Non-neutralizing SARS-CoV-2 N-terminal domain antibodies protect mice against severe disease using Fc-mediated effector functions.

Antibodies perform both neutralizing and non-neutralizing effector functions that protect against certain pathogen-induced diseases. A human antibody directed at the SARS-CoV-2 Spike N-terminal domain (NTD), DH1052, was recently shown to be non-neutralizing, yet it protected mice and cynomolgus macaques from severe disease. The mechanisms of NTD non-neutralizing antibody-mediated protection are unknown. Here we show that Fc effector functions mediate NTD non-neutralizing antibody (non-nAb) protection against SARS-CoV-2 MA10 viral challenge in mice. Though non-nAb prophylactic infusion did not suppress infectious viral titers in the lung as potently as neutralizing antibody (nAb) infusion, disease markers including gross lung discoloration were similar in nAb and non-nAb groups. Fc functional knockout substitutions abolished non-nAb protection and increased viral titers in the nAb group. Fc enhancement increased non-nAb protection relative to WT, supporting a positive association between Fc functionality and degree of protection from SARS-CoV-2 infection. For therapeutic administration of antibodies, non-nAb effector functions contributed to virus suppression and lessening of lung discoloration, but the presence of neutralization was required for optimal protection from disease. This study demonstrates that non-nAbs can utilize Fc-mediated mechanisms to lower viral load and prevent lung damage due to coronavirus infection.

A mouse-adapted model of SARS-CoV-2 to test COVID-19 countermeasures.

Coronaviruses are prone to transmission to new host species, as recently demonstrated by the spread to humans of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the coronavirus disease 2019 (COVID-19) pandemic1. Small animal models that recapitulate SARS-CoV-2 disease are needed urgently for rapid evaluation of medical countermeasures2,3. SARS-CoV-2 cannot infect wild-type laboratory mice owing to inefficient interactions between the viral spike protein and the mouse orthologue of the human receptor, angiotensin-converting enzyme 2 (ACE2)4. Here we used reverse genetics5 to remodel the interaction between SARS-CoV-2 spike protein and mouse ACE2 and designed mouse-adapted SARS-CoV-2 (SARS-CoV-2 MA), a recombinant virus that can use mouse ACE2 for entry into cells. SARS-CoV-2 MA was able to replicate in the upper and lower airways of both young adult and aged BALB/c mice. SARS-CoV-2 MA caused more severe disease in aged mice, and exhibited more clinically relevant phenotypes than those seen in Hfh4-ACE2 transgenic mice, which express human ACE2 under the control of the Hfh4 (also known as Foxj1) promoter. We demonstrate the utility of this model using vaccine-challenge studies in immune-competent mice with native expression of mouse ACE2. Finally, we show that the clinical candidate interferon-λ1a (IFN-λ1a) potently inhibits SARS-CoV-2 replication in primary human airway epithelial cells in vitro-both prophylactic and therapeutic administration of IFN-λ1a diminished SARS-CoV-2 replication in mice. In summary, the mouse-adapted SARS-CoV-2 MA model demonstrates age-related disease pathogenesis and supports the clinical use of pegylated IFN-λ1a as a treatment for human COVID-196.

Genomewide CRISPR knockout screen identified PLAC8 as an essential factor for SADS-CoVs infection.

