Expansion of Candidate HPV-Specific T Cells in the Tumor Microenvironment during Chemoradiotherapy Is Prognostic in HPV16+ Cancers.

Human papillomavirus (HPV) infection causes 600,000 new cancers worldwide each year. HPV-related cancers express the oncogenic proteins E6 and E7, which could serve as tumor-specific antigens. It is not known whether immunity to E6 and E7 evolves during chemoradiotherapy or affects survival. Using T cells from 2 HPV16+ patients, we conducted functional T-cell assays to identify candidate HPV-specific T cells and common T-cell receptor motifs, which we then analyzed across 86 patients with HPV-related cancers. The HPV-specific clones and E7-related T-cell receptor motifs expanded in the tumor microenvironment over the course of treatment, whereas non-HPV-specific T cells did not. In HPV16+ patients, improved recurrence-free survival was associated with HPV-responsive T-cell expansion during chemoradiotherapy.

A recombinant bovine adenoviral mucosal vaccine expressing mycobacterial antigen-85B generates robust protection against tuberculosis in mice.

Although the BCG vaccine offers partial protection, tuberculosis remains a leading cause of infectious disease death, killing ∼1.5 million people annually. We developed mucosal vaccines expressing the autophagy-inducing peptide C5 and mycobacterial Ag85B-p25 epitope using replication-defective human adenovirus (HAdv85C5) and bovine adenovirus (BAdv85C5) vectors. BAdv85C5-infected dendritic cells (DCs) expressed a robust transcriptome of genes regulating antigen processing compared to HAdv85C5-infected DCs. BAdv85C5-infected DCs showed enhanced galectin-3/8 and autophagy-dependent in vitro Ag85B-p25 epitope presentation to CD4 T cells. BCG-vaccinated mice were intranasally boosted using HAdv85C5 or BAdv85C5 followed by infection using aerosolized Mycobacterium tuberculosis (Mtb). BAdv85C5 protected mice against tuberculosis both as a booster after BCG vaccine (>1.4-log10 reduction in Mtb lung burden) and as a single intranasal dose (>0.5-log10 reduction). Protection was associated with robust CD4 and CD8 effector (TEM), central memory (TCM), and CD103+/CD69+ lung-resident memory (TRM) T cell expansion, revealing BAdv85C5 as a promising mucosal vaccine for tuberculosis.

Gut microbiome diversity is an independent predictor of survival in cervical cancer patients receiving chemoradiation.

Diversity of the gut microbiome is associated with higher response rates for cancer patients receiving immunotherapy but has not been investigated in patients receiving radiation therapy. Additionally, current studies investigating the gut microbiome and outcomes in cancer patients may not have adjusted for established risk factors. Here, we sought to determine if diversity and composition of the gut microbiome was independently associated with survival in cervical cancer patients receiving chemoradiation. Our study demonstrates that the diversity of gut microbiota is associated with a favorable response to chemoradiation. Additionally, compositional variation among patients correlated with short term and long-term survival. Short term survivor fecal samples were significantly enriched in Porphyromonas, Porphyromonadaceae, and Dialister, whereas long term survivor samples were significantly enriched in Escherichia Shigella, Enterobacteriaceae, and Enterobacteriales. Moreover, analysis of immune cells from cervical tumor brush samples by flow cytometry revealed that patients with a high microbiome diversity had increased tumor infiltration of CD4+ lymphocytes as well as activated subsets of CD4 cells expressing ki67+ and CD69+ over the course of radiation therapy. Modulation of the gut microbiota before chemoradiation might provide an alternative way to enhance treatment efficacy and improve treatment outcomes in cervical cancer patients.

Human Biofield Therapy Modulates Tumor Microenvironment and Cancer Stemness in Mouse Lung Carcinoma.

Studies have demonstrated that purported biofield therapy emitted from humans can inhibit the proliferation of cancer cells and suppress tumor growth in various cancers. We explored the effects of biofield therapy on tumor growth in the Lewis lung carcinoma and expanded mechanistic outcomes. We found biofield therapy did not inhibit tumor growth. However, the experimental (Ex) condition exposed tumors had a significantly higher percentage of necrosis (24.4 ± 6.8%) compared with that of the Control condition (6.5 ± 2.7%; P < .02) and cleaved caspase-3 positive cells were almost 2.3-fold higher (P < .05). Similarly, tumor-infiltrating lymphocytes profiling showed that CD8+/CD45+ immune cell population was significantly increased by 2.7-fold in Ex condition (P < .01) whereas the number of intratumoral FoxP3+/CD4+ (T-reg cells) was 30.4% lower than that of the Control group (P = .01), leading to a significant 3.1-fold increase in the ratio of CD8+/T-reg cells (P < .01). Additionally, there was a 51% lower level of strongly stained CD68+ cells (P < .01), 57.9% lower level of F4/80high/CD206+ (M2 macrophages; P < .02) and a significant 1.8-fold increase of the ratio of M1/M2 macrophages (P < .02). Furthermore, Ex exposure resulted in a 15% reduction of stem cell marker CD44 and a significant 33% reduction of SOX2 compared with that of the Controls (P < .02). The Ex group also engaged in almost 50% less movement throughout the session than the Controls. These findings suggest that exposure to purported biofields from a human is capable of enhancing cancer cell death, in part mediated through modification of the tumor microenvironment and stemness of tumor cells in mouse Lewis lung carcinoma model. Future research should focus on defining the optimal treatment duration, replication with different biofield therapists, and exploring the mechanisms of action.

