Combinatorial Regimens Augment Drug Monotherapy for SARS-CoV-2 Clearance in Mice (preprint)

Direct acting antivirals (DAAs) represent critical tools for combating SARS-CoV-2 variants of concern (VOCs) that evolve to escape spike-based immunity and future coronaviruses with pandemic potential. Here, we used bioluminescence imaging to evaluate therapeutic efficacy of DAAs that target SARS-CoV-2 RNA-dependent RNA polymerase (favipiravir, molnupiravir) or Main protease (nirmatrelvir) against Delta or Omicron VOCs in K18-hACE2 mice. Nirmatrelvir displayed the best efficacy followed by molnupiravir and favipiravir in suppressing viral loads in the lung. Unlike neutralizing antibody treatment, DAA monotherapy did not eliminate SARS-CoV-2 in mice. However, targeting two viral enzymes by combining molnupiravir with nirmatrelvir resulted in superior efficacy and virus clearance. Furthermore, combining molnupiravir with Caspase-1/4 inhibitor mitigated inflammation and lung pathology whereas combining molnupiravir with COVID-19 convalescent plasma yielded rapid virus clearance and 100% survival. Thus, our study provides insights into treatment efficacies of DAAs and other effective combinations to bolster COVID-19 therapeutic arsenal.

Vaccinia Virus Strain Mva Expressing a Prefusion-Stabilized SARS-CoV-2 Spike Glycoprotein Induces Robust Protection and Prevents Brain Infection in Mouse and Hamster Models (preprint)

The COVID-19 pandemic has underscored the importance of swift responses and the necessity of dependable technologies for vaccine development. Our team previously developed a fast cloning system for the modified vaccinia virus Ankara (MVA) vaccine platform. In this study, we report the construction and preclinical testing of a recombinant MVA vaccine obtained using this system. We obtained recombinant MVA expressing the unmodified full-length SARS-CoV-2 spike (S) protein containing the D614G amino acid substitution (MVA-Sdg) and a version expressing a modified S protein containing amino acid substitutions designed to stabilize the protein a in a pre-fusion conformation (MVA-Spf). S protein expressed by MVA-Sdg was found to be expressed and correctly processed and transported to the cell surface, where it efficiently produced cell-cell fusion. Version Spf, however, was not proteolytically processed and despite being transported to the plasma membrane, it failed to induce cell-cell fusion. We assessed both vaccine candidates in prime-boost regimens in the susceptible transgenic K18-human angiotensin converting enzyme 2 (K18-hACE2) mice and in golden Syrian hamsters. Robust immunity and protection from disease was induced with either vaccine in both animal models. Remarkably, the MVA-Spf vaccine candidate produced higher levels of antibodies, a stronger T cell response, and a higher degree of protection from challenge. In addition, the levels of SARS-CoV-2 in the brain of MVA-Spf inoculated mice was decreased to undetectable levels. Those results add to our current experience and range of vaccine vectors and technologies for developing a safe and effective COVID-19 vaccine

Molecular basis for antiviral activity of pediatric neutralizing antibodies targeting SARS-CoV-2 Spike receptor binding domain (preprint)

Neutralizing antibodies (NAbs) hold great promise for clinical interventions against SARS-CoV-2 variants of concern (VOCs). Understanding NAb epitope-dependent antiviral mechanisms is crucial for developing vaccines and therapeutics against VOCs. Here we characterized two potent NAbs, EH3 and EH8, isolated from an unvaccinated pediatric patient with exceptional plasma neutralization activity. EH3 and EH8 cross-neutralize the early VOCs and mediate strong Fc-dependent effector activity in vitro. Structural analyses of EH3 and EH8 in complex with the receptor-binding domain (RBD) revealed the molecular determinants of the epitope-driven protection and VOC-evasion. While EH3 represents the prevalent IGHV3-53 NAb whose epitope substantially overlaps with the ACE2 binding site, EH8 recognizes a narrow epitope exposed in both RBD-up and RBD-down conformations. When tested in vivo, a single-dose prophylactic administration of EH3 fully protected stringent K18-hACE2 mice from lethal challenge with Delta VOC. Our study demonstrates that protective NAbs responses converge in pediatric and adult SARS-CoV-2 patients.

The Fc-effector function of COVID-19 convalescent plasma contributes to SARS-CoV-2 treatment efficacy in mice (preprint)

COVID-19 convalescent plasmas (CCPs) are chosen for plasma therapy based on neutralizing titers and anti-Spike immunoglobulin levels. However, specific CCP characteristics that promote SARS-CoV-2 control in recipients are complex and incompletely defined. Using an in vivo imaging approach, we demonstrate that CCPs with low neutralizing and high Fc-effector activity, in contrast to those with poor Fc-function, afford effective prophylaxis and therapy in K18-hACE2 mice lethally challenged with SARS-CoV-2-nLuc. Macrophages and neutrophils significantly contributed to CCP effects during therapy but to a reduced extent under prophylaxis. Both IgG and Ig(M+A) were required during therapy, but the IgG fraction alone was sufficient during prophylaxis. Finally, despite neutralizing poorly, SARS-CoV-2 Wuhan-elicited CCPs delayed Delta and Beta variants of concern (VOC)-induced mortality in mice illustrating the contribution of polyclonal Fc-effector functions in immunity against VOCs. Thus, in addition to neutralization, Fc-effector activity is a significant criterion for CCP selection for therapeutic applications.

