carcinoma (SCNEC) of the uterine cervix is a rare but extremely aggressive tumor. While high-risk human papillomavirus (HPV) is involved at an early stage of oncogenesis in many tumors, additional driving events have been postulated to facilitate the progression of SCNECs. Identification of oncogenic drivers could guide targeted therapy of this neoplasm. Clinicopathologic features of 10 cervical SCNECs are reported. Analyses included immunohistochemical evaluation of p16, p53, synaptophysin, and chromogranin expression; in situ hybridizations and polymerase chain reaction for high-risk HPV and/or HPV 18; and next-generation sequencing based on a 637-gene panel. The patients ranged in age from 28 to 68 years (mean, 45.6 y; median, 40.5 y). All tumors had diffuse p16 and synaptophysin expression. All but 1 tumor was positive for chromogranin (extent of staining ranged from focal to diffuse). HPV 18 was detected in 6 tumors and HPV 35 in 1 tumor. At least 1 driver mutation was detected in 8 tumors. Four cases harbored TP53 somatic mutations, 3 of which correlated with an aberrant p53 staining pattern. Four PIK3CA mutations (p.G106A, p.N345T, p.E545K, and p.E545D) were detected in 3 tumors, 2 of which also harbored TP53 mutations. Oncogenic driver mutations involving KRAS, Erbb2, c-Myc, NOTCH1, BCL6, or NCOA3 were detected in 4 tumors. Mutations in caretaker tumor suppressors PTEN, RB1, BRCA1, BRCA2, and ARID1B were also identified in 4 tumors that commonly coharbored activating oncogenic mutations. Targeted next-generation gene sequencing identified genetic alterations involving the MAPK, PI3K/AKT/mTOR, and TP53/BRCA pathways in SCNECs. The presence of genetic alterations that are amenable to targeted therapy in SCNECs offers the potential for individualized management strategies for treatment of this aggressive tumor.

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.

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.

Malignant ascites (MA) is a major cause of morbidity that occurs in 37% of ovarian cancer patients. The accumulation of MA in the peritoneal cavity due to cancer results in debilitating symptoms and extremely poor quality of life. There is an urgent unmet need to expand the understanding of MA to design effective treatment strategies, and to improve MA diagnosis. Our purpose here is to contribute to a better characterization of MA metabolic composition in ovarian cancer. We determined the metabolic composition of ascitic fluids resulting from orthotopic growth of two ovarian cancer cell lines, the mouse ID8- vascular endothelial growth factor (VEGF)-Defb29 cell line and the human OVCAR3 cell line using high-resolution 1H MRS. ID8-VEGF-Defb29 tumors induce large volumes of ascites, while OVCAR3 tumors induce ascites less frequently and at smaller volumes. To better understand the factors driving the metabolic composition of the fluid, we characterized the metabolism of these ovarian cancer cells in culture by analyzing cell lysates and conditioned culture media with 1H NMR. Distinct metabolite patterns were detected in ascitic fluid collected from OVCAR3 and ID8-VEGF-Defb29 tumor bearing mice that were not reflected in the corresponding cell culture or conditioned medium. High-resolution 1H NMR metabolic markers of MA can be used to improve characterization and diagnosis of MA. Metabolic characterization of MA can provide new insights into how MA fluid supports cancer cell growth and resistance to treatment, and has the potential to identify metabolic targeting strategies to reduce or eliminate the formation of MA.

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, NRasG12V, 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, NRasG12V, 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.