Zoonotic transmission of coronaviruses poses an ongoing threat to human populations. Endemic outbreaks of swine acute diarrhea syndrome coronavirus (SADS-CoV) have caused severe economic losses in the pig industry and have the potential to cause human outbreaks. Currently, there are no vaccines or specific antivirals against SADS-CoV, and our limited understanding of SADS-CoV host entry factors could hinder prompt responses to a potential human outbreak. Using a genomewide CRISPR knockout screen, we identified placenta-associated 8 protein (PLAC8) as an essential host factor for SADS-CoV infection. Knockout of PLAC8 abolished SADS-CoV infection, which was restored by complementing PLAC8 from multiple species, including human, rhesus macaques, mouse, pig, pangolin, and bat, suggesting a conserved infection pathway and susceptibility of SADS-CoV among mammals. Mechanistically, PLAC8 knockout does not affect viral entry; rather, knockout cells displayed a delay and reduction in viral subgenomic RNA expression. In a swine primary intestinal epithelial culture (IEC) infection model, differentiated cultures have high levels of PLAC8 expression and support SADS-CoV replication. In contrast, expanding IECs have low levels of PLAC8 expression and are resistant to SADS-CoV infection. PLAC8 expression patterns translate in vivo; the immunohistochemistry of swine ileal tissue revealed high levels of PLAC8 protein in neonatal compared to adult tissue, mirroring the known SADS-CoV pathogenesis in neonatal piglets. Overall, PLAC8 is an essential factor for SADS-CoV infection and may serve as a promising target for antiviral development for potential pandemic SADS-CoV.

Impact Of Low-Frequency HIV-1 Drug Resistance Mutations On Antiretroviral Therapy Outcomes.

BACKGROUND: The association between low-frequency HIV-1 drug resistance mutations (DRMs) and treatment failure (TF) is controversial. We explore this association using NGS methods that accurately sample low-frequency DRMs. METHODS: We enrolled women with HIV-1 in Malawi who were either ART naïve (A), had ART failure (B), or had discontinued ART (C). At entry, A and C began an NNRTI-based regimen and B started a PI-based regimen. We used Primer ID MiSeq to identify regimen-relevant DRMs in entry and TF plasma samples, and a Cox proportional hazards model to calculate hazard ratios (HRs) for entry DRMs. Low-frequency DRMs were defined as ≤ 20%. RESULTS: We sequenced 360 participants. Cohort B and C participants were more likely to have TF than Cohort A participants. The presence of K103N at entry significantly increased TF risk among A and C participants at both high and low frequency, with HR of 3.12 [1.58-6.18, 95% CI] and 2.38 [1.00-5.67, 95% CI] respectively. At TF, 45% of participants showed selection of DRMs while in the remaining participants there was an apparent lack of selective pressure from ART. CONCLUSIONS: Using accurate NGS for DRM detection may benefit an additional 10% of the patients by identifying low-frequency K103N mutations.

DROIDS 3.0-Detecting Genetic and Drug Class Variant Impact on Conserved Protein Binding Dynamics.

The application of statistical methods to comparatively framed questions about the molecular dynamics (MD) of proteins can potentially enable investigations of biomolecular function beyond the current sequence and structural methods in bioinformatics. However, the chaotic behavior in single MD trajectories requires statistical inference that is derived from large ensembles of simulations representing the comparative functional states of a protein under investigation. Meaningful interpretation of such complex forms of big data poses serious challenges to users of MD. Here, we announce Detecting Relative Outlier Impacts from Molecular Dynamic Simulation (DROIDS) 3.0, a method and software package for comparative protein dynamics that includes maxDemon 1.0, a multimethod machine learning application that trains on large ensemble comparisons of concerted protein motions in opposing functional states generated by DROIDS and deploys learned classifications of these states onto newly generated MD simulations. Local canonical correlations in learning patterns generated from independent, yet identically prepared, MD validation runs are used to identify regions of functionally conserved protein dynamics. The subsequent impacts of genetic and/or drug class variants on conserved dynamics can also be analyzed by deploying the classifiers on variant MD simulations and quantifying how often these altered protein systems display opposing functional states. Here, we present several case studies of complex changes in functional protein dynamics caused by temperature, genetic mutation, and binding interactions with nucleic acids and small molecules. We demonstrate that our machine learning algorithm can properly identify regions of functionally conserved dynamics in ubiquitin and TATA-binding protein (TBP). We quantify the impact of genetic variation in TBP and drug class variation targeting the ATP-binding region of Hsp90 on conserved dynamics. We identify regions of conserved dynamics in Hsp90 that connect the ATP binding pocket to other functional regions. We also demonstrate that dynamic impacts of various Hsp90 inhibitors rank accordingly with how closely they mimic natural ATP binding.

