Oncolytic herpes simplex viruses designed for targeted treatment of EGFR-bearing tumors.

Oncolytic herpes simplex viruses (oHSVs) have emerged as leading cancer therapeutic agents. Effective oHSV virotherapy may ultimately require both intratumoral and systemic vector administration to target the primary tumor and distant metastases. An attractive approach to enhancing oHSV tumor specificity is engineering the virus envelope glycoproteins for selective recognition of and infection via tumor-specific cell surface proteins. We previously demonstrated that oHSVs could be retargeted to EGFR-expressing cells by the incorporation of a single-chain antibody (scFv) at the N terminus of glycoprotein D (gD). Here, we compared retargeted oHSVs generated by the insertion of scFv, affibody molecule, or VHH antibody ligands at different positions within the N terminus of gD. When compared to the scFv-directed oHSVs, VHH and affibody molecules mediated enhanced EGFR-specific tumor cell entry, spread and cell killing in vitro, and enabled long-term tumor-specific virus replication following intravenous delivery in vivo. Moreover, oHSVs retargeted via a VHH ligand reduced tumor growth upon intravenous injection and achieved complete tumor destruction after intratumoral injection. Systemic oHSV delivery is important for the treatment of metastatic disease, and our enhancements in targeted oHSV design are a critical step in creating an effective tumor-specific oHSVs for safe administration via the bloodstream.

Novel mutations in UL24 and gH rescue efficient infection of an HSV vector retargeted to TrkA.

Transductional targeting of herpes simplex virus (HSV)-based gene therapy vectors offers the potential for improved tissue-specific delivery and can be achieved by modification of the viral entry machinery to incorporate ligands that bind the desired cell surface proteins. The interaction of nerve growth factor (NGF) with tropomyosin receptor kinase A (TrkA) is essential for survival of sensory neurons during development and is involved in chronic pain signaling. We targeted HSV infection to TrkA-bearing cells by replacing the signal peptide and HVEM binding domain of glycoprotein D (gD) with pre-pro-NGF. This TrkA-targeted virus (KNGF) infected cells via both nectin-1 and TrkA. However, infection through TrkA was inefficient, prompting a genetic search for KNGF mutants showing enhanced infection following repeat passage on TrkA-expressing cells. These studies revealed unique point mutations in envelope glycoprotein gH and in UL24, a factor absent from mature particles. Together these mutations rescued efficient infection of TrkA-expressing cells, including neurons, and facilitated the production of a completely retargeted KNGF derivative. These studies provide insight into HSV vector improvements that will allow production of replication-defective TrkA-targeted HSV for delivery to the peripheral nervous system and may be applied to other retargeted vector studies in the central nervous system.

Evolution of the SARS-CoV-2 proteome in three dimensions (3D) during the first 6 months of the COVID-19 pandemic.

Understanding the molecular evolution of the SARS-CoV-2 virus as it continues to spread in communities around the globe is important for mitigation and future pandemic preparedness. Three-dimensional structures of SARS-CoV-2 proteins and those of other coronavirusess archived in the Protein Data Bank were used to analyze viral proteome evolution during the first 6 months of the COVID-19 pandemic. Analyses of spatial locations, chemical properties, and structural and energetic impacts of the observed amino acid changes in >48 000 viral isolates revealed how each one of 29 viral proteins have undergone amino acid changes. Catalytic residues in active sites and binding residues in protein-protein interfaces showed modest, but significant, numbers of substitutions, highlighting the mutational robustness of the viral proteome. Energetics calculations showed that the impact of substitutions on the thermodynamic stability of the proteome follows a universal bi-Gaussian distribution. Detailed results are presented for potential drug discovery targets and the four structural proteins that comprise the virion, highlighting substitutions with the potential to impact protein structure, enzyme activity, and protein-protein and protein-nucleic acid interfaces. Characterizing the evolution of the virus in three dimensions provides testable insights into viral protein function and should aid in structure-based drug discovery efforts as well as the prospective identification of amino acid substitutions with potential for drug resistance.

Undergraduate structural biology education: A shift from users to developers of computation and simulation tools.

The use of theory and simulation in undergraduate education in biochemistry, molecular biology, and structural biology is now common, but the skills students need and the curriculum instructors have to train their students are evolving. The global pandemic and the immediate switch to remote instruction forced instructors to reconsider how they can use computation to teach concepts previously approached with other instructional methods. In this review, we survey some of the curricula, materials, and resources for instructors who want to include theory, simulation, and computation in the undergraduate curriculum. There has been a notable progression from teaching students to use discipline-specific computational tools to developing interactive computational tools that promote active learning to having students write code themselves, such that they view computation as another tool for solving problems. We are moving toward a future where computational skills, including programming, data analysis, visualization, and simulation, will no longer be considered an optional bonus for students but a required skill for the 21st century STEM (Science, Technology, Engineering, and Mathematics) workforce; therefore, all physical and life science students should learn to program in the undergraduate curriculum.

Treatment of glioblastoma with current oHSV variants reveals differences in efficacy and immune cell recruitment.

