Bovine Herpes Virus Type 1 Oncolytic Therapy Elicits Direct Cytotoxic Effects and Immune Microenvironment Reshaping in Multiple Myeloma
Authors: Raimondi Vincenzo , Storti Paola , Vescovini Rosanna , Franceschi Valentina , Pozzi Giulia , Toscani Denise , Iannozzi Nicolas Thomas , Lungu Oxana , Dalla Palma Anna Benedetta , Pelagatti Laura , Librale Federica , Dessena Mattia , Ricci Stefania , Mirandola Prisco , Donofrio Gaetano , Giuliani Nicola 
Published: December 2024

Multiple myeloma (MM) is a plasma cell (PC) malignancy characterized by a high tendency to relapse and develop drug resistance. In this context, immune dysregulation is a defining characteristic of MM, with interactions between malignant PCs and immune cells within the bone marrow (BM) microenvironment playing a crucial role in creating a tumor-promoting environment. Recently, therapeutic strategies have shown promise to overcome the immunosuppressive tumor microenvironment and activate the host immune system.

Oncolytic viruses (OVs) are an emerging class of immunotherapy, exploiting tumor-specific oncolysis and generating a robust anti-tumor immune response. Several OVs have demonstrated efficacy in preclinical MM studies, with some undergoing early clinical trials. However, many of these viruses are of human origin, limiting their use due to pre-existing humoral immunity and/or potential pathogenicity. To overcome these issues, non-human OVs that are not pathogenic to humans are being considered. To date, it is known that Bovine Herpes Virus Type 1 (BoHV-1) has antitumor activity in several solid tumors, but its efficacy in hematological malignancies is unknown. Indeed, this study aims to explore the BoHV-1 direct oncolytic effect on MM cells and the putative indirect stimulation of the immune cells in an ex vivo setting.

Firstly, by flow cytometry, we confirmed that human myeloma cell lines (HMCLs), JJN3 and MM1.S expressed high levels of the putative cellular receptors for BoHV-1 entry (CD111, CD155, and CD138). Moreover, the direct oncolytic effect of BoHV-1 was assessed on the same HMCLs after 24, 48, and 72h of treatment at 1 and 2 multiplicity of infection (MOI). Flow cytometry analysis on HMCLs treated with BoHV-1 showed a significant increase in cell mortality, as a percentage of 7-AAD+ cells, and in the expression of Apo2.7, an apoptotic marker, after 48 and 72h. Additionally, it was found that the BoHV-1 treatment of HMCLs led to caspase-3 activation by western blot analysis. Next, we conducted an ex vivo study to investigate the effect of BoHV-1 treatment on BM mononuclear cells (MNCs) from a cohort of newly diagnosed (N=15) and relapsed/refractory MM patients (N=7). The preliminary analysis of BoHV-1 entry receptors on primary MM PCs confirmed their expression by all patients' PCs, although with notable heterogeneity in expression level. In parallel, the patients' BM MNCs were treated with BoHV-1 at 1 and 2 MOI for 48, 72, and 96h. At each time point, flow cytometry analysis showed a significant decrease in viable CD138+CD38+ MM PCs among virus-treated compared with untreated BM MNCs.

Secondly, the role of immune effector cells in enhancing the efficacy of BoHV-1 oncolytic therapy in MM cells was evaluated. In vitro, JJN3 cells were pre-treated with BoHV-1 for 24h, after virus removal, a 4h cytotoxicity assay was performed in a co-culture with NK-92, a stabilized human natural killer cell line. We found that BoHV-1 pre-treatment markedly enhanced the cytotoxicity of NK-92 cells against JJN3. Moreover, ex vivo, we investigated how the BoHV-1 treatment impacts the immune microenvironment cells. Flow cytometry analysis showed that BoHV-1-treated BM MNCs exhibited a significant increase in the expression of CD107a, a degranulation marker, on NK cells and a significant upregulation of CD69 expression on NK, CD8+ T cells, and monocytes. Interestingly, BoHV-1 treatment induced a switch in monocyte polarization from an immunosuppressive M2 phenotype (CD14+CD16+) to a pro-inflammatory and anti-tumoral M1 phenotype (CD14+CD16-). Furthermore, throughout the BoHV-1 treatment, there was a significant upregulation of CD38 expression, observed not only in all main immune cell subsets but also in MM PCs. Based on this finding, we evaluated the efficacy of the CD38-targeted monoclonal antibody, daratumumab, on patients' BM MNCs pre-treated for 48h with the virus. The results demonstrated that the combination of BoHV-1 treatment and daratumumab led to a statistically significant reduction in the MM PCs viability compared to the effects of either treatment alone.

Overall, our data highlight that BoHV-1 not only exerts direct cytotoxic effects on MM cells but also reshapes the immune microenvironment modulating the surrounding immune landscape. These findings suggest its potential as a novel alternative anti-MM virotherapy strategy, either alone or in combination with immunotherapeutic drugs.