Structure-Function Studies of the Antibiotic Target l,l-Diaminopimelate Aminotransferase from Verrucomicrobium spinosum Reveal an Unusual Oligomeric Structure.

While humans lack the biosynthetic pathways for meso-diaminopimelate and l-lysine, they are essential for bacterial survival and are therefore attractive targets for antibiotics. It was recently discovered that members of the Chlamydia family utilize a rare aminotransferase route of the l-lysine biosynthetic pathway, thus offering a new enzymatic drug target. Here we characterize diaminopimelate aminotransferase from Verrucomicrobium spinosum (VsDapL), a nonpathogenic model bacterium for Chlamydia trachomatis. Complementation experiments verify that the V. spinosum dapL gene encodes a bona fide diaminopimelate aminotransferase, because the gene rescues an Escherichia coli strain that is auxotrophic for meso-diaminopimelate. Kinetic studies show that VsDapL follows a Michaelis-Menten mechanism, with a KMapp of 4.0 mM toward its substrate l,l-diaminopimelate. The kcat (0.46 s-1) and the kcat/KM (115 s-1 M-1) are somewhat lower than values for other diaminopimelate aminotransferases. Moreover, whereas other studied DapL orthologs are dimeric, sedimentation velocity experiments demonstrate that VsDapL exists in a monomer-dimer self-association, with a KD2-1 of 7.4 µM. The 2.25 Å resolution crystal structure presents the canonical dimer of chalice-shaped monomers, and small-angle X-ray scattering experiments confirm the dimer in solution. Sequence and structural alignments reveal that active site residues important for activity are conserved in VsDapL, despite the lower activity compared to those of other DapL homologues. Although the dimer interface buries 18% of the total surface area, several loops that contribute to the interface and active site, notably the L1, L2, and L5 loops, are highly mobile, perhaps explaining the unstable dimer and lower catalytic activity. Our kinetic, biophysical, and structural characterization can be used to inform the development of antibiotics.

Global Proteomic Profiling of Salmonella Infection by a Giant Phage.

The 240-kb Salmonella phage SPN3US genome encodes 264 gene products, many of which are functionally uncharacterized. We have previously used mass spectrometry to define the proteomes of wild-type and mutant forms of the SPN3US virion. In this study, we sought to determine whether this technique was suitable for the characterization of the SPN3US proteome during liquid infection. Mass spectrometry of SPN3US-infected cells identified 232 SPN3US and 1,994 Salmonella proteins. SPN3US proteins with related functions, such as proteins with roles in DNA replication, transcription, and virion formation, were coordinately expressed in a temporal manner. Mass spectral counts showed the four most abundant SPN3US proteins to be the major capsid protein, two head ejection proteins, and the functionally unassigned protein gp22. This high abundance of gp22 in infected bacteria contrasted with its absence from mature virions, suggesting that it might be the scaffold protein, an essential head morphogenesis protein yet to be identified in giant phages. We identified homologs to SPN3US gp22 in 45 related giant phages, including ϕKZ, whose counterpart is also abundant in infected bacteria but absent in the virion. We determined the ϕKZ counterpart to be cleaved in vitro by its prohead protease, an event that has been observed to promote head maturation of some other phages. Our findings are consistent with a scaffold protein assignment for SPN3US gp22, although direct evidence is required for its confirmation. These studies demonstrate the power of mass spectral analyses for facilitating the acquisition of new knowledge into the molecular events of viral infection.IMPORTANCE "Giant" phages with genomes >200 kb are being isolated in increasing numbers from a range of environments. With hosts such as Salmonella enterica, Pseudomonas aeruginosa, and Erwinia amylovora, these phages are of interest for phage therapy of multidrug-resistant pathogens. However, our understanding of how these complex phages interact with their hosts is impeded by the proportion (∼80%) of their gene products that are functionally uncharacterized. To develop the repertoire of techniques for analysis of phages, we analyzed a liquid infection of Salmonella phage SPN3US (240-kb genome) using third-generation mass spectrometry. We observed the temporal production of phage proteins whose genes collectively represent 96% of the SPN3US genome. These findings demonstrate the sensitivity of mass spectrometry for global proteomic profiling of virus-infected cells, and the identification of a candidate for a major head morphogenesis protein will facilitate further studies into giant phage head assembly.