Engineered ACE2-Fc counters murine lethal SARS-CoV-2 infection through direct neutralization and Fc-effector activities (preprint)

Soluble Angiotensin-Converting Enzyme 2 (ACE2) constitutes an attractive antiviral receptor decoy that targets the vulnerable site on SARS-CoV-2 spike (S). Here, using structure-guided approaches, we developed divalent ACE2 molecules by grafting the extracellular ACE2-domain onto a human IgG1 or IgG3 (ACE2-Fc). These ACE2-Fcs harbored structurally validated mutations that enhance S binding and remove enzymatic activity. The lead variant bound tightly to S, mediated in vitro neutralization of SARS-CoV-2 variants of concern (VOCs) with sub-nanomolar IC50 and was capable of robust Fc-effector functions, including antibody-dependent cellular cytotoxicity, phagocytosis and complement deposition. When tested in a stringent K18-hACE2 mouse model, the lead variant prevented or delayed lethal SARS-CoV-2 infection in a prophylactic or therapeutic setting utilizing the combined effect of neutralization and Fc-effector functions. These data confirm the utility of ACE2-Fcs as valuable agents in preventing and eliminating SARS-CoV-2 infection and demonstrate that ACE2-Fc therapeutic activity require Fc-effector functions.

An anti-SARS-CoV-2 non-neutralizing antibody with Fc-effector function defines a new NTD epitope and delays neuroinvasion and death in K18-hACE2 mice (preprint)

Emerging evidence in animal models indicate that both neutralizing activity and Fc- mediated effector functions of neutralizing antibodies contribute to protection against SARS-CoV-2. It is unclear if antibody effector functions alone could protect against SARS-CoV-2. Here we isolated CV3-13, a non-neutralizing antibody from a convalescent individual with potent Fc-mediated effector functions that targeted the N- terminal domain (NTD) of SARS-CoV-2 Spike. The cryo-EM structure of CV3-13 in complex with SAR-CoV-2 spike revealed that the antibody bound from a distinct angle of approach to a novel NTD epitope that partially overlapped with a frequently mutated NTD supersite in SARS-CoV-2 variants. While CV3-13 did not alter the replication dynamics of SARS-CoV-2 in a K18-hACE2 transgenic mouse model, an Fc-enhanced CV3-13 significantly delayed neuroinvasion and death in prophylactic settings. Thus, we demonstrate that efficient Fc-mediated effector functions can contribute to the in vivo efficacy of anti-SARS-CoV-2 monoclonal antibodies in the absence of neutralization.

Structural Basis and Mode of Action for Two Broadly Neutralizing Antibodies Against SARS-CoV-2 Emerging Variants of Concern (preprint)

Emerging variants of concern for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can transmit more efficiently and partially evade protective immune responses, thus necessitating continued refinement of antibody therapies and immunogen design. Here we elucidate the structural basis and mode of action for two potent SARS-CoV-2 Spike (S) neutralizing monoclonal antibodies CV3-1 and CV3-25 that remained effective against emerging variants of concern in vitro and in vivo. CV3-1 bound to the (485-GFN-487) loop within the receptor-binding domain (RBD) in the RBD-up position and triggered potent shedding of the S1 subunit. In contrast, CV3-25 inhibited membrane fusion by binding to an epitope in the stem helix region of the S2 subunit that is highly conserved among beta-coronaviruses. Thus, vaccine immunogen designs that incorporate the conserved regions in RBD and stem helix region are candidates to elicit pan-coronavirus protective immune responses.

Live imaging of SARS-CoV-2 infection in mice reveals neutralizing antibodies require Fc function for optimal efficacy (preprint)

Neutralizing antibodies (NAbs) are effective in treating COVID-19 but the mechanism of immune protection is not fully understood. Here, we applied live bioluminescence imaging (BLI) to monitor the real-time effects of NAb treatment in prophylaxis and therapy of K18-hACE2 mice intranasally infected with SARS-CoV-2-nanoluciferase. We visualized sequential spread of virus from the nasal cavity to the lungs followed by systemic spread to various organs including the brain, culminating in death. Highly potent NAbs from a COVID-19 convalescent subject prevented, and also effectively resolved, established infection when administered within three days of infection. In addition to direct neutralization, in vivo efficacy required Fc effector functions of NAbs, with contributions from monocytes, neutrophils and natural killer cells, to dampen inflammatory responses and limit immunopathology. Thus, our study highlights the requirement of both Fab and Fc effector functions for an optimal in vivo efficacy afforded by NAbs against SARS-CoV-2.

Real-time conformational dynamics of SARS-CoV-2 spikes on virus particles (preprint)

SARS-CoV-2 spike (S) mediates entry into cells and is critical for vaccine development against COVID-19. Structural studies have revealed distinct conformations of S, but real-time information that connects these structures, is lacking. Here we apply single-molecule Forster Resonance Energy Transfer (smFRET) imaging to observe conformational dynamics of S on virus particles. Virus-associated S dynamically samples at least four distinct conformational states. In response to hACE2, S opens sequentially into the hACE2-bound S conformation through at least one on-path intermediate. Conformational preferences of convalescent plasma and antibodies suggest mechanisms of neutralization involving either competition with hACE2 for binding to RBD or allosteric interference with conformational changes required for entry. Our findings inform on mechanisms of S recognition and conformations for immunogen design.