The current COVID-19 pandemic has claimed hundreds of thousands of lives and its causative agent, SARS-CoV-2, has infected millions, globally. The highly contagious nature of this respiratory virus has spurred massive global efforts to develop vaccines at record speeds. In addition to enhanced immunogen delivery, adjuvants may greatly impact protective efficacy of a SARS-CoV-2 vaccine. To investigate adjuvant suitability, we formulated protein subunit vaccines consisting of the recombinant S1 domain of SARS-CoV-2 Spike protein alone or in combination with either CoVaccine HT™ or Alhydrogel. CoVaccine HT™ induced high titres of antigen-binding IgG after a single dose, facilitated affinity maturation and class switching to a greater extent than Alhydrogel and elicited potent cell-mediated immunity as well as virus neutralizing antibody titres. Data presented here suggests that adjuvantation with CoVaccine HT™ can rapidly induce a comprehensive and protective immune response to SARS-CoV-2.

Filoviruses such as Ebola virus (EBOV) cause outbreaks of viral hemorrhagic fevers for which no FDA-approved vaccines or drugs are available. The 2014–2016 EBOV outbreak in West Africa infected approximately 30,000 people, killing more than 11,000 and affecting thousands more in areas still suffering from the effects of civil wars. Sierra Leone and Liberia reported EBOV cases in every county demonstrating the efficient spread of this highly contagious virus in the well-connected societies of West Africa. In communities, canines are often in contact with people while scavenging for food, which may include sickly bush animals or, as reported from the outbreak, EBOV infected human bodies and excrement. Therefore, dogs may serve as sentinel animals for seroprevalence studies of emerging infectious viruses. Further, due to their proximity to humans, they may have important One Health implications while offering specimens, which may be easier to obtain than human serum samples. Previous reports on detecting EBOV exposure in canines have been limited. Herein we describe a pilot project to detect IgG-responses directed against multiple filovirus and Lassa virus (LASV) antigens in dogs from EBOV affected communities in Liberia. We used a multiplex Luminex-based microsphere immunoassay (MIA) to detect dog IgG binding to recombinant filovirus antigens or LASV glycoprotein (GP) in serum from dogs that were old enough to be present during the EBOV outbreak. We identified 47 (73%) of 64 dog serum samples as potentially exposed to filoviruses and up to 100% of the dogs from some communities were found to have elevated levels of EBOV antigen-binding IgG titers. The multiplex MIA described in this study provides evidence for EBOV IgG antibodies present in dogs potentially exposed to the virus during the 2014–16 outbreak in Liberia. These data support the feasibility of canines as EBOV sentinels and provides evidence that seroprevalence studies in dogs can be conducted using suitable assays even under challenging field conditions. Further studies are warranted to collect data and to define the role canines may play in transmission or detection of emerging infectious diseases.

Galectin-9 has emerged as a promising biological target for cancer immunotherapy due to its role as a regulator of macrophage and T-cell differentiation. In addition, its expression in tumor cells modulates tumor cell adhesion, metastasis, and apoptosis. Malignant mesothelioma (MM) is an aggressive neoplasm of the mesothelial cells lining the pleural and peritoneal cavities, and in this study, we found that both human MM tissues and mouse MM cells express high levels of galectin-9. Using a novel monoclonal antibody (mAb) (Clone P4D2) that binds the C-terminal carbohydrate recognition domain (CRD) of galectin-9, we demonstrate unique agonistic properties resulting in MM cell apoptosis. Furthermore, the P4D2 mAb reduced tumor-associated macrophages differentiation toward a protumor phenotype. Importantly, these effects exerted by the P4D2 mAb were observed in both human and mouse in vitro experiments and not observed with another antigalectin-9 specific mAb (clone P1D9) that engages the N-terminus CRD of galectin-9. In syngeneic murine models of MM, P4D2 mAb treatment inhibited tumor growth and improved survival, with tumors from P4D2-treated mice exhibited reduced infiltration of tumor-associated M2 macrophages. This was consistent with an increased production of inducible nitric oxide synthase, which is a major enzyme-regulating macrophage inflammatory response to cancer. These data suggest that using an antigalectin 9 mAb with agonistic properties similar to those exerted by galectin-9 may provide a novel multitargeted strategy for the treatment of mesothelioma and possibly other galectin-9 expressing tumors. ARTICLE HISTORY

