Greater Durability and Protection against Herpes Simplex Viral Disease following Immunization of Mice with Single-Cycle ΔgD-2 Compared to an Adjuvanted Glycoprotein D Protein Vaccine.

Herpes simplex viruses (HSV) cause chronic infections with significant morbidity. Prior vaccines, designed to generate neutralizing antibodies (nAbs) targeting glycoprotein D (gD), failed to provide durable protection. We adopted a different strategy and evaluated a single-cycle virus deleted in gD (ΔgD-2). ΔgD-2elicits antibodies that primarily mediate antibody-dependent cell mediated cytolysis (ADCC) and provides complete protection against clinical isolates of HSV in multiple lethal mouse models. To assess durability, we vaccinated mice (2 doses administered intramuscularly) with ΔgD-2, adjuvanted recombinant gD-2 (rgD-2/Alum-MPL), or uninfected cells as a control, and quantified antibody responses over one year. Mice (n = 5/group) were lethally challenged at 2, 4, 6, 8, and 10-months post-boost. ΔgD-2-vaccinated mice elicited a durable ADCC-mediating response, which provided complete protection against challenge at all timepoints. In contrast, rgD-2/Alum-MPL elicited only nAbs, which declined significantly within 6 months, provided only partial protection at early timepoints, and no protection after 6 months. Serum sampling after viral challenge showed that infection elicited low levels of ADCC-mediating antibodies in rgD-2/Alum-MPL-vaccinated mice and boosted the nAb response, but only after 6 months. Conversely, infection significantly and consistently boosted both the ADCC and nAbs responses in ΔgD-2-vaccinated mice. Results recapitulate clinical trial outcomes with gD vaccines, highlight the importance of ADCC, and predict that ΔgD-2 will elicit durable responses in humans.

A targetable 'rogue' neutrophil-subset, [CD11b+DEspR+] immunotype, is associated with severity and mortality in acute respiratory distress syndrome (ARDS) and COVID-19-ARDS.

Neutrophil-mediated secondary tissue injury underlies acute respiratory distress syndrome (ARDS) and progression to multi-organ-failure (MOF) and death, processes linked to COVID-19-ARDS. This secondary tissue injury arises from dysregulated neutrophils and neutrophil extracellular traps (NETs) intended to kill pathogens, but instead cause cell-injury. Insufficiency of pleiotropic therapeutic approaches delineate the need for inhibitors of dysregulated neutrophil-subset(s) that induce subset-specific apoptosis critical for neutrophil function-shutdown. We hypothesized that neutrophils expressing the pro-survival dual endothelin-1/VEGF-signal peptide receptor, DEspR, are apoptosis-resistant like DEspR+ cancer-cells, hence comprise a consequential pathogenic neutrophil-subset in ARDS and COVID-19-ARDS. Here, we report the significant association of increased peripheral DEspR+CD11b+ neutrophil-counts with severity and mortality in ARDS and COVID-19-ARDS, and intravascular NET-formation, in contrast to DEspR[-] neutrophils. We detect DEspR+ neutrophils and monocytes in lung tissue patients in ARDS and COVID-19-ARDS, and increased neutrophil RNA-levels of DEspR ligands and modulators in COVID-19-ARDS scRNA-seq data-files. Unlike DEspR[-] neutrophils, DEspR+CD11b+ neutrophils exhibit delayed apoptosis, which is blocked by humanized anti-DEspR-IgG4S228P antibody, hu6g8, in ex vivo assays. Ex vivo live-cell imaging of Rhesus-derived DEspR+CD11b+ neutrophils showed hu6g8 target-engagement, internalization, and induction of apoptosis. Altogether, data identify DEspR+CD11b+ neutrophils as a targetable 'rogue' neutrophil-subset associated with severity and mortality in ARDS and COVID-19-ARDS.

Increased Neutrophil-Subset Associated With Severity/Mortality In ARDS And COVID19-ARDS Expresses The Dual Endothelin-1/VEGFsignal-Peptide Receptor (DEspR): An Actionable Therapeutic Target.