DROIDS 1.20: A GUI-Based Pipeline for GPU-Accelerated Comparative Protein Dynamics.

Traditional informatics in comparative genomics work only with static representations of biomolecules (i.e., sequence and structure), thereby ignoring the molecular dynamics (MD) of proteins that define function in the cell. A comparative approach applied to MD would connect this very short timescale process, defined in femtoseconds, to one of the longest in the universe: molecular evolution measured in millions of years. Here, we leverage advances in graphics-processing-unit-accelerated MD simulation software to develop a comparative method of MD analysis and visualization that can be applied to any two homologous Protein Data Bank structures. Our open-source pipeline, DROIDS (Detecting Relative Outlier Impacts in Dynamic Simulations), works in conjunction with existing molecular modeling software to convert any Linux gaming personal computer into a "comparative computational microscope" for observing the biophysical effects of mutations and other chemical changes in proteins. DROIDS implements structural alignment and Benjamini-Hochberg-corrected Kolmogorov-Smirnov statistics to compare nanosecond-scale atom bond fluctuations on the protein backbone, color mapping the significant differences identified in protein MD with single-amino-acid resolution. DROIDS is simple to use, incorporating graphical user interface control for Amber16 MD simulations, cpptraj analysis, and the final statistical and visual representations in R graphics and UCSF Chimera. We demonstrate that DROIDS can be utilized to visually investigate molecular evolution and disease-related functional changes in MD due to genetic mutation and epigenetic modification. DROIDS can also be used to potentially investigate binding interactions of pharmaceuticals, toxins, or other biomolecules in a functional evolutionary context as well.

The oral nucleoside prodrug GS-5245 is efficacious against SARS-CoV-2 and other endemic, epidemic, and enzootic coronaviruses.

Despite the wide availability of several safe and effective vaccines that prevent severe COVID-19, the persistent emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) that can evade vaccine-elicited immunity remains a global health concern. In addition, the emergence of SARS-CoV-2 VOCs that can evade therapeutic monoclonal antibodies underscores the need for additional, variant-resistant treatment strategies. Here, we characterize the antiviral activity of GS-5245, obeldesivir (ODV), an oral prodrug of the parent nucleoside GS-441524, which targets the highly conserved viral RNA-dependent RNA polymerase (RdRp). We show that GS-5245 is broadly potent in vitro against alphacoronavirus HCoV-NL63, SARS-CoV, SARS-CoV-related bat-CoV RsSHC014, Middle East respiratory syndrome coronavirus (MERS-CoV), SARS-CoV-2 WA/1, and the highly transmissible SARS-CoV-2 BA.1 Omicron variant. Moreover, in mouse models of SARS-CoV, SARS-CoV-2 (WA/1 and Omicron B1.1.529), MERS-CoV, and bat-CoV RsSHC014 pathogenesis, we observed a dose-dependent reduction in viral replication, body weight loss, acute lung injury, and pulmonary function with GS-5245 therapy. Last, we demonstrate that a combination of GS-5245 and main protease (Mpro) inhibitor nirmatrelvir improved outcomes in vivo against SARS-CoV-2 compared with the single agents. Together, our data support the clinical evaluation of GS-5245 against coronaviruses that cause or have the potential to cause human disease.

Adjuvant-dependent impact of inactivated SARS-CoV-2 vaccines during heterologous infection by a SARS-related coronavirus.