Human papillomavirus (HPV) has long been recognized as the causative agent of cervical cancer. High-risk HPV types 16 and 18 alone are responsible for over 70% of all cases of cervical cancers. More recently, HPV has been identified as an etiological factor for several other forms of cancers, including oropharyngeal, anogenital, and skin. Thus, the association of HPV with these malignancies creates an opportunity to control these HPV lesions and HPV-associated malignancies through immunization. Strategies to prevent or to therapeutically treat HPV infections have been developed and are still pushing innovative boundaries. Currently, commercial prophylactic HPV vaccines are widely available, but they are not able to control established infections or lesions. As a result, there is an urgent need for the development of therapeutic HPV vaccines, to treat existing infections, and to prevent the development of HPV-associated cancers. In particular, DNA vaccination has emerged as a promising form of therapeutic HPV vaccine. DNA vaccines have great potential for the treatment of HPV infections and HPV-associated cancers due to their safety, stability, simplicity of manufacturability, and ability to induce antigen-specific immunity. This review focuses on the current state of therapeutic HPV DNA vaccines, including results from recent and ongoing clinical trials, and outlines different strategies that have been employed to improve their potencies. The continued progress and improvements made in therapeutic HPV DNA vaccine development holds great potential for innovative ways to effectively treat HPV infections and HPV-associated diseases.

The host immune system plays a pivotal role in the emergence of tumor cells that are refractory to multiple clinical interventions including immunotherapy, chemotherapy, and radiotherapy. Here, we examined the molecular mechanisms by which the immune system triggers cross-resistance to these interventions. By examining the biological changes in murine and tumor cells subjected to sequential rounds of in vitro or in vivo immune selection via cognate cytotoxic T lymphocytes, we found that multimodality resistance arises through a core metabolic reprogramming pathway instigated by epigenetic loss of the ATP synthase subunit ATP5H, which leads to ROS accumulation and HIF-1α stabilization under normoxia. Furthermore, this pathway confers to tumor cells a stem-like and invasive phenotype. In vivo delivery of antioxidants reverses these phenotypic changes and resensitizes tumor cells to therapy. ATP5H loss in the tumor is strongly linked to failure of therapy, disease progression, and poor survival in patients with cancer. Collectively, our results reveal a mechanism underlying immune-driven multimodality resistance to cancer therapy and demonstrate that rational targeting of mitochondrial metabolic reprogramming in tumor cells may overcome this resistance. We believe these results hold important implications for the clinical management of cancer.

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.Preclinical scientific investigation.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.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.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.NA. Laryngoscope, 127:2713-2720, 2017.

DNA vaccination has emerged as an attractive immunotherapeutic approach against cancer due to its simplicity, stability, and safety. Results from numerous clinical trials have demonstrated that DNA vaccines are well tolerated by patients and do not trigger major adverse effects. DNA vaccines are also very cost effective and can be administered repeatedly for long-term protection. Despite all the practical advantages, DNA vaccines face challenges in inducing potent antigen specific cellular immune responses as a result of immune tolerance against endogenous self-antigens in tumors. Strategies to enhance immunogenicity of DNA vaccines against self-antigens have been investigated including encoding of xenogeneic versions of antigens, fusion of antigens to molecules that activate T cells or trigger associative recognition, priming with DNA vectors followed by boosting with viral vector, and utilization of immunomodulatory molecules. This review will focus on discussing strategies that circumvent immune tolerance and provide updates on findings from recent clinical trials.

Local delivery of chemotherapeutics in the cervicovaginal tract using nanoparticles may reduce adverse side effects associated with systemic chemotherapy, while improving outcomes for early-stage cervical cancer. It is hypothesized here that drug-loaded nanoparticles that rapidly penetrate cervicovaginal mucus (CVM) lining the female reproductive tract will more effectively deliver their payload to underlying diseased tissues in a uniform and sustained manner compared with nanoparticles that do not efficiently penetrate CVM. Paclitaxel-loaded nanoparticles are developed, composed entirely of polymers used in FDA-approved products, which rapidly penetrate human CVM and provide sustained drug release with minimal burst effect. A mouse model is further employed with aggressive cervical tumors established in the cervicovaginal tract to compare paclitaxel-loaded poly(lactic-co-glycolic acid) (PLGA) nanoparticles (conventional particles, or CP) and similar particles coated with Pluronic F127 (mucus-penetrating particles, or MPP). CP are mucoadhesive and, thus, aggregated in mucus, while MPP achieve more uniform distribution and close proximity to cervical tumors. Paclitaxel-MPP suppress tumor growth more effectively and prolong median survival of mice compared with unencapsulated paclitaxel or paclitaxel-CP. Histopathological studies demonstrate minimal toxicity to the cervicovaginal epithelia, suggesting paclitaxel-MPP may be safe for intravaginal use. These results demonstrate the in vivo advantages of polymer-based MPP for treatment of tumors localized to a mucosal surface.