Neutrophil-mediated secondary tissue injury underlies acute respiratory distress syndrome (ARDS) and progression to multi-organ-failure (MOF) and death, processes linked to severe COVID19. This 'innocent bystander' tissue injury arises in dysregulated hyperinflammatory states from neutrophil functions and neutrophil extracellular traps (NETs) intended to kill pathogens, but injure cells instead, causing MOF. Insufficiency of prior therapeutic approaches suggest need to identify dysregulated neutrophil-subset(s) and induce subset-specific apoptosis critical for neutrophil function-shutdown and clearance. We hypothesized that neutrophils expressing the pro-survival dual endothelin-1/signal peptide receptor, DEspR, are apoptosis-resistant just like DEspR+ cancer cells, hence comprise a consequential pathogenic neutrophil-subset in ARDS and COVID19-ARDS. Here, we report correlation of circulating DEspR+CD11b+ activated neutrophils (DESpR+actNs) and NETosing-neutrophils with severity in ARDS and in COVID19-ARDS, increased DEspR+ neutrophils and monocytes in post-mortem ARDS-patient lung sections, and neutrophil DEspR/ET1 receptor/ligand autocrine loops in severe COVID19. Unlike DEspR[-] neutrophils, ARDS patient DEspR+actNs exhibit apoptosis-resistance, which decreased upon ex vivo treatment with humanized anti-DEspR-IgG4S228P antibody, hu6g8. Ex vivo live-cell imaging of non-human primate DEspR+actNs showed hu6g8 target-engagement, internalization, and induction of apoptosis. Altogether, data differentiate DEspR+actNs as a targetable neutrophil-subset associated with ARDS and COVID19-ARDS severity, and suggest DEspR-inhibition as a potential therapeutic paradigm.

Humanized anti-DEspR IgG4S228P antibody increases overall survival in a pancreatic cancer stem cell-xenograft peritoneal carcinomatosis ratnu/nu model.

BACKGROUND: Pancreatic peritoneal carcinomatosis (PPC), with the worst median overall-survival (mOS), epitomizes the incurability of metastatic cancer. Cancer stem cells (CSCs) underpin this incurability. However, inhibitors of CSC-stemness fail to increase mOS in cancer patients despite preclinical tumor-reduction. This shortfall reinforces that preclinical efficacy should be defined by increased mOS in the presence of cancer comorbidities, CSC-heterogeneity and plasticity. The primary objectives of this study are: to test the dual endothelin-1/signal peptide receptor, DEspR, as a nodal therapeutic target in PPC, given DEspR induction in anoikis-resistant pancreatic CSCs, and to validate humanized anti-DEspR antibody, hu-6g8, as a potential therapeutic for PPC. METHODS: We used heterogeneous pools of CSCs selected for anoikis resistance from reprogrammed Panc1 and MiaPaCa2 tumor cells (TCs), and adherent TCs reprogrammed from CSCs (cscTCs). We used multiple anti-DEspR blocking antibodies (mAbs) with different epitopes, and a humanized anti-DEspR recombinant mAb cross-reactive in rodents and humans, to test DEspR inhibition effects. We measured DEspR-inhibition efficacy on multiple prometastatic CSC-functions in vitro, and on tumorigenesis and overall survival in a CSC-derived xenograft (CDX) nude rat model of PPC with comorbidities. RESULTS: Here we show that DEspR, a stress-survival receptor, is present on subsets of PDAC Panc1-TCs, TC-derived CSCs, and CSC-differentiated TCs (cscTCs), and that DESpR-inhibition decreases apoptosis-resistance and pro-metastatic mesenchymal functions of CSCs and cscTCs in vitro. We resolve the DNA-sequence/protein-function discordance by confirming ADAR1-RNA editing-dependent DEspR-protein expression in Panc1 and MiaPaCa2 TCs. To advance DEspR-inhibition as a nodal therapeutic approach for PPC, we developed and show improved functionality of a recombinant, humanized anti-DEspR IgG4S228P antibody, hu-6g8, over murine precursor anti-DEspR mabs. Hu-6g8 internalizes and translocates to the nucleus colocalized with cyto-nuclear shuttling galectins-1/3, and induces apoptotic cell changes. DEspR-inhibition blocks transperitoneal dissemination and progression to peritoneal carcinomatosis of heterogeneous DEspR±/CD133 ± Panc1-derived CSCs in xenografted nude rats, improving mOS without chemotherapy-like adverse effects. Lastly, we show DEspR expression in Stage II-IV primary and invasive TCs in the stroma in PDAC-patient tumor arrays. CONCLUSION: Collectively, the data support humanized anti-DEspR hu-6g8 as a potential targeted antibody-therapeutic with promising efficacy, safety and prevalence profiles for PPC patients.