Whole virus-based inactivated SARS-CoV-2 vaccines adjuvanted with aluminum hydroxide have been critical to the COVID-19 pandemic response. Although these vaccines are protective against homologous coronavirus infection, the emergence of novel variants and the presence of large zoonotic reservoirs harboring novel heterologous coronaviruses provide significant opportunities for vaccine breakthrough, which raises the risk of adverse outcomes like vaccine-associated enhanced respiratory disease. Here, we use a female mouse model of coronavirus disease to evaluate inactivated vaccine performance against either homologous challenge with SARS-CoV-2 or heterologous challenge with a bat-derived coronavirus that represents a potential emerging disease threat. We show that inactivated SARS-CoV-2 vaccines adjuvanted with aluminum hydroxide can cause enhanced respiratory disease during heterologous infection, while use of an alternative adjuvant does not drive disease and promotes heterologous viral clearance. In this work, we highlight the impact of adjuvant selection on inactivated vaccine safety and efficacy against heterologous coronavirus infection.

Efficacy of different AV7909 dose regimens in a nonclinical model of pulmonary anthrax.

Pulmonary anthrax caused by exposure to inhaled Bacillus anthracis, the most lethal form of anthrax disease, is a continued military and public health concern for the United States. The vaccine AV7909, consisting of the licensed anthrax drug substance AVA adjuvanted with CpG7909, induces high levels of toxin neutralizing antibodies in healthy adults using fewer doses than AVA. This study compares the ability of one- or two-dose regimens of AV7909 to induce a protective immune response in guinea pigs challenged with a lethal dose of aerosolized B. anthracis spores 6 weeks after the last vaccine dose. The results indicated that AV7909 was less effective when delivered as a single dose compared to the two-dose regimen that resulted in dose-dependent protection against death. The toxin neutralizing assay (TNA) titer and anti-PA IgG responses were proportional to the protective efficacy, with a 50% TNA neutralizing factor (NF50) greater than 0.1 associated with survival in animals receiving two doses of vaccine. The strong protection at relatively low TNA NF50 titers in this guinea pig model supports the exploration of lower doses in clinical trials to determine if these protective levels of neutralizing antibodies can be achieved in humans; however, protection with a single dose may not be feasible.

Non-neutralizing SARS-CoV-2 N-terminal domain antibodies protect mice against severe disease using Fc-mediated effector functions.

Antibodies perform both neutralizing and non-neutralizing effector functions that protect against certain pathogen-induced diseases. A human antibody directed at the SARS-CoV-2 Spike N-terminal domain (NTD), DH1052, was recently shown to be non-neutralizing yet it protected mice and cynomolgus macaques from severe disease. The mechanisms of this non-neutralizing antibody-mediated protection are unknown. Here we show that Fc effector functions mediate non-neutralizing antibody (non-nAb) protection against SARS-CoV-2 MA10 viral challenge in mice. Though non-nAb infusion did not suppress infectious viral titers in the lung as potently as NTD neutralizing antibody (nAb) infusion, disease markers including gross lung discoloration were similar in nAb and non-nAb groups. Fc functional knockout substitutions abolished non-nAb protection and increased viral titers in the nAb group. Finally, Fc enhancement increased non-nAb protection relative to WT, supporting a positive association between Fc functionality and degree of protection in SARS-CoV-2 infection. This study demonstrates that non-nAbs can utilize Fc-mediated mechanisms to lower viral load and prevent lung damage due to coronavirus infection.

Extremely potent pan-sarbecovirus neutralizing antibodies generated by immunization of macaques with an AS03-adjuvanted monovalent subunit vaccine against SARS-CoV-2.