The identification of HMGB1 as a late mediator in sepsis has highlighted HMGB1 as a promising therapeutic target for sepsis treatment. Recent studies have revealed that annexin A5, a 35 kDa Ca2+-dependent phospholipid binding protein, exerts antiinflammatory effect by inhibiting LPS binding to TLR4/MD2 complex. Annexin A5 administration has been shown to protect against endotoxin lethality even when the treatment was given after the early cytokine response, which prompted our group to suspect that annexin A5 may inhibit the binding of HMGB1, as well as endotoxin, to TLR4. Here we suggest annexin A5 as a new inhibitor of HMGB1-mediated proinflammatory cytokine production and coagulation in sepsis. We first confirmed the inhibitory role of annexin A5 in LPS-induced production of proinflammatory cytokines both in vitro and in vivo. We observed that annexin A5 protects against tissue damage and organ dysfunction during endotoxemia in vivo. We then assessed the inhibiting role of annexin A5 in HMGB1/TLR4 interaction, and showed that annexin A5 treatment reduces HMGB1-mediated cytokines IL6 and TNFα both in vitro and in vivo. Finally, we confirmed that anticoagulant property of annexin A5 persists in various septic conditions including elevated HMGB1. Overall, we suggest annexin A5 as an alternative therapeutic approach for controlling HMGB1-mediated proinflammation and coagulation in patients with sepsis.

Cervical cancer is the fourth most lethal women's cancer worldwide. Current treatments against cervical cancer include surgery, radiotherapy, chemotherapy, and anti-angiogenic agents. However, despite the various treatments utilized for the treatment of cervical cancer, its disease burden remains a global issue. Persistent infection of human papillomavirus (HPV) has been identified as an essential step of pathogenesis of cervical cancer and many other cancers, and nation-wide HPV screening as well as preventative HPV vaccination program have been introduced globally. However, even though the commercially available prophylactic HPV vaccines, Gardasil (Merck) and Cervarix (GlaxoSmithKline), are effective in blocking the entry of HPV into the epithelium of cervix through generation of HPV-specific neutralizing antibodies, they cannot eliminate the pre-existing HPV infection. For these reason, other immunotherapeutic options against HPV-associated diseases, including therapeutic vaccines, have been continuously explored. Therapeutic HPV vaccines enhance cell-mediated immunity targeting HPV E6 and E7 antigens by modulating primarily dendritic cells and cytotoxic T lymphocyte. Our review will cover various therapeutic vaccines in development for the treatment of HPV-associated lesions and cancers. Furthermore, we will discuss the potential of immune checkpoint inhibitors that have recently been adopted and tested for their treatment efficacy against HPV-induced cervical cancer.

Approximately 10% to 15% of human cancers lack detectable telomerase activity, and a subset of these maintain telomere lengths by the telomerase-independent telomere maintenance mechanism termed alternative lengthening of telomeres (ALT). The ALT phenotype, relatively common in subtypes of sarcomas and astrocytomas, has rarely been reported in epithelial malignancies. However, the prevalence of ALT has not been thoroughly assessed across all cancer types. We therefore comprehensively surveyed the ALT phenotype in a broad range of human cancers. In total, two independent sets comprising 6110 primary tumors from 94 different cancer subtypes, 541 benign neoplasms, and 264 normal tissue samples were assessed by combined telomere-specific fluorescence in situ hybridization and immunofluorescence labeling for PML protein. Overall, ALT was observed in 3.73% (228/6110) of all tumor specimens, but was not observed in benign neoplasms or normal tissues. This is the first report of ALT in carcinomas arising from the bladder, cervix, endometrium, esophagus, gallbladder, kidney, liver, and lung. Additionally, this is the first report of ALT in medulloblastomas, oligodendrogliomas, meningiomas, schwannomas, and pediatric glioblastoma multiformes. Previous studies have shown associations between ALT status and prognosis in some tumor types; thus, further studies are warranted to assess the potential prognostic significance and unique biology of ALT-positive tumors. These findings may have therapeutic consequences, because ALT-positive cancers are predicted to be resistant to anti-telomerase therapies.