Effect of Antibiotics on Gut and Vaginal Microbiomes Associated with Cervical Cancer Development in Mice.

Antibiotics affect microbial diversity in the gut, leading to dysbiosis and impaired immunity. However, the impact of antibiotics on microbial communities at other sites, such as vagina is less understood. It is also not clear whether changes induced by antibiotics in both microbiomes affect the development of cervical cancer. In this study, we utilized the murine model to evaluate these questions. We show that oral application of broad-spectrum antibiotics in mice changed not only diversity, but composition and sharing of gut and vaginal microbiomes in mice and influenced cervical cancer development in an orthotopic tumor model. Antibiotics decreased richness and diversity indexes in the gut but increased them in the vagina. Some beneficial taxa, such as Bacteroides, Ruminococcaceae, and Lachnospiraceae increased their abundance in the vagina while other pathogenic species, such as Proteobacteria, were decreased. As a result of the changes, mice with greater richness and diversity of the vaginal microbiome after antibiotics exposure were less likely developed tumors. No association between richness and diversity of the gut microbiome and tumor development was identified.

Intranasal Therapeutic Peptide Vaccine Promotes Efficient Induction and Trafficking of Cytotoxic T Cell Response for the Clearance of HPV Vaginal Tumors.

Human papillomavirus (HPV)-induced cancers continue to affect millions of women around the world, and the five year survival rate under the current standard of care for these cancers is less than 60% in some demographics. Therefore there is still an unmet need to develop an effective therapy that can be easily administered to treat established HPV cervical cancer lesions. We sought to investigate the potential of an intranasal HPV peptide therapeutic vaccine incorporating the combination of α-Galactosylceramide (α-GalCer) and CpG-ODN adjuvants (TVAC) against established HPV genital tumors in a syngeneic C57BL/6J mouse model. We obtained evidence to show that TVAC, delivered by the mucosal intranasal route, induced high frequencies of antigen-specific CD8 T cells concurrent with significant reduction in the immunosuppressive regulatory T cells and myeloid derived suppressor cells in the tumor microenvironment (TME), correlating with sustained elimination of established HPV genital tumors in over 85% of mice. Inclusion of both the adjuvants in the vaccine was necessary for significant increase of antigen-specific CD8 T cells to the tumor and antitumor efficacy because vaccination incorporating either adjuvant alone was inefficient. These results strongly support the utility of the TVAC administered by needle-free intranasal route as a safe and effective strategy for the treatment of established genital HPV tumors.

Targeting interferon signaling and CTLA-4 enhance the therapeutic efficacy of anti-PD-1 immunotherapy in preclinical model of HPV+ oral cancer.

BACKGROUND: The US is experiencing an epidemic of HPV+ oropharyngeal cancers (OPC), the rates and burden of which now exceed that for cervical cancer. Immunotherapy targeting programmed death 1 (PD-1) on tumor-infiltrating lymphocytes and/or its ligand PD-L1 on tumor cells, which was effective in several cancers has however, showed efficacy in only less than 15% of patients. METHODS: We used a preclinical HPV+ oral tumor model, mEER, consisting of mouse tonsil derived epithelial cells expressing HPV-16 E6 and E7 genes, along with the H-ras oncogene to test strategies for enhancing the efficacy of anti-PD-1 therapy. RESULTS: Monotherapy with PD-1 blocking antibody was ineffective against flank-implanted tumors, but induced regression in 54% of mice bearing orthotopic tongue tumors that correlated with higher CD8 T cell responses. Since the CD8+ T cells derived from tongue tumors also showed high levels of the immune checkpoint inhibitory receptor CTLA-4, we tested combination immunotherapy targeting both CTLA-4 and PD-1 together and observed 93.3% survival of mice bearing tumors in the tongue for the duration of our 100-day study. Protective immunity correlated with a significant decrease in immunosuppressive lymphoid and myeloid populations within the tumor microenvironment. Consistent with the reported capacity of interferon-driven PD-L1/PD-1 pathway induction to serve as a biomarker of response to PD-1 blockade, we observed elevated interferon signaling and significantly higher levels of PD-1/PD-L1 in tongue-implanted mEER tumors compared to those growing on the flank correlating with their preferential responsiveness to PD-1 blockade. More importantly, in a pseudometastasic mouse model bearing both flank and tongue tumors to represent metastatic disease, delivery of Stimulator of Interferon Induced Genes (STING) agonist into the flank tumors combined with systemic treatment with α-PD-1 and α-CTLA-4 antibodies resulted in sustained tumor regression in 71% of mice. In this case, productive abscopal anti-tumor immunity was associated with robust increases in the ratios of cytotoxic CD8+ T cells (CTL) versus regulatory T cells (Treg) and versus functional myeloid-derived suppressor cells (MDSC). CONCLUSIONS: These results support combining α-PD-1 therapy with induction of IFN-α/ß signaling via provision of STING agonist and/or through CTLA-4 blockade as potential treatment option for HNSCC patients, especially, those not responding to α-PD-1 monotherapy.

