Murine cytomegalovirus (MCMV) immediate-early protein 3 (IE3) is essential for successful viral infection. This study developed MCMVs with an EGFP-fused IE3 gene in order to study IE3 gene expression, subnuclear distribution and biological function, as well as to examine the interaction of IE3 with cellular and viral proteins. The generated viruses included MCMVIE3gfp, in which IE1 was completely removed by the in-frame fusion of exons 3 and 5 and the C terminus of IE3 was tagged with EGFP, and MCMVIE1/3gfp, in which IE1 was kept intact and EGFP was also fused to the C terminus of IE3. Unlike human CMV (HCMV), whose growth was significantly reduced when IE2 (the HCMV homologue of IE3 in MCMV) was tagged with EGFP, MCMVs with IE3–EGFP presented an unchanged replication profile. Using these new constructs, the distribution of IE3 was revealed as well as its interaction with viral and cellular proteins, especially proteins pertaining to DNA replication (M44 and E1) and cellular intrinsic defence [promyelocytic leukemia protein and histone deacetylases (HDACs)]. It was also shown that IE3 domains co-localize with DNA replication domains, and IE3 attracted other required proteins into IE3 domains via protein–protein interactions. In addition, IE3 was shown to interact with HDAC2 and to eliminate the inhibitory effect of HDAC2 on early viral gene production. Together, these results suggest that IE3 acts as a key protein for viral DNA replication by establishing pre-replication domains via recruitment of the required viral and cellular proteins, and by reducing host defences.

No abstract

Background-Kaposi's sarcoma-associated herpesvirus (KSHV; also known as Human herpesvirus 8) has been determined to be the most frequent cause of malignancies in AIDS patients. It is associated primarily with Kaposi's sarcoma (KS) and primary effusion lymphoma (PEL), as well as with multicentric Castleman's disease (2). The switch from the latent to the lytic stage is important both in maintenance of malignancy and viral infection. So far, the mechanism of its reactivation has not been fully understood.

Immunotherapy is currently one of the primary focuses of cancer research and has the potential to become the standard treatment for oncologic patients over the next decades. Promising clinical responses have been observed in patients treated with a variety of immune-targeted therapies such as checkpoint inhibitors, modified dendritic cells, and T cell or NK cell-based immunotherapies. However, the efficacy of these treatments is still very limited due to multiple factors related to tumor nature and microenvironment. Hence, combination therapies aimed at modulating the strong tumor immunosuppressive microenvironment and stimulating T-cell infiltration into the tumor lesions are urgently needed to overcome cancer intrinsic resistance mechanisms. Preclinical and clinical studies have shown the efficacy of oncolytic virotherapy to elicit antitumor immune responses and to increase infiltration of both innate and adaptive immune cells. These capabilities of the oncolytic viruses can be enhanced and exploited to improve response of patients to different immunotherapies. Here we tested the ability of two modified oncolytic adenoviruses termed Delta-24-RGDOX and Delta-24-GREAT, expressing OX40L and GITRL respectively, to modify the tumor microenvironment in different murine triple-negative breast cancer models. We hypothesized that intratumoral injection of these armed oncolytic adenoviruses will decrease immunosuppression within the tumor microenvironment. To this end, mice harboring fat pad tumors were treated with intratumoral doses of these armed oncolytic adenoviruses. Leukocytes from spleen and tumor tissues were analyzed for the relative presence of lymphocyte and myeloid cell subsets. In all mice treated with Delta-24-RGDOX or Delta-24-GITRL, CD8+ T-cell populations were significantly higher compared with those observed in the control mice. Significantly lower percentages of cell populations with immunosuppressive phenotypes, such as PD-1+ T cells (P<0.0001, Student t-test), CD4+CD25+ Foxp3+ regulatory T cells (P<0.0001, Student t-test) and MDSC (P<0.0001, Student t-test) were found in the mice treated with the armed oncolytic adenoviruses. Taken together, our results confirmed the strong immunomodulatory effect of enhanced oncolytic viruses, which can be easily combined with immune-modulating checkpoints, for the treatment of deadliest human cancers. Citation Format: Francisco W. Puerta Martinez, Yisel A. Rivera, Teresa Nguyen, Xuejun Fan, Rehnuma Shifat, Mohammad Belayat Hossain, Hong Jiang, Chandra Bartholomeusz, Lang F. Frederick, Juan Fueyo, Candelaria Gomez-Manzano. Partial reversion of the tumor immunosuppressed environment by oncolytic adenoviruses armed with positive stimulators of the immune synapsis in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4053.

The extent to which immune cell phenotypes in the peripheral blood reflect intratumoral immune activity prior to and early in cancer immunotherapy is unclear. Here we studied the population dynamics of immune cells and immune phenotypic changes, using clinical blood samples and a multicolor flow cytometry analysis. These samples were serially obtained from a cohort of high-grade glioma canine patients enrolled in a trial with an immunotherapy that targets the CD200/CD200R immunological synapse. This therapy consists of an alternative checkpoint inhibitor peptide (CD200AR-L) that modulates the CD200 immune checkpoint, currently being used in an adult phase I safety trial for recurrent glioblastoma trial (NCT04642937) and a canine high-grade spontaneous glioma trial (U01CA224160-01). We found evidence of a strong effect of the therapy on peripheral immune cells in responder versus nonresponder dogs. Upon initiation of therapy, immune effector cell proliferation increased in responsive patients, whereas in nonresponders immune cell populations either didn’t change significantly or an increase in immunosuppressive phenotypes was observed. Cell populations such as MDSC, regulatory T cells and exhausted T cells were predominantly higher in nonresponder dogs. These data showed that peripheral immune cell phenotypes were linked to the outcome of the immunotherapy, thus dogs with better immune cell profiles, including increased cytotoxic differentiation, had a better prognosis and longer overall survival rates. Taken together, these results suggest that variation in patient responsiveness can be explained by differences in peripheral immune cell proliferation and differentiation soon after the initiation of immunotherapy. Citation Format: Francisco W. Puerta Martinez, Sathya Schnell, Susan Arnold, Elizabeth G. Pluhar, Michael Olin. Circulating immune biomarkers in peripheral blood reflect the effect of a CD200 immune checkpoint targeted immunotherapy in pet dogs with high-grade glioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1266.