Ovarian cancer is currently the most lethal gynecologic cancer in the United States. There is an urgent need for the development of innovative therapies against ovarian cancer, such as immunotherapy. The toll-like receptor 3 ligand, polyriboinosinic:polyribocytidylic acid (poly(I:C), has emerged as a promising adjuvant for activating the host immune responses for the control of tumors. We reasoned that a strategy to enhance the intracellular uptake of poly(I:C) will likely improve the poly(I:C) adjuvant effect. Since polyethylenimine (PEI) has been shown to increase the transfection efficiency of nucleic acids, we characterized the antitumor effects in mouse ovarian surface epithelial cells (MOSEC) tumor-bearing mice treated intraperitoneally with poly(I:C) and PEI. We observed that tumor-bearing mice treated with poly(I:C) and PEI generated significantly better therapeutic antitumor effects against MOSEC tumors compared with treatment with poly(I:C) alone. Furthermore, we found that NK cells play a significant role in the antitumor effects generated by treatment with poly(I:C) in combination with PEI. Intraperitoneal administration of poly(I:C) with PEI led to the uptake of poly(I:C) mainly by CD11b+ macrophages, resulting in the high expression of MHC class II and IL-12 (M1 phenotype). In addition, adoptive transfer of CD11b+ macrophages from mice treated with poly(I:C) and PEI was found to lead to increased number of activated NK cells in the recipient mice. Taken together, our data indicate that PEI can potentially be used to improve the uptake of poly(I:C) by CD11b+ macrophages, leading to the activation of NK cells and the control of murine ovarian tumors.

Dendritic cell (DC)-based vaccines are recognized as a promising immunotherapeutic strategy against cancer. Various adjuvants are often incorporated to enhance the modest immunogenicity of DC vaccines. More specifically, many of the commonly used adjuvants are derived from bacteria. In the current study, we evaluate the use of apoptosis inhibitor 5 (API5), a damage-associated molecular pattern expressed by many human cancer cells, as a novel DC vaccine adjuvant. We showed that API5 can prompt activation and maturation of DCs and activate NFkB by stimulating the Toll-like receptor signaling pathway. We also demonstrated that vaccination with API5-treated DCs pulsed with OVA, E7, or AH1-A5 peptides led to the generation of OVA, E7, or AH1-A5-specific CD8 + T cells and memory T cells, which is associated with long term tumor protection and antitumor effects in mice, against EG.7, TC-1, and CT26 tumors. Additionally, we determined that API5-mediated DC activation and immune stimulation are dependent on TLR4. Lastly, we showed that the API5 protein sequence fragment that is proximal to its leucine zipper motif is responsible for the adjuvant effects exerted by API5. Our data provide evidence that support the use of API5 as a promising adjuvant for DC-based therapies, which can be applied in combination with other cancer therapies. Most notably, our results further support the continued investigation of human-based adjuvants.

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.