The rapid emergence of SARS-CoV-2 variants that evade immunity to vaccination has placed a global health imperative on the development of therapeutic countermeasures that provide broad protection against SARS-CoV-2 and related sarbecoviruses. Here, we identified extremely potent pan-sarbecovirus antibodies from non-human primates vaccinated with an AS03 adjuvanted subunit vaccine against SARS-CoV-2 that recognize conserved epitopes in the receptor binding domain (RBD) with femtomolar affinities. Longitudinal analysis revealed progressive accumulation of somatic mutation in the immunoglobulin genes of antigen-specific memory B cells for at least one year following primary vaccination. 514 monoclonal antibodies (mAbs) were generated from antigen-specific memory B cells. Antibodies isolated at 5 to 12 months following vaccination displayed greater potency and breadth, relative to those identified at 1.4 months. Notably, 15 out of 338 (∼4.4%) antibodies isolated at 1.4∼6 months after the primary vaccination showed extraordinary neutralization potency against SARS-CoV-2 omicron BA.1, despite the absence of BA.1 neutralization in serum. Two of them, 25F9 and 20A7, neutralized authentic clade Ia sarbecoviruses (SARS-CoV, WIV-1, SHC014) and clade Ib sarbecoviruses (SARS-CoV-2 D614G, SARS-CoV-2 BA.1, Pangolin-GD) with half-maximal inhibition concentrations of (0.85 ng/ml, 3 ng/ml, 6 ng/ml, 6 ng/ml, 42 ng/ml, 6 ng/ml) and (13 ng/ml, 2 ng/ml, 18 ng/ml, 9 ng/ml, 6 ng/ml, 345 ng/ml), respectively. Furthermore, 20A7 and 27A12 showed potent neutralization against all SARS-CoV-2 variants of concern and multiple Omicron sublineages, including BA.1, BA.2, BA.3, BA.4/5, BQ.1, BQ.1.1 and XBB variants. X-ray crystallography studies revealed the molecular basis of broad and potent neutralization through targeting conserved RBD sites. In vivo prophylactic protection of 25F9, 20A7 and 27A12 was confirmed in aged Balb/c mice. Notably, administration of 25F9 provided complete protection against SARS-CoV-2, SARS-CoV-2 BA.1, SARS-CoV, and SHC014 challenge, underscoring that these mAbs are promising pan-sarbecovirus therapeutic antibodies. One Sentence Summary: Extremely potent pan-sarbecovirus neutralizing antibodies.

Adjuvant-dependent effects on the safety and efficacy of inactivated SARS-CoV-2 vaccines during heterologous infection by a SARS-related coronavirus.

Inactivated whole virus SARS-CoV-2 vaccines adjuvanted with aluminum hydroxide (Alum) are among the most widely used COVID-19 vaccines globally and have been critical to the COVID-19 pandemic response. Although these vaccines are protective against homologous virus infection in healthy recipients, the emergence of novel SARS-CoV-2 variants and the presence of large zoonotic reservoirs provide significant opportunities for vaccine breakthrough, which raises the risk of adverse outcomes including vaccine-associated enhanced respiratory disease (VAERD). To evaluate this possibility, we tested the performance of an inactivated SARS-CoV-2 vaccine (iCoV2) in combination with Alum against either homologous or heterologous coronavirus challenge in a mouse model of coronavirus-induced pulmonary disease. Consistent with human results, iCoV2 + Alum protected against homologous challenge. However, challenge with a heterologous SARS-related coronavirus, Rs-SHC014-CoV (SHC014), up to at least 10 months post-vaccination, resulted in VAERD in iCoV2 + Alum-vaccinated animals, characterized by pulmonary eosinophilic infiltrates, enhanced pulmonary pathology, delayed viral clearance, and decreased pulmonary function. In contrast, vaccination with iCoV2 in combination with an alternative adjuvant (RIBI) did not induce VAERD and promoted enhanced SHC014 clearance. Further characterization of iCoV2 + Alum-induced immunity suggested that CD4+ T cells were a major driver of VAERD, and these responses were partially reversed by re-boosting with recombinant Spike protein + RIBI adjuvant. These results highlight potential risks associated with vaccine breakthrough in recipients of Alum-adjuvanted inactivated vaccines and provide important insights into factors affecting both the safety and efficacy of coronavirus vaccines in the face of heterologous virus infections.