Mucosal HPV E6/E7 Peptide Vaccination in Combination with Immune Checkpoint Modulation Induces Regression of HPV+ Oral Cancers.

High-risk human papillomavirus (HPV)-associated squamous cell carcinomas of the oropharynx (SCCOP) are among the fastest growing cancers. After standard-of-care treatment, however, patients with HPV+ SCCOP have better overall and disease-specific survival than patients with HPV- SCCOP, suggesting the importance of HPV-specific immunity. We reasoned that therapeutic vaccination targeting the HPV-16 E6 and E7 oncogenes could elicit high-affinity, high-frequency tumor antigen-specific T-cell responses, which could then be augmented and shielded from suppression in the tumor microenvironment by immune checkpoint modulation. In this study, we used a preclinical syngeneic mouse model of oral cancer comprised of mouse tonsil-derived epithelial cells stably expressing HPV-16 E6 and E7 genes along with H-ras oncogene (mEER) to identify combinations of vaccination and checkpoint antibodies capable of promoting tumor regression. Intranasal HPV E6/E7 peptide vaccination and single checkpoint antibodies failed to elicit responses in more than half of animals; however, 4-1BB agonist antibody along with either CD40 agonist antibody or CTLA-4 blockade eliminated the majority of established mEER tumors. The combination of intranasal HPV peptide vaccine and α4-1BB and αCTLA-4 antibodies produced curative efficacy and a better safety profile against orally implanted mEER tumors. Correlates of protective immunity included enhanced intratumoral levels of CD8 T cells relative to immunosuppressive regulatory T cells and myeloid-derived suppressor cells. Overall, our results demonstrate combination vaccine-immunotherapy modalities as novel treatment options for HPV+ SCCOP.Significance: Combinations of vaccine and checkpoint modulation are effective and safe treatment options for HPV+ oral cancers. Cancer Res; 78(18); 5327-39. ©2018 AACR.

Kinetics of Intratumoral Immune Cell Activation During Chemoradiation for Cervical Cancer.

PURPOSE: Radiation therapy has direct cytotoxic effects on tumor-infiltrating lymphocytes, but it also has immune stimulatory effects that increase immune cell infiltration. The dynamics of these competing effects on immune cells at the site of the tumor are poorly characterized during chemoradiation treatment (CRT) because of the difficulty of obtaining consecutive tumor biopsies. We used a minimally invasive cervical cytobrushing method to analyze the kinetics of intratumoral immune cell changes in patients with cervical cancer during CRT. METHODS AND MATERIALS: Cervical brushings were obtained from 20 patients with cervical cancer at baseline and during fractionated radiation therapy and cisplatin (weeks 1, 3, and 5). Matching peripheral blood mononuclear cells were obtained from 9 patients at the same time points. Cells were analyzed using multispectral flow cytometry to identify T cell and myeloid cell subsets and their activation status. Changes in immune cell subsets throughout treatment were calculated using matched-pair analysis with Wilcoxon rank sum test. RESULTS: We observed a significant decline in CD3+ total T cells, as well as CD8+ and CD4+ T-cell subsets in the first week of treatment from baseline, followed by variable expansion at weeks 3 and 5. This coincided with higher levels of proliferating CD8+ T cells expressing Ki67 at week 3 of treatment. The percentages of activated CD8+ T cells expressing CD69 continuously increased over the course of treatment, whereas the percentage of activated CD11c+CD11b- dendritic cells was highest during the first week. Many of these changes were not observed in the blood. CONCLUSIONS: Our results identified immune dynamic changes during CRT, indicating that CRT may be immune activating at the site of the tumor. This study also suggests the importance of sequential analyses of the local tumor microenvironment in addition to peripheral blood.