The discovery of human papillomavirus (HPV) as a necessary etiological factor for cervical cancer has spurred the development of preventive and therapeutic HPV vaccines for the control of HPV-associated malignancies including cervical, vulvar, vaginal, and a subset of head and neck cancers. The commercial preventive HPV vaccines, Gardasil and Cervarix, use HPV virus-like particles to generate neutralizing antibodies against HPV major capsid protein L1. However, they do not exert therapeutic effects on existing lesions and are unlikely to have an immediate impact on the prevalence of cervical cancer due to their cost and limited availability in developing countries, which account for more than 80% of cer- vical cancers. Thus, there is an urgent need for therapeutic HPV vaccines. Therapeutic HPV vaccines can eliminate pre- existing lesions and infections by generating cellular immunity against HPV-infected cells. HPV E6 and E7 oncoproteins represent ideal targets for therapeutic intervention because of their constitutive expression in HPV-associated tumors and their crucial role in the induction and maintenance of HPV-associated disease. This review discusses the current progress of various therapeutic HPV vaccine approaches, including live vector-based, peptide/protein-based, nucleic acid-based and cell-based vaccines targeting E6 and/or E7 antigens, and their future prospects for the control of HPV-associated ma- lignancies.

Tumor immune escape is a major obstacle in cancer immunotherapy, but the mechanisms involved remain poorly understood. We have previously developed an immune evasion tumor model using an in vivo immune selection strategy and revealed Akt-mediated immune resistance to antitumor immunity induced by various cancer immunotherapeutic agents. In the current study, we used microarray gene analysis to identify an Akt-activating candidate molecule overexpressed in immune-resistant tumors compared with parental tumors. X-linked lymphocyte-regulated protein pM1 (XLR) gene was the most upregulated in immune-resistant tumors compared with parental tumor cells. Furthermore, the retroviral transduction of XLR in parental tumor cells led to activation of Akt, resulting in upregulation of antiapoptotic proteins and the induction of immune resistance phenotype in parental tumor cells. In addition, we found that transduction of parental tumor cells with other homologous genes from the mouse XLR family, such as synaptonemal complex protein 3 (SCP3) and XLR-related, meiosis-regulated protein (XMR) and its human counterpart of SCP3 (hSCP3), also led to activation of Akt, resulting in the upregulation of antiapoptotic proteins and induction of immune resistance phenotype. Importantly, characterization of a panel of human cervical cancers revealed relatively higher expression levels of hSCP3 in human cervical cancer tissue compared with normal cervical tissue. Thus, our data indicate that ectopic expression of XLR and its homologues in tumor cells represents a potentially important mechanism for tumor immune evasion and serves as a promising molecular target for cancer immunotherapy. Cancer Res; 70(8); 3062–70. ©2010 AACR.

The high-risk types of human papillomavirus (HPV) have been found to be associated with most cervical cancers and play an essential role in the pathogenesis of the disease. Despite recent advances in preventive HPV vaccine development, such preventive vaccines are unlikely to reduce the prevalence of HPV infections within the next few years, due to their cost and limited availability in developing countries. Furthermore, preventive HPV vaccines may not be capable of treating established HPV infections and HPV-associated lesions, which account for high morbidity and mortality worldwide. Thus, it is important to develop therapeutic HPV vaccines for the control of existing HPV infection and associated malignancies. Therapeutic vaccines are quite different from preventive vaccines in that they require the generation of cell-mediated immunity, particularly T cell-mediated immunity, instead of the generation of neutralizing antibodies. The HPV-encoded early proteins, E6 and E7 oncoproteins, form ideal targets for therapeutic HPV vaccines since they are consistently expressed in HPV-associated cervical cancer and its precursor lesions and thus play crucial roles in the generation and maintenance of HPV-associated disease. Our review will cover the various therapeutic HPV vaccines for cervical cancer, including live vector-based, peptide or protein-based, nucleic acid-based, and cell-based vaccines targeting the HPV E6 and/or E7 antigens. Furthermore, we will review the studies using therapeutic HPV vaccines in combination with other therapeutic modalities and review the latest clinical trials on therapeutic HPV vaccines.