Oncolytic herpes simplex viruses (oHSVs) have demonstrated efficient lytic replication in human glioblastoma tumors using immunodeficient mouse models, but early-phase clinical trials have reported few complete responses. Potential reasons for the lack of efficacy are limited vector potency and the suppressive glioma tumor microenvironment (TME). Here we compare the oncolytic activity of two HSV-1 vectors, a KOS-strain derivative KG4:T124 and an F-strain derivative rQNestin34.5v.1, in the CT2A and GL261N4 murine syngeneic glioma models. rQNestin34.5v1 generally demonstrated a greater in vivo viral burden compared to KG4:T124. However, both vectors were rapidly cleared from CT2A tumors, while virus remained ensconced in GL261N4 tumors. Immunological evaluation revealed that the two vectors induced similar changes in immune cell recruitment to either tumor type at 2 days after infection. However, at 7 days after infection, the CT2A microenvironment displayed the phenotype of an untreated tumor, while GL261N4 tumors exhibited macrophage and CD4+/CD8+ T cell accumulation. Furthermore, the CT2A model was completely resistant to virus therapy, while in the GL261N4 model rQNestin34.5v1 treatment resulted in enhanced macrophage recruitment, impaired tumor progression, and long-term survival of a few animals. We conclude that prolonged intratumoral viral presence correlates with immune cell recruitment, and both are needed to enhance anti-tumor immunity.

Evolution of the SARS-CoV-2 proteome in three dimensions (3D) during the first six months of the COVID-19 pandemic.

Three-dimensional structures of SARS-CoV-2 and other coronaviral proteins archived in the Protein Data Bank were used to analyze viral proteome evolution during the first six months of the COVID-19 pandemic. Analyses of spatial locations, chemical properties, and structural and energetic impacts of the observed amino acid changes in >48,000 viral proteome sequences showed how each one of the 29 viral study proteins have undergone amino acid changes. Structural models computed for every unique sequence variant revealed that most substitutions map to protein surfaces and boundary layers with a minority affecting hydrophobic cores. Conservative changes were observed more frequently in cores versus boundary layers/surfaces. Active sites and protein-protein interfaces showed modest numbers of substitutions. Energetics calculations showed that the impact of substitutions on the thermodynamic stability of the proteome follows a universal bi-Gaussian distribution. Detailed results are presented for six drug discovery targets and four structural proteins comprising the virion, highlighting substitutions with the potential to impact protein structure, enzyme activity, and functional interfaces. Characterizing the evolution of the virus in three dimensions provides testable insights into viral protein function and should aid in structure-based drug discovery efforts as well as the prospective identification of amino acid substitutions with potential for drug resistance.

Generation of an Oncolytic Herpes Simplex Viral Vector Completely Retargeted to the GDNF Receptor GFRα1 for Specific Infection of Breast Cancer Cells.

Oncolytic herpes simplex viruses (oHSV) are under development for the treatment of a variety of human cancers, including breast cancer, a leading cause of cancer mortality among women worldwide. Here we report the design of a fully retargeted oHSV for preferential infection of breast cancer cells through virus recognition of GFRα1, the cellular receptor for glial cell-derived neurotrophic factor (GDNF). GFRα1 displays a limited expression profile in normal adult tissue, but is upregulated in a subset of breast cancers. We generated a recombinant HSV expressing a completely retargeted glycoprotein D (gD), the viral attachment/entry protein, that incorporates pre-pro-GDNF in place of the signal peptide and HVEM binding domain of gD and contains a deletion of amino acid 38 to eliminate nectin-1 binding. We show that GFRα1 is necessary and sufficient for infection by the purified recombinant virus. Moreover, this virus enters and spreads in GFRα1-positive breast cancer cells in vitro and caused tumor regression upon intratumoral injection in vivo. Given the heterogeneity observed between and within individual breast cancers at the molecular level, these results expand our ability to deliver oHSV to specific tumors and suggest opportunities to enhance drug or viral treatments aimed at other receptors.

Point Mutations in Retargeted gD Eliminate the Sensitivity of EGFR/EGFRvIII-Targeted HSV to Key Neutralizing Antibodies.

Effective oncolytic virotherapy may require systemic delivery, tumor targeting, and resistance to virus-neutralizing (VN) antibodies. Since herpes simplex virus (HSV) glycoprotein D (gD) is the viral attachment/entry protein and predominant VN target, we examined the impact of gD retargeting alone and in combination with alterations in dominant VN epitopes on virus susceptibility to VN antibodies. We compared the binding of a panel of anti-gD monoclonal antibodies (mAbs) that mimic antibody specificities in human HSV-immune sera to the purified ectodomains of wild-type and retargeted gD, revealing the retention of two prominent epitopes. Substitution of a key residue in each epitope, separately and together, revealed that both substitutions (1) blocked retargeted gD recognition by mAbs to the respective epitopes, and, in combination, caused a global reduction in mAb binding; (2) protected against fusion inhibition by VN mAbs reactive with each epitope in virus-free cell-cell fusion assays; and (3) increased the resistance of retargeted HSV-1 to these VN mAbs. Although the combined modifications of retargeted gD allowed bona fide retargeting, incorporation into virions was partially compromised. Our results indicate that stacking of epitope mutations can additively block retargeted gD recognition by VN antibodies but also that improvements in gD incorporation into virus particles may be required.