Host range, transmissibility and antigenicity of a pangolin coronavirus.

The pathogenic and cross-species transmission potential of SARS-CoV-2-related coronaviruses (CoVs) remain poorly characterized. Here we recovered a wild-type pangolin (Pg) CoV GD strain including derivatives encoding reporter genes using reverse genetics. In primary human cells, PgCoV replicated efficiently but with reduced fitness and showed less efficient transmission via airborne route compared with SARS-CoV-2 in hamsters. PgCoV was potently inhibited by US Food and Drug Administration approved drugs, and neutralized by COVID-19 patient sera and SARS-CoV-2 therapeutic antibodies in vitro. A pan-Sarbecovirus antibody and SARS-CoV-2 S2P recombinant protein vaccine protected BALB/c mice from PgCoV infection. In K18-hACE2 mice, PgCoV infection caused severe clinical disease, but mice were protected by a SARS-CoV-2 human antibody. Efficient PgCoV replication in primary human cells and hACE2 mice, coupled with a capacity for airborne spread, highlights an emergence potential. However, low competitive fitness, pre-immune humans and the benefit of COVID-19 countermeasures should impede its ability to spread globally in human populations.

Efficacy of the oral nucleoside prodrug GS-5245 (Obeldesivir) against SARS-CoV-2 and coronaviruses with pandemic potential.

Despite the wide availability of several safe and effective vaccines that can prevent severe COVID-19 disease, the emergence of SARS-CoV-2 variants of concern (VOC) that can partially evade vaccine immunity remains a global health concern. In addition, the emergence of highly mutated and neutralization-resistant SARS-CoV-2 VOCs such as BA.1 and BA.5 that can partially or fully evade (1) many therapeutic monoclonal antibodies in clinical use underlines the need for additional effective treatment strategies. Here, we characterize the antiviral activity of GS-5245, Obeldesivir (ODV), an oral prodrug of the parent nucleoside GS-441524, which targets the highly conserved RNA-dependent viral RNA polymerase (RdRp). Importantly, we show that GS-5245 is broadly potent in vitro against alphacoronavirus HCoV-NL63, severe acute respiratory syndrome coronavirus (SARS-CoV), SARS-CoV-related Bat-CoV RsSHC014, Middle East Respiratory Syndrome coronavirus (MERS-CoV), SARS-CoV-2 WA/1, and the highly transmissible SARS-CoV-2 BA.1 Omicron variant in vitro and highly effective as antiviral therapy in mouse models of SARS-CoV, SARS-CoV-2 (WA/1), MERS-CoV and Bat-CoV RsSHC014 pathogenesis. In all these models of divergent coronaviruses, we observed protection and/or significant reduction of disease metrics such as weight loss, lung viral replication, acute lung injury, and degradation in pulmonary function in GS-5245-treated mice compared to vehicle controls. Finally, we demonstrate that GS-5245 in combination with the main protease (Mpro) inhibitor nirmatrelvir had increased efficacy in vivo against SARS-CoV-2 compared to each single agent. Altogether, our data supports the continuing clinical evaluation of GS-5245 in humans infected with COVID-19, including as part of a combination antiviral therapy, especially in populations with the most urgent need for more efficacious and durable interventions.

Broadly neutralizing antibodies against sarbecoviruses generated by immunization of macaques with an AS03-adjuvanted COVID-19 vaccine.

