1088. A Whole-Body Quantitative System Pharmacology Physiologically-Based Pharmacokinetic (QSP/PBPK) Model to Support Dose Selection of ADG20: an Extended Half-Life Monoclonal Antibody Being Developed for the Treatment of Coronavirus Disease (COVID-19)

Background ADG20 is a fully human IgG1 monoclonal antibody engineered to have potent and broad neutralization against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other SARS-like CoVs with pandemic potential and an extended half-life. ADG20 is administered intramuscularly (IM). A QSP/PBPK model was constructed to support dose selection for a Phase 2/3 trial of ambulatory patients with mild to moderate COVID-19 (STAMP: NCT04805671). Methods A QSP/PBPK model was used to simulate receptor occupancy (RO) and drug exposure in the upper airway (nasopharyngeal/oropharyngeal epithelial lining fluid [ELF] compartment). RO was linked to an existing viral dynamic model to enable the prediction of the natural time course of viral load and the effect of ADG20 on viral clearance and infectivity rate. RO was calculated using: 1) in vitro ADG20–SARS-CoV-2 binding kinetics (association rate constant (kon) of 1.52E+06 M-1•s1 and dissociation rate constant (koff) of 2.81E-04 s-1 from a Biacore assay; 2) time course of ADG20 concentrations in ELF; and 3) time course of viral load following ADG20 administration. Molar SARS-CoV-2 viral binding site capacity was calculated assuming 40 spike proteins per virion, 3 binding sites per spike, and an initial viral load of log 107 copies/mL for all patients. The QSP/PBPK model and a 2018 CDC reference body weight distribution (45–150 kg) were used to simulate 1000 concentration-time profiles for a range of candidate ADG20 regimens. ADG20 regimens were evaluated against 2 criteria: 1) ability to attain near complete ( >90%), and durable (28-day) SARS-CoV-2 RO in the ELF; and 2) ability to maintain ELF ADG20 concentrations relative to a concentration (0.5 mg/L) associated with 100% viral growth suppression in an in vitro post-infection assay. Results A single 300 mg IM ADG20 dose met the dose selection criteria in terms of RO (Figure A) and viral growth suppression (Figure B). Conclusion These data support the evaluation of an ADG20 300 mg IM dose for the treatment of mild to moderate COVID-19. ADG20 is forecasted to attain near complete ( >90%) SARS-CoV-2 RO in the ELF and maintain ELF ADG20 concentrations above that associated with 100% viral growth suppression in vitro. Figure. QSP/PBPK model forecast of ADG20 300 mg IM in adults (A) Predicted RO expressed as percent occupancy with the dotted line representing the threshold for 90% RO. (B) Predicted median concentration of ADG20 relative to a concentration (0.5 mg/L) associated with 100% viral growth suppression as indicated by the dotted line; the shaded area represents the 90% prediction interval. Disclosures Evan D. Tarbell, PhD, Adagio Therapeutics, Inc. (Independent Contractor) Scott A. Van Wart, PhD, Adagio Therapeutics, Inc. (Independent Contractor) Laura M. Walker, PhD, Adagio Therapeutics, Inc. (Other Financial or Material Support, Laura M. Walker is an inventor on a patent application submitted by Adagio Therapeutics, Inc., describing the engineered SARS-CoV-2 antibody.) Andrew Santulli, PhD, Adagio Therapeutics, Inc. (Independent Contractor) Lynn E. Connolly, MD, PhD, Adagio Therapeutics, Inc. (Employee) Donald E Mager, PharmD, PhD, Adagio Therapeutics, Inc. (Independent Contractor) Paul G. Ambrose, PharmD, Adagio Therapeutics, Inc. (Employee)

Respiratory Syncytial Virus Phosphoprotein Residue S156 Plays a Role in Regulating Genome Transcription and Replication