The current COVID-19 pandemic has claimed hundreds of thousands of lives and its causative agent, SARS-CoV-2, has infected millions, globally. The highly contagious nature of this respiratory virus has spurred massive global efforts to develop vaccines at record speeds. In addition to enhanced immunogen delivery, adjuvants may greatly impact protective efficacy of a SARS-CoV-2 vaccine. To investigate adjuvant suitability, we formulated protein subunit vaccines consisting of the recombinant S1 domain of SARS-CoV-2 Spike protein alone or in combination with either CoVaccine HT™ or Alhydrogel. CoVaccine HT™ induced high titres of antigen-binding IgG after a single dose, facilitated affinity maturation and class switching to a greater extent than Alhydrogel and elicited potent cell-mediated immunity as well as virus neutralising antibody titres. Data presented here suggests that adjuvantation with CoVaccine HT™ can rapidly induce a comprehensive and protective immune response to SARS-CoV-2.

Cutaneous melanoma is a highly invasive skin cancer. Mortality for melanoma is low if caught before metastasis occurs, but is poor if metastasis has occurred at diagnosis. Melanomas are among the most mutated cancer genomes and have a high neoantigen load, which is related to better response to immunotherapies. A subset of melanomas contains mutations in BRAF(V600E) and respond initially to BRAF inhibitors, though they become resistant through escape pathways. Focused development of BRAF and immunotherapies has resulted in the clinical use of targeted therapies against the oncogenic BRAF/MEK/ERK pathway and immune checkpoint inhibitors for the treatment of metastatic melanoma. Although some patients with metastatic melanoma benefit from these therapies, others either do not respond or become resistant. Because of this, new targets and drugs are needed for patients who are either intrinsically resistant or have acquired resistance to targeted therapies and immunotherapies. RSKs are serine/threonine kinases downstream of BRAF/ERK that activate transcription, proliferation and cell survival. Thus RSKs are potential new targets and the availability of a RSK inhibitor called PMD-026, now in Phase 1b clinical trials for breast cancer, provides a tool to explore this possibility in pre-clinical models of melanoma. PMD-026 is an ATP-competitive inhibitor of the N-terminal kinase domain. This inhibitor is highly stable and effective in cell-based assays with an average IC50 of 2nM and 200-400 nM in soft agar and inhibits tumor growth in multiple mouse models of breast cancer given 70-100 mg/kg given orally. It is currently the only RSK inhibitor that can be given to mice orally with demonstrated in vivo activity. To explore the opportunity for RSK inhibitors in melanoma, we screened 1,959 PDx models for RSK1-4 expression, which was evaluated based on RNAseq. In melanoma, RSK1 and RSK2 were highly expressed, unlike RSK3 and RSK4. In a study of 21 cases of melanoma, RSK2 was activated in 52% of the cases based on nuclear localization using our companion diagnostic. Using FRET based assays, PMD-026 inhibited RSK2 binding with an IC50=14 nM compared to BID1870 with IC50=94 nM demonstrating competitive in vitro potency. Importantly, PMD-026 inhibits viability of melanoma cells including those that are resistant to the BRAF inhibitor. Surprisingly, we also found that it significantly reduces PD-L1 expression at the cell surface of several melanoma lines. This is through down-regulation of PD-L1 transcription and subsequent reduced PD-L1 cell surface expression. As expected, in our melanoma models, PMD-026 inhibited RSK signaling through pYB-1, a transcription factor known to bind to the PD-L1 promoter. Testing in pre-clinical mouse models is in progress. We propose that PMD-026 may therefore potentiate melanoma immunotherapy approaches and also improve response of patients to BRAFV600E targeted therapies. Citation Format: Joe William Ramos, Stephanie Si, Takeo Fujii, Brien Haun, Won Seok Yang, Aarthi Jayanthan, Sandra E. Dunn. Targeting B-RAF and checkpoint inhibitor resistant melanoma with RSK inhibitor PMD-026 in pre-clinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1218.