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Human papillomavirus (HPV) is the most common sexually transmitted infection, currently affecting close to 80 million Americans. Importantly, HPV infection is recognized as the etiologic factor for numerous cancers, including cervical, vulval, vaginal, penile, anal, and a subset of oropharyngeal cancers. The prevalence of HPV infection and its associated diseases are a significant problem, affecting millions of individuals worldwide. Likewise, the incidence of HPV infection poses a significant burden on individuals and the broader healthcare system. Between 2011 and 2015, there were an estimated 42,700 new cases of HPV-associated cancers each year in the United States alone. Similarly, the global burden of HPV is high, with around 630,000 new cases of HPV-associated cancer occurring each year. In the last decade, a total of three preventive major capsid protein (L1) virus-like particle-based HPV vaccines have been licensed and brought to market as a means to prevent the spread of HPV infection. These prophylactic vaccines have been demonstrated to be safe and efficacious in preventing HPV infection. The most recent iteration of the preventive HPV vaccine, a nanovalent, L1-VLP vaccine, protects against a total of nine HPV types (seven high-risk and two low-risk HPV types), including the high-risk types HPV16 and HPV18, which are responsible for causing the majority of HPV-associated cancers. Although current prophylactic HPV vaccines have demonstrated huge success in preventing infection, existing barriers to vaccine acquisition have limited their widespread use, especially in low- and middle-income countries, where the burden of HPV-associated diseases is highest. Prophylactic vaccines are unable to provide protection to individuals with existing HPV infections or HPV-associated diseases. Instead, therapeutic HPV vaccines capable of generating T cell-mediated immunity against HPV infection and associated diseases are needed to ameliorate the burden of disease in individuals with existing HPV infection. To generate a cell-mediated immune response against HPV, most therapeutic vaccines target HPV oncoproteins E6 and E7. Several types of therapeutic HPV vaccine candidates have been developed including live-vector, protein, peptide, dendritic cell, and DNA-based vaccines. This chapter will review the commercially available prophylactic HPV vaccines and discuss the recent progress in the development of therapeutic HPV vaccines.

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.

BackgroundType I interferons (IFN) promote dendritic cells maturation and subsequently enhance generation of antigen-specific CD8 +T cell for the control of tumor. Using type I interferons as an adjuvant to vaccination could prove to be a potent strategy. However, type I interferons have a short half-life. Albumin linked to a protein will prolong the half-life of the linked protein.MethodsIn this study, we explored the fusion of albumin to IFNβ (Alb-IFNβ) for its functional activity both in vitro and in vivo. We determined the half-life of Alb-IFNβ following treatment in the serum, tumor, and tumor draining lymph nodes in both wild type and FcRn knockout mice. We characterized the ability of Alb-IFNβ to enhance antigen-specific CD8+ T cells using ovalbumin (OVA) or human papillomavirus (HPV) E7 long peptides. Next, we evaluated the therapeutic antitumor effect of coadministration of AlbIFNβ with antigenic peptides against HPVE7 expressing tumor and the treatment’s ability to generate HPVE7 antigen specific CD8+ T cells. The contribution of the antitumor effect by lymphocytes was also examined by an antibody depletion experiment. The ability of Alb-IFNβ to serve as an adjuvant was tested using clinical grade therapeutic protein-based HPV vaccine, TACIN.ResultsAlb-IFNβ retains biological function and does not alter the biological activity of IFNβ. In addition, Alb-IFNβ extends half-life of IFNβ in serum, lymph nodes and tumor. The coadministration of Alb-IFNβ with OVA or HPVE7 antigenic peptides enhances antigen-specific CD8 +T cell immunity, and in a TC-1 tumor model results in a significant therapeutic antitumor effect. We found that CD8 +T cells and dendritic cells, but not CD4 +T cells, are important for the observed antitumor therapeutic effect mediated by Alb-IFNβ. Finally, Alb-IFNβ served as a potent adjuvant for TA-CIN for the treatment of HPV antigen expressing tumors.ConclusionsOverall, Alb-IFNβ serves as a potent adjuvant for enhancement of strong antigen-specific CD8 +T cell antitumor immunity, reduction of tumor burden, and increase in overall survival. Alb-IFNβ potentially can serve as an innovative adjuvant for the development of vaccines for the control of infectious disease and cancer.

ASC-J9® is a recently-developed androgen receptor (AR)-degradation enhancer that effectively suppresses castration resistant prostate cancer (PCa) cell proliferation and invasion. The optimal half maximum inhibitory concentrations (IC50) of ASC-J9® at various PCa cell confluences (20%, 50%, and 100%) were assessed via both short-term MTT growth assays and long-term clonogenic proliferation assays. Our results indicate that the IC50 values for ASC-J9® increased with increasing cell confluency. The IC50 values were significantly decreased in PCa AR-positive cells compared to PCa AR-negative cells or in normal prostate cells. This suggests that ASC-J9® may function mainly via targeting the AR-positive PCa cells with limited unwanted side-effects to suppress the surrounding normal prostate cells. Mechanism dissection indicated that ASC-J9® might function via altering the apoptosis signals to suppress the PCa AR-negative PC-3 cells. Preclinical studies using multiple in vitro PCa cell lines and an in vivo mouse model with xenografted castration-resistant PCa CWR22Rv1 cells demonstrated that ASC-J9® has similar AR degradation effects when dissolved in FDA-approved solvents, including DMSO, PEG-400:Tween-80 (95:5), DMA:Labrasol:Tween-80 (10:45:45), and DMA:Labrasol:Tween-20 (10:45:45). Together, results from preclinical studies suggest a potential new therapy with AR-degradation enhancer ASC-J9® may potentially be ready to be used in human clinical trials in order to better suppress PCa at later castration resistant stages.