Respiratory syncytial virus (RSV) is a single-stranded, negative-sense, RNA virus in the family Pneumoviridae and genus Orthopneumoviridae that can cause severe disease in infants, immunocompromised adults, and the elderly. The RSV viral RNA-dependent RNA polymerase (vRdRp) complex is composed of the phosphoprotein (P) and the large polymerase protein (L). The P protein is constitutively phosphorylated by host kinases and has 41 serine (S) and threonine (T) residues as potential phosphorylation sites. To identify important phosphorylation residues in the P protein, we systematically and individually mutated all serine S and T residues to alanine (A) and first analyzed their effect on genome transcription and replication using a minigenome system. We found that the mutation of eight residues resulted in significantly reduced minigenome activity compared to wild-type P. We then incorporated these mutations (T210A, S203A, T151A, S156A, T160A, S23A, T188A, and T105A) into full-length genome cDNA to rescue recombinant RSV. We were able to recover four recombinant viruses (T151A, S156A, T160A, and S23A), suggesting RSV-P residues T210, S203, T188, and T105 are essential for viral RNA replication. Among the four rescued, rRSV-T160A caused a minor growth defect compared to its parental virus while rRSV-S156A had severely restricted replication due to decreased levels of genomic RNA. During infection, P-S156A phosphorylation was decreased, and when passaged, the S156A virus acquired a known compensatory mutation in L (L795I) that enhanced both WT-P and P-S156A minigenome activity and was able to partially rescue the S156A viral growth defect. This work demonstrates that residues T210, S203, T188, and T105 are critical for RSV replication, and S156 plays a critical role in viral RNA synthesis. Importance RSV-P is a heavily phosphorylated protein that is required for RSV replication. In this study, we identified several residues, including P-S156, as phosphorylation sites that play critical roles in efficient viral growth and genome replication. Future studies to identify the specific kinase(s) that phosphorylate these residues can lead to kinase inhibitors and anti-viral drugs for this important human pathogen.

Basic reproduction numbers of three strains of mouse hepatitis viruses in mice

Mouse hepatitis virus (MHV) is a murine coronavirus and one of the most important pathogens in laboratory mice. Although various strains of MHV have been isolated, they are generally excreted in the feces and transmitted oronasally via aerosols and contaminated bedding. In this study, we attempted to determine the basic reproduction numbers of three strains of MHV to improve our understanding of MHV infections in mice. Five-week-old female C57BL/6J mice were inoculated intranasally with either the Y, NuU, or JHM variant strain of MHV and housed with two naive mice. After 4 weeks, the presence or absence of anti-MHV antibody in the mice was determined by the enzyme-linked immunosorbent assay. We also examined the distribution of MHV in the organs of Y, NuU, or JHM variant-infected mice. Our data suggest that the transmissibility of MHV is correlated with viral growth in the gastrointestinal tract of infected mice. To the best of our knowledge, this is the first report to address the basic reproduction numbers among pathogens in laboratory animals.

Possible Effects of Air Temperature on COVID-19 Disease Severity and Transmission Rates

Currently available data are consistent with increased severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replication at temperatures encountered in the upper airways (25–33°C when breathing room temperature air, 25°C) compared to those in the lower airways (37°C). One factor that may contribute to more rapid viral growth in the upper airways is the exponential increase in SARS-CoV-2 stability that occurs with reductions in temperature, as measured in vitro. Because SARS-CoV-2 frequently initiates infection in the upper airways before spreading through the body, increased upper airway viral growth early in the disease course may result in more rapid progression of disease and potentially contribute to more severe outcomes. Similarly, higher SARS-CoV-2 viral titer in the upper airways likely supports more efficient transmission. Conversely, the possible significance of air temperature to upper airway viral growth suggests that prolonged delivery of heated air might represent a preventative measure and prophylactic treatment for coronavirus disease 2019.

Key amino acids of M1-41 and M2-27 determine growth and pathogenicity of chimeric H17 bat influenza virus in cells and in mice

Based on our previous studies, we show that M gene is critical for viral replication and pathogenicity of the chimeric H17 bat influenza virus (Bat09:mH1mN1) by replacing bat M gene with those from human and swine influenza A viruses. However, the key amino acids of M1 and/or M2 proteins responsible for virus replication and pathogenicity remain unknown. In this study, the Eurasian avian-like M gene from the A/California/04/2009 pandemic H1N1 virus significantly decreased viral replication in both mammalian and avian cells in the background of chimeric H17 bat influenza virus by replacing the PR8 M gene. Further studies revealed that the M1 was more crucial for viral growth and pathogenicity in contrast to the M2, and amino acid residues of M1-41V and M2-27A were responsible for these characteristics in cells and in mice. These key residues of M1 and M2 proteins identified in this study might be important for influenza virus surveillance and used to produce live attenuated vaccines in the future. Importance The M1 and M2 proteins influence the morphology, replication, virulence and transmissibility of influenza viruses. Although a few key residues in M1/M2 proteins have been identified, whether other residues of M1/M2 proteins involved in viral replication and pathogenicity need to be discovered. In the background of chimeric H17 bat influenza virus, the Eurasian avian-like M gene from A/California/04/2009 significantly decreased viral growth in mammalian and avian cells. Further study showed that M1 was implicated more than M2 for viral growth and pathogenicity in vitro and in vivo , and the key amino acid residues of M1-41V and M2-27A were responsible for these characteristics in cells and in mice. These key residues of M1 and M2 proteins could be used for influenza virus surveillance and live attenuated vaccine application in the future. These findings provide important information for knowledge on the genetic basis of virulence of influenza viruses.