DNA vaccines have recently emerged at the forefront of approaches to harness the immune system in the prevention and treatment of viral infections, as well as the prevention and treatment of cancers. However, these vaccines suffer from limited efficacy since they often fail to produce significant antigen-specific CD8+T-cell responses. We report here a novel concept for DNA vaccine design that exploits the unique and powerful ability of viral fusogenic membrane glycoproteins (FMGs) to couple concentrated antigen transfer to dendritic cells (DCs) with local induction of the acute inflammatory response. Intramuscular administration into mice by electroporation technology of a plasmid containing the FMG gene from vesicular stomatitis virus (VSV-G)—together with DNA encoding the E7 protein of human papillomavirus type 16, a model cervical cancer antigen—elicited robust E7-specific CD8+T-cell responses, as well as therapeutic control of E7-expressing tumors. This effect could potentially be mediated through the immunogenic form of cellular fusion and necrosis induced by VSV-G, which in a concerted fashion provokes leukocyte infiltration into the inoculation site, enhances cross-presentation of antigen to DCs, and stimulates them to mature efficiently. Thus, the incorporation of FMGs into DNA vaccines holds promise for the successful control of viral infections and cancers in the clinic.

Introduction: Traditional biochemical and pharmaceutical methods employed today encounter problems of clinical side effects and drug resistance, and their use is becoming limited. Therefore, it has become important and necessary to develop new, alternative strategies to combat viral diseases.Materials and Method: A variety of viruses including M13 bacetriophage (nonenveloped ssDNA), tobacco mosaic virus (nonenveloped ssRNA), human papillomavirus (nonenveloped dsDNA) and human immunodeficiency virus (enveloped ssRNA), together with human red blood cells, Jurkat T-cells and mouse dendritic cells in their buffer solutions have been irradiated with near-infrared subpicosecond laser pulses in vitro.Results: A window of laser power density, approximately between 1 GW/cm2 and 10 GW/cm2, has been observed that allows killing the viral particles while leaving mammalian cells unharmed.Conclusion: The ultrashort pulsed laser technology may have great potential for disinfection of blood components.

Essentially all squamous cervical cancers and their precursor lesions, high grade cervical intraepithelial neoplasia (CIN2/3), are caused by persistent human papil-lomavirus (HPV) infection. However, not all CIN2/3 lesions progress to cancer. In a brief, observational study window monitoring subjects with CIN2/3 from protocol entry (biopsy diagnosis) to definitive therapy (cervical conization) at week 15, in a cohort of 50 subjects, we found that 26% of CIN2/3 lesions associated with HPV16, the genotype most commonly associated with disease, underwent complete histologic regression. Nonetheless, HPV16-specific T cell responses measured in peripheral blood obtained at the time of study entry and at the time of conization were marginally detectable directly ex vivo, and did not correlate with lesion regression. This finding suggests that, in the setting of natural infection, immune responses which are involved in elimination of cervical dysplastic epithelium are not represented to any great extent in the systemic circulation.

Intramuscular administration of DNA vaccines can lead to the generation of antigen-specific immune responses through cross-priming mechanisms. We propose a strategy that is capable of leading to local inflammation and enhancing cross-priming, thus resulting in improved antigen-specific immune responses. Therefore, in this study, we evaluated the immunological responses elicited through electroporation-mediated intramuscular administration of a DNA vaccine encoding calreticulin (CRT) linked to human papillomavirus type 16 E7 (CRT-E7) in combination with DNA expressing HLA-A2 as compared with CRT-E7 DNA vaccination alone. We found that the co-administration of a DNA vaccine in conjunction with a DNA encoding a xenogenic major histocompatibility complex (MHC) molecule could significantly enhance the E7-specific CD8+ T-cell immune responses and antitumor effects against an E7-expressing tumor, TC-1, in C57BL/6 tumor-bearing mice. Furthermore, a similar enhancement in E7-specific immune responses was observed by the co-administration of CRT-E7 DNA with DNA encoding other types of xenogenic MHC class-I molecules. This strategy was also applicable to another antigenic system, ovalbumin. Further characterization of the injection site revealed that the co-administration of HLA-A2 DNA led to a significant increase in the number of infiltrating CD8+ T lymphocytes and CD11b/c+ antigen-presenting cells. Furthermore, the E7-specific immune responses generated by intramuscular co-administration of CRT-E7 with HLA-A2 DNA were reduced in HLA-A2 transgenic mice. Thus, our data suggest that intramuscular co-administration of DNA encoding xenogenic MHC class-I can further improve the antigen-specific immune responses, as well as antitumor effects generated by DNA vaccines through enhancement of cross-priming mechanisms.