Objective: The objective of the current service evaluation was to assess whether classical music positively impacts upon exercise intensity in patients undergoing a cardiovascular prevention and rehabilitation programme. Design: A crossover study design was used, in which patients were exposed to both classical music and no music over 2 consecutive weeks, determined through a random order. Setting: This took place in cardiovascular prevention and rehabilitation exercise sessions at two Imperial College NHS Healthcare sites. Patients: The sample consisted of 30 cardiac rehabilitation patients undergoing secondary prevention. Main outcome measures: Comparison of heart rate (average and peak) and Borg Rating of Perceived Exertion (RPE) recorded in each patient under both music and non-music conditions. Results: Average heart rate, Borg RPE, and energy expenditure showed minimal differences between exercise modes. However, peak heart rate increased on average by 7 beats per minute when comparing exercising under classical to no music. Conclusions: The study found that classical music significantly increased an individual's peak heart rate during a cardiac exercise session, thereby increasing physical activity intensity.

A long duration of treatment and emerging drug resistance pose significant challenges for global tuberculosis (TB) eradication efforts. Therefore, there is an urgent need to develop novel strategies to shorten TB treatment regimens and to treat drug-resistant TB. Using an albumin-fusion strategy, we created a novel albumin-fused granulocyte-macrophage colony-stimulating factor (albGM-CSF) molecule that harnesses albumin’s long half-life and targeting abilities to enhance the biostability of GM-CSF and direct it to the lymph nodes, where the effects of GM-CSF can increase dendritic cell populations crucial for eliciting a potent immune response. In this study, we demonstrate that albGM-CSF serves as a novel immunotherapy for chronic Mycobacterium tuberculosis (Mtb) infections by enhancing GM-CSF biostability in serum. Specifically, albumin is very safe, stable, and has a long half-life, thereby enhancing the biostability of GM-CSF. In the lungs and draining lymph nodes, albGM-CSF is able to increase the numbers of dendritic cells, which are crucial for the activation of naive T cells and for eliciting potent immune responses. Subcutaneous administration of albGM-CSF alone reduced the mean lung bacillary burden in mice with chronic tuberculosis infection. While GM-CSF administration was associated with IL-1β release from Mtb-infected dendritic cells and macrophages, higher IL-1β levels were observed in albGM-CSF-treated mice with chronic tuberculosis infection than in mice receiving GM-CSF. Albumin fusion with GM-CSF represents a promising strategy for the control of chronic lung tuberculosis infections and serves as a novel therapeutic vaccination platform for other infectious diseases and malignancies.

A major factor impeding the success of numerous therapeutic approaches in cancer is the immunosuppressive nature of the tumor microenvironment (TME). Hence, methods capable of reverting tumor immunosuppression through depletion or reprogramming of myeloid-derived suppressive cells (MDSCs) and regulatory T cells (Tregs) are of great clinical need. Here, we explore NKG2D-Fc as a modality to modulate antitumor immunity through the depletion of immunosuppressive MDSCs and Tregs in the TME. We have generated the NKG2D-Fc fusion protein and characterized its potential to mediate tumor control and overall survival in LL2 and MC38 murine models. Upon treatment of LL2 or MC38 tumor-bearing mice with NKG2D-Fc, we observe significant tumor control and enhanced survival compared to Fc control. When characterizing MDCSs and Tregs from tumor-bearing mice, we observe clear expression of NKG2D-ligand RAE1γ and subsequent binding of NKG2D-Fc fusion protein to both MDSCs and Tregs. Examining the immune profile of mice treated with NKG2D-Fc reveals significant depletion of MDSCs and Tregs in the TME, as well as an increase in NK cells likely due to the reversed suppressive TME. In conclusion, NKG2D-Fc induces antitumor immunity and tumor control through the depletion of MDSCs and Tregs, subsequently providing a niche for the infiltration and expansion of proinflammatory cells, such as NK cells. Strategies capable of modulating the immunosuppressive state in cancer are in high clinical demand. NKG2D-Fc is a simple, single tool capable of depleting both MDSCs and Tregs and should be further investigated as a therapeutic agent for the treatment of cancer.

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.

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.

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.

ASC-J9

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.

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.

Estrogen and estrogen receptor (ER)-mediated signaling pathways play important roles in the etiology and progression of human breast, endometrial, and ovarian cancers. Attenuating ER activities by natural products and their derivatives is a relatively practical strategy to control and reduce breast, endometrial, and ovarian cancer risk. Here, we found 3-butoxy-1,8,9-trihydroxy-6H-benzofuro[3,2-c]benzopyran-6-one (BTB), a new derivative of wedelolactone, could effectively inhibit the 17-estradiol (E2)-induced ER transactivation and suppress the growth of breast cancer as well as endometrial and ovarian cancer cells. Our results indicate that 2.5 μM BTB effectively suppresses ER-positive, but not ER-negative, breast, endometrial, and ovarian cancer cells. Furthermore, our data indicate that BTB can modulate ER transactivation and suppress the expression of E2-mediated ER target genes (Cyclin D1, E2F1, and TERT) in the ER-positive MCF-7, Ishikawa, and SKOV-3 cells. Importantly, this BTB mediated inhibition of ER activity is selective since BTB does not suppress the activities of other nuclear receptors, including glucocorticoid receptor and progesterone receptor, suggesting that BTB functions as a selective ER signaling inhibitor with the potential to treat breast, endometrial, and ovarian cancers.