Characteristics of Helicase-primase Inhibitor Amenamevir-resistant Herpes Simplex Virus

The antiherpetic drug amenamevir (AMNV) inhibits the helicase-primase complex of herpes simplex virus type 1 (HSV-1), HSV-2 and varicella-zoster virus directly as well as inhibiting the replication of these viruses. Although several mutated HSV viruses resistant to helicase-primase inhibitors have been reported, the mutations contributing to the resistance remain unclear as recombinant viruses containing a single mutation have not been analyzed. We obtained AMNV-resistant viruses with amino acid substitutions by several passages under AMNV-treatment. Twenty HSV-1 and 19 HSV-2 mutants with mutation(s) in UL5 helicase and/or UL52 primase, but not in co-factor UL8, were isolated. The mutations in UL5 were located downstream of motif IV, with UL5 K356N in HSV-1 and K355N in HSV-2, in particular, identified as having the highest frequency: 9/20 and 9/19, respectively. We generated recombinant AMNV-resistant HSV-1 with a single amino acid substitution using BAC mutagenesis. As a result, G352C in UL5 helicase and F360C/V and N902T in UL52 primase were identified as novel mutations. The virus with K356N in UL5 showed 10-fold higher AMNV resistance than did other mutants, and showed equivalent viral growth in vitro and virulence in vivo as the parent HSV-1, although other mutants showed attenuated virulence. All recombinant viruses were susceptible to the other antiherpetic drugs, acyclovir and foscarnet. In conclusion, based on BAC mutagenesis, this study identified for the first time mutations in UL5 and UL52 that contributed to AMNV resistance, and found that a mutant with the most frequent K356N mutation in HSV-1 maintained viral growth and virulence equivalent to the parent virus.

Intrinsic innate immune responses control viral growth and protect against neuronal death in an ex vivo model of WNV-induced CNS disease.

Recruitment of immune cells from the periphery is critical for controlling West Nile virus (WNV) growth in the central nervous system (CNS) and preventing subsequent WNV-induced CNS disease. Neuroinflammatory responses, including the release of pro-inflammatory cytokines and chemokines by CNS cells, influence the entry and function of peripheral immune cells that infiltrate the CNS. However these same cytokines and chemokines contribute to tissue damage in other models of CNS injury. Rosiglitazone is a Peroxisome Proliferator-Activated Receptor gamma (PPAR-γ) agonist that inhibits neuroinflammation. We used rosiglitazone in WNV-infected ex vivo brain slice cultures (BSC) to investigate the role of neuroinflammation within the CNS in the absence of peripheral immune cells. Rosiglitazone treatment inhibited WNV-induced expression of pro-inflammatory chemokines and cytokines, interferon (IFN)-ß and IFN stimulated genes (ISG), and also decreased WNV-induced activation of microglia. These decreased neuroinflammatory responses were associated with activation of astrocytes, robust viral growth, increased activation of caspase 3, and increased neuronal loss. Rosigitazone had a similar effect on in vivo WNV infection causing increased viral growth, tissue damage, and disease severity in infected mice, even though the number of infiltrating peripheral immune cells was higher in rosiglitazone-treated WNV-infected mice than in untreated, infected controls. These results indicate that local neuroinflammatory responses are capable of controlling viral growth within the CNS and limiting neuronal loss and may function to keep the virus in check prior to the infiltration of peripheral immune cells, limiting both virus- and immune-mediated neuronal damage. Importance. West Nile Virus is the most common cause of epidemic encephalitis in the US and can result in debilitating CNS disease. There are no effective vaccines or treatments for WNV-induced CNS disease in humans. The peripheral immune response is critical for protection against WNV CNS infections. We now demonstrate that intrinsic immune responses also control viral growth and limit neuronal loss. These findings have important implications for developing new therapies for WNV-induced CNS disease.

A unified route for flavivirus structures uncovers essential pocket factors conserved across pathogenic viruses

The epidemic emergence of relatively rare and geographically isolated flaviviruses adds to the ongoing disease burden of viruses such as dengue. Structural analysis is key to understand and combat these pathogens. Here, we present a chimeric platform based on an insect-specific flavivirus for the safe and rapid structural analysis of pathogenic viruses. We use this approach to resolve the architecture of two neurotropic viruses and a structure of dengue virus at 2.5 Å, the highest resolution for an enveloped virion. These reconstructions allow improved modelling of the stem region of the envelope protein, revealing two lipid-like ligands within highly conserved pockets. We show that these sites are essential for viral growth and important for viral maturation. These findings define a hallmark of flavivirus virions and a potential target for broad-spectrum antivirals and vaccine design. We anticipate the chimeric platform to be widely applicable for investigating flavivirus biology.