Mammalian orthoreovirus (reovirus) is under development as a cancer virotherapy. Clinical trials demonstrate that reovirus-based therapies are safe and tolerated in patients with a wide variety of cancers. Although reovirus monotherapy has proven largely ineffective, reovirus sensitizes cancer cells to existing chemotherapeutic agents and radiation. Clinical trials are underway to test the efficacy of reovirus in combination with chemotherapeutic and radiation regimens and to evaluate the effectiveness of reovirus in conjunction with immunotherapies. Central to the use of reovirus to treat cancer is its capacity to directly kill cancer cells and alter the cellular environment to augment other therapies. Apoptotic cell death is a prominent mechanism of reovirus cancer cell killing. However, reoviruses can also kill cancer cells through nonapoptotic mechanisms. Here, we describe mechanisms of reovirus cancer cell killing, highlight how reovirus is used in combination with existing cancer treatments, and discuss what is known as to how reovirus modulates cancer immunotherapy.

Breast cancer is the second leading cause of cancer-related deaths in women in the United States. Triple-negative breast cancer constitutes a subset of breast cancer that is associated with higher rates of relapse, decreased survival, and limited therapeutic options for patients afflicted with this type of breast cancer. Mammalian orthoreovirus (reovirus) selectively infects and kills transformed cells, and a serotype 3 reovirus is in clinical trials to assess its efficacy as an oncolytic agent against several cancers. It is unclear if reovirus serotypes differentially infect and kill triple-negative breast cancer cells and if reovirus-induced cytotoxicity of breast cancer cells can be enhanced by modulating the activity of host molecules and pathways. Here, we generated reassortant reoviruses by forward genetics with enhanced infective and cytotoxic properties in triple-negative breast cancer cells. From a high-throughput screen of small-molecule inhibitors, we identified topoisomerase inhibitors as a class of drugs that enhance reovirus infectivity and cytotoxicity of triple-negative breast cancer cells. Treatment of triple-negative breast cancer cells with topoisomerase inhibitors activates DNA damage response pathways, and reovirus infection induces robust production of type III, but not type I, interferon (IFN). Although type I and type III IFNs can activate STAT1 and STAT2, triple-negative breast cancer cellular proliferation is only negatively affected by type I IFN. Together, these data show that reassortant viruses with a novel genetic composition generated by forward genetics in combination with topoisomerase inhibitors more efficiently infect and kill triple-negative breast cancer cells. IMPORTANCE Patients afflicted by triple-negative breast cancer have decreased survival and limited therapeutic options. Reovirus infection results in cell death of a variety of cancers, but it is unknown if different reovirus types lead to triple-negative breast cancer cell death. In this study, we generated two novel reoviruses that more efficiently infect and kill triple-negative breast cancer cells. We show that infection in the presence of DNA-damaging agents enhances infection and triple-negative breast cancer cell killing by reovirus. These data suggest that a combination of a genetically engineered oncolytic reovirus and topoisomerase inhibitors may provide a potent therapeutic option for patients afflicted with triple-negative breast cancer.

Several approaches have produced an effective vaccine against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Since millions of people are exposed to influenza virus and SARS-CoV-2, it is of great interest to develop a two-in-one vaccine that will be able to protect against infection of both viruses. We have developed a hybrid vaccine for SARS-CoV-2 and influenza viruses using influenza virus-like particles (VLP) incorporated by protein transfer with glycosylphosphatidylinositol (GPI)-anchored SARS-CoV-2 RBD fused to GM-CSF as an adjuvant. GPI-RBD-GM-CSF fusion protein was expressed in CHO-S cells, purified and incorporated onto influenza VLPs to develop the hybrid vaccine. Our results show that the hybrid vaccine induced a strong antibody response and protected mice from both influenza virus and mouse-adapted SARS-CoV-2 challenges, with vaccinated mice having significantly lower lung viral titers compared to naive mice. These results suggest that a hybrid vaccine strategy is a promising approach for developing multivalent vaccines to prevent influenza A and SARS-CoV-2 infections.

Breast cancer is the second leading cause of cancer-related deaths in women in the United States. The triple-negative breast cancer (TNBC) subtype associates with higher rates of relapse, shorter overall survival, and aggressive metastatic disease. Hormone therapy is ineffective against TNBC, leaving patients with limited therapeutic options. Mammalian orthoreovirus (reovirus) preferentially infects and kills transformed cells, and a genetically engineered reassortant reovirus infects and kills TNBC cells more efficiently than prototypical strains. Reovirus oncolytic efficacy is further augmented by combination with topoisomerase inhibitors, including the frontline chemotherapeutic doxorubicin. However, long-term doxorubicin use correlates with toxicity to healthy tissues. Here, we conjugated doxorubicin to reovirus (reo-dox) to control drug delivery and enhance reovirus-mediated oncolysis. Our data indicate that conjugation does not impair viral biology and enhances reovirus oncolytic capacity in TNBC cells. Reo-dox infection promotes innate immune activation, and crosslinked doxorubicin retains DNA-damaging properties within infected cells. Importantly, reovirus and reo-dox significantly reduce primary TNBC tumor burden in vivo , with greater reduction in metastatic burden after reo-dox inoculation. Together, these data demonstrate that crosslinking chemotherapeutic agents to oncolytic viruses facilitates functional drug delivery to cells targeted by the virus, making it a viable approach for combination therapy against TNBC.

20Breast cancer is the second-leading cause of cancer-related deaths in women in the 21 United States. Triple-negative breast cancer constitutes a subset of breast cancer that is 22 Importance 38Patients afflicted by triple-negative breast cancer have decreased survival and limited 39 therapeutic options. Reovirus infection results in cell death of a variety of cancers, but it is 40 unknown if different reovirus types lead to triple-negative breast cancer cell death. In this study, 41 we generated two novel reoviruses that more efficiently infect and kill triple-negative breast 42 cancer cells. We show that infection in the presence of DNA-damaging agents enhances 43 infection and triple-negative breast cancer cell killing by reovirus. These data suggest that a 44 combination of a genetically engineered oncolytic reovirus and topoisomerase inhibitors may 45 provide a potent therapeutic option for patients afflicted with triple-negative breast cancer. 46 47Breast cancer is the leading cause of cancer and second leading cause of deaths by cancer 48 in women in the United States (1). Triple-negative breast cancer (TNBC) constitutes 49 approximately 15% of breast cancers, has a higher rate of relapse, and shorter overall survival 50 after metastasis than other subtypes of breast cancer (2). TNBC more frequently affects the 51 young, is more prevalent in African American women, and tumors are larger in size and 52 biologically more aggressive (3). TNBC is characterized by the lack of expression of estrogen 53 receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 54 (HER2/neu) and can be classified into seven subtypes based on their genetic signature (3). 55