Immune evasion is an important reason why the immune system cannot control tumor growth. To elucidate the mechanism for tumor immune evasion, we generated an immune-resistant human papillomavirus type 16 (HPV-16) E7-expressing tumor cell line by subjecting a susceptible tumor cell line to multiple rounds of in vivo immune selection with an E7-specific vaccine. Comparison of parental and immune-resistant tumors revealed that Akt is highly activated in the immune-resistant tumors. Retroviral transfer of a constitutively active form of Akt into the parental tumor significantly increased its resistance against E7-specific CD8(+) T-cell mediated apoptosis. The observed resistance against apoptosis was found to be associated with the upregulation of antiapoptotic molecules. We also observed that intratumoral injection of an Akt inhibitor enhanced the therapeutic efficacy of E7-specific vaccine or E7-specific CD8(+) T-cell adoptive transfer against the immune-resistant tumors. Thus, our data indicate that the activation of PI3K/Akt pathway represents a new mechanism of immune escape and has important implications for the development of a novel strategy in cancer immunotherapy against immune-resistant tumor cells.

Purpose: To evaluate the safety and immunogenicity of a therapeutic human papillomavirus (HPV)16 DNA vaccine administered to women with HPV16+cervical intraepithelial neoplasia (CIN)2/3. Experimental Design: This phase I trial incorporated the standard ′3+3″ dose-escalation design with an additional 6 patients allocated to the maximally tolerated dose. Healthy adult women with colposcopically directed, biopsy-proven HPV16+ CIN2/3 received 3 i.m. vaccinations (0.5, 1, or 3 mg) of a plasmid expressing a Sig-E7(detox)-heat shock protein 70 fusion protein on days 0, 28, and 56, and underwent standard therapeutic resection of the cervical squamocolumnar junction at day 105 (week 15). The safety and immunogenicity of the vaccine and histologic outcome based on resection at week 15 were assessed. Results: Fifteen patients were evaluable (3 each at 0.5 and 1mg, 9 at 3 mg). The vaccine was well tolerated: most adverse events were mild, transient injection-site discomfort; no dose-limiting toxicities were observed. Although HPVE7-specific T-cell responses to E7 detected by enzyme-linked immunospot assays (IFN-γ) were of low frequency and magnitude, detectable increases in response subsequent to vaccination were identified in subjects in the second and third cohorts. Complete histologic regression occurred in 3 of 9 (33%; 7-70% confidence interval) individuals in the highest-dose cohort. Although the difference is not significant, it is slightly higher than would be expected in an unvaccinated cohort (25%). Conclusions: This HPV16 DNA vaccine was safe and well tolerated. Whereas it seems possible to elicit HPV-specific T-cell responses in patients with established dysplastic lesions, other factors are likely to play a role in lesion regression.

Current therapeutic approaches to treatment of patients with bulky cervical cancer are based on conventional in situ ablative modalities including cisplatin-based chemotherapy and radiation therapy. The 5-year survival of patients with nonresectable disease is dismal. Because over 99% of squamous cervical cancer is caused by persistent infection with an oncogenic strain of human papillomavirus (HPV), particularly type 16 and viral oncoproteins E6 and E7 are functionally required for disease initiation and persistence, HPV-targeted immune strategies present a compelling opportunity in which to demonstrate proof of principle. Sublethal doses of radiation and chemotherapeutic agents have been shown to have synergistic effect in combination with either vaccination against cancer-specific antigens, or with passive transfer of tumor-specific cytotoxic T lymphocytes (CTLs). Here, we explored the combination of low-dose radiation therapy with DNA vaccination with calreticulin (CRT) linked to the mutated form of HPV-16 E7 antigen (E7(detox)), CRT/E7(detox) in the treatment of E7-expressing TC-1 tumors. We observed that TC-1 tumor-bearing mice treated with radiotherapy combined with CRT/E7(detox) DNA vaccination generated significant therapeutic antitumor effects and the highest frequency of E7-specific CD8(+) T cells in the tumors and spleens of treated mice. Furthermore, treatment with radiotherapy was shown to render the TC-1 tumor cells more susceptible to lysis by E7-specific CTLs. In addition, we observed that treatment with radiotherapy during the second DNA vaccination generated the highest frequency of E7-specific CD8(+) T cells in the tumors and spleens of TC-1 tumor-bearing mice. Finally, TC-1 tumor-bearing mice treated with the chemotherapy in combination with radiation and CRT/E7(detox) DNA vaccination generate significantly enhanced therapeutic antitumor effects. The clinical implications of the study are discussed.