The rapid emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants that evade immunity elicited by vaccination has placed an imperative on the development of countermeasures that provide broad protection against SARS-CoV-2 and related sarbecoviruses. Here, we identified extremely potent monoclonal antibodies (mAbs) that neutralized multiple sarbecoviruses from macaques vaccinated with AS03-adjuvanted monovalent subunit vaccines. Longitudinal analysis revealed progressive accumulation of somatic mutation in the immunoglobulin genes of antigen-specific memory B cells (MBCs) for at least 1 year after primary vaccination. Antibodies generated from these antigen-specific MBCs at 5 to 12 months after vaccination displayed greater potency and breadth relative to those identified at 1.4 months. Fifteen of the 338 (about 4.4%) antibodies isolated at 1.4 to 6 months after the primary vaccination showed potency against SARS-CoV-2 BA.1, despite the absence of serum BA.1 neutralization. 25F9 and 20A7 neutralized authentic clade 1 sarbecoviruses (SARS-CoV, WIV-1, SHC014, SARS-CoV-2 D614G, BA.1, and Pangolin-GD) and vesicular stomatitis virus-pseudotyped clade 3 sarbecoviruses (BtKY72 and PRD-0038). 20A7 and 27A12 showed potent neutralization against all SARS-CoV-2 variants and multiple Omicron sublineages, including BA.1, BA.2, BA.3, BA.4/5, BQ.1, BQ.1.1, and XBB. Crystallography studies revealed the molecular basis of broad and potent neutralization through targeting conserved sites within the RBD. Prophylactic protection of 25F9, 20A7, and 27A12 was confirmed in mice, and administration of 25F9 particularly provided complete protection against SARS-CoV-2, BA.1, SARS-CoV, and SHC014 challenge. These data underscore the extremely potent and broad activity of these mAbs against sarbecoviruses.

Fc-mediated pan-sarbecovirus protection after alphavirus vector vaccination.

Group 2B ß-coronaviruses (sarbecoviruses) have caused regional and global epidemics in modern history. Here, we evaluate the mechanisms of cross-sarbecovirus protective immunity, currently less clear yet important for pan-sarbecovirus vaccine development, using a panel of alphavirus-vectored vaccines covering bat to human strains. While vaccination does not prevent virus replication, it protects against lethal heterologous disease outcomes in both severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and clade 2 bat sarbecovirus challenge models. The spike vaccines tested primarily elicit a highly S1-specific homologous neutralizing antibody response with no detectable cross-virus neutralization. Rather, non-neutralizing antibody functions, mechanistically linked to FcgR4 and spike S2, mediate cross-protection in wild-type mice. Protection is lost in FcR knockout mice, further supporting a model for non-neutralizing, protective antibodies. These data highlight the importance of FcR-mediated cross-protective immune responses in universal pan-sarbecovirus vaccine designs.

Generation of Mature DENVs via Genetic Modification and Directed Evolution.

Maturation of dengue viruses (DENVs) alters the structure, immunity, and infectivity of the virion and highly mature particles represent the dominant form in vivo. The production of highly mature virions principally relies on the structure and function of the viral premature membrane protein (prM) and its cleavage by the host protease furin. We redeveloped a reliable clonal cell line (VF1) which produces single-round mature DENVs without the need for DENV reverse genetics. More importantly, using protein engineering and directed evolution of the prM cleavage site, we engineered genetically stable mature DENVs in all serotypes independent of cell or host, usually with minimal impact on viral yield. Using these complementary strategies to regulate maturation, we demonstrate that the resulting mature DENVs are antigenically distinct from their isogenic partially mature forms. Given the clinical importance of mature DENVs in immunity, our study provides reliable strategies and reagents for the production of stable, high-titer mature DENVs for DENV antibody neutralization and vaccination immunity studies. Biologically, our data from directed evolution across host species reveals distinct maturation-dependent selective pressures between mammalian and insect cells, verifying the substrate preference between mammalian and insect furin, while hinting at an evolutionary equilibrium of DENV prM cleavage site between its host and vector in nature. IMPORTANCE Mature DENVs represent the dominant form in vivo and are the target for vaccine development. Here, we used multiple strategies, including protein engineering and natural and directed evolution to generate DENV1, -2, -3, and -4 variants that are highly mature without compromising replication efficiency compared to the parental strains. Given the clinical importance of mature DENVs in immunity, this work provides a roadmap for engineering highly mature DENV that could apply to future vaccine development. Our directed-evolution data also shed light on the divergent evolutionary relationship of DENVs between its host and vector.