There is an urgent need to develop new innovative therapies for the control of cancer. Antigen-specific immunotherapy and the employment of proteasome inhibitors have emerged as two potentially plausible approaches for the control of cancer. In the current study, we explored the combination of the DNA vaccine encoding calreticulin (CRT) linked to human papillomavirus type 16 E7 antigen (CRT/E7) with the proteasome inhibitor, bortezomib, for their ability to generate E7-specific immune responses and antitumor effects in vaccinated mice. We found that the combination of treatment with bortezomib and CRT/E7(detox) DNA generated more potent E7-specific CD8+ T cell immune responses and better therapeutic effects against TC-1 tumors in tumor-bearing mice compared to monotherapy. Furthermore, we found that treatment with bortezomib led to increased apoptosis of TC-1 tumor cells and could render the TC-1 tumor cells more susceptible to lysis by E7-specific CD8+ T cells. Our data have significant implications for future clinical translation.

Multimodality treatments that combine conventional cancer therapies with antigen-specific immunotherapy have emerged as promising approaches for the control of cancer. In the current study, we have explored the effect of doxorubicin on the antigen-specific immune responses generated in mice vaccinated with calreticulin (CRT)/E6 and/or Ii-PADRE DNA. We observed that pretreatment with doxorubicin suppressed the E6-specific CD8+ T-cell immune responses generated by CRT/E6 DNA vaccination in vaccinated mice. In contrast, pretreatment with doxorubicin enhanced the PADRE-specific CD4+ T-cell immune responses generated by Ii-PADRE DNA vaccination. Furthermore, coadministration of Ii-PADRE DNA could not only reverse the suppression, but also enhanced the E6-specific CD8+ T-cell responses in CRT/E6-vaccinated mice pretreated with doxorubicin. Finally, treatment with doxorubicin followed by CRT/E6 combined with Ii-PADRE DNA vaccination led to enhanced antitumor effects and prolonged survival in TC-1 tumor-bearing mice. The clinical implications of the current study are discussed.

We contrast the efforts to treat ovarian cancer and cervical cancer through vaccination because of their different pathobiology. A plethora of approaches have been developed for therapeutic vaccination against cancer, many of which target defined tumor-associated antigens (TAAs). Persistent infection with oncogenic human papillomavirus (HPV) types is necessary cause of cervical cancer. Furthermore, cervical cancer patients frequently mount both humoral and T cell immune responses to the HPV E6 and E7 oncoproteins, whose expression is required for the transformed phenotype. Numerous vaccine studies target these viral TAAs, including recent trials that may enhance clearance of pre-malignant disease. By contrast little is known about the etiology of epithelial ovarian cancer. Although it is clear that p53 mutation or loss is a critical early event in the development of epithelial ovarian cancer, no precursor lesion has been described for the most common serous histotype, and even the location of its origin is debated. These issues have complicated the selection of appropriate ovarian TAAs and the design of vaccines. Here we focus on mesothelin as a promising ovarian TAA because it is overexpressed and immunogenic at high frequency in patients, is displayed on the cell surface and potentially contributes to ovarian cancer biology.