Broadly neutralizing anti-S2 antibodies protect against all three human betacoronaviruses that cause severe disease

Pan-betacoronavirus neutralizing antibodies may hold the key to developing broadly protective vaccines against coronaviruses that cause severe disease, for anticipating novel pandemic-causing viruses, and to respond more effectively to SARS-CoV-2 variants. The emergence of the Omicron variant of SARS-CoV-2 has illustrated the limitations of solely targeting the receptor binding domain (RBD) of the envelope Spike (S)-protein. Here, we isolated a large panel of broadly neutralizing antibodies (bnAbs) from SARS-CoV-2 recovered-vaccinated donors that target a conserved S2 region in the fusion machinery on betacoronavirus spikes. Select bnAbs show broad in vivo protection against all three pathogenic betacoronaviruses, SARS-CoV-1, SARS-CoV-2 and MERS-CoV, that have spilled over into humans in the past 20 years to cause severe disease. The bnAbs provide new opportunities for antibody-based interventions and key insights for developing pan-betacoronavirus vaccines.

Potent Neutralization of Omicron and other SARS-CoV-2 Variants of Concern by Biparatopic Human VH Domains

The emergence of SARS-CoV-2 variants of concern (VOCs) requires the development of next-generation biologics that are effective against a variety of strains of the virus. Herein, we characterize a human VH domain, F6, which we generated by sequentially panning large phage displayed VH libraries against receptor binding domains (RBDs) containing VOC mutations. Cryo-EM analyses reveal that F6 has a unique binding mode that spans a broad surface of the RBD and involves the antibody framework region. Attachment of an Fc region to a fusion of F6 and ab8, a previously characterized VH domain, resulted in a construct (F6-ab8-Fc) that neutralized Omicron pseudoviruses with a half-maximal neutralizing concentration (IC50) of 4.8 nM in vitro. Additionally, prophylactic treatment using F6-ab8-Fc reduced live Beta (B.1.351) variant viral titers in the lungs of a mouse model. Our results provide a new potential therapeutic against SARS-CoV-2 VOCs - including the recently emerged Omicron variant - and highlight a vulnerable epitope within the spike protein RBD that may be exploited to achieve broad protection against circulating variants.

Breadth of SARS-CoV-2 Neutralization and Protection Induced by a Nanoparticle Vaccine

Coronavirus vaccines that are highly effective against SARS-CoV-2 variants are needed to control the current pandemic. We previously reported a receptor-binding domain (RBD) sortase A-conjugated ferritin nanoparticle (RBD-scNP) vaccine that induced neutralizing antibodies against SARS-CoV-2 and pre-emergent sarbecoviruses and protected monkeys from SARS-CoV-2 WA-1 infection. Here, we demonstrate SARS-CoV-2 RBD-scNP immunization induces potent neutralizing antibodies in non-human primates (NHPs) against all eight SARS-CoV-2 variants tested including the Beta, Delta, and Omicron variants. The Omicron variant was neutralized by RBD-scNP-induced serum antibodies with a mean of 10.6-fold reduction of ID50 titers compared to SARS-CoV-2 D614G. Immunization with RBD-scNPs protected NHPs from SARS-CoV-2 WA-1, Beta, and Delta variant challenge, and protected mice from challenges of SARS-CoV-2 Beta variant and two other heterologous sarbecoviruses. These results demonstrate the ability of RBD-scNPs to induce broad neutralization of SARS-CoV-2 variants and to protect NHPs and mice from multiple different SARS-related viruses. Such a vaccine could provide the needed immunity to slow the spread of and reduce disease caused by SARS-CoV-2 variants such as Delta and Omicron.

Therapeutic efficacy of an oral nucleoside analog of remdesivir against SARS-CoV-2 pathogenesis in mice.

The COVID-19 pandemic remains uncontrolled despite the rapid rollout of safe and effective SARS-CoV-2 vaccines, underscoring the need to develop highly effective antivirals. In the setting of waning immunity from infection and vaccination, breakthrough infections are becoming increasingly common and treatment options remain limited. Additionally, the emergence of SARS-CoV-2 variants of concern with their potential to escape therapeutic monoclonal antibodies emphasizes the need to develop second-generation oral antivirals targeting highly conserved viral proteins that can be rapidly deployed to outpatients. Here, we demonstrate the in vitro antiviral activity and in vivo therapeutic efficacy of GS-621763, an orally bioavailable prodrug of GS-441524, the parental nucleoside of remdesivir, which targets the highly conserved RNA-dependent RNA polymerase. GS-621763 exhibited significant antiviral activity in lung cell lines and two different human primary lung cell culture systems. The dose-proportional pharmacokinetic profile observed after oral administration of GS-621763 translated to dose-dependent antiviral activity in mice infected with SARS-CoV-2. Therapeutic GS-621763 significantly reduced viral load, lung pathology, and improved pulmonary function in COVID-19 mouse model. A direct comparison of GS-621763 with molnupiravir, an oral nucleoside analog antiviral currently in human clinical trial, proved both drugs to be similarly efficacious. These data demonstrate that therapy with oral prodrugs of remdesivir can significantly improve outcomes in SARS-CoV-2 infected mice. Thus, GS-621763 supports the exploration of GS-441524 oral prodrugs for the treatment of COVID-19 in humans.

Targeted isolation of panels of diverse human broadly neutralizing antibodies against SARS-like viruses

The emergence of current SARS-CoV-2 variants of concern (VOCs) and potential future spillovers of SARS-like coronaviruses into humans pose a major threat to human health and the global economy 1-7. Development of broadly effective coronavirus vaccines that can mitigate these threats is needed 8, 9. Notably, several recent studies have revealed that vaccination of recovered COVID-19 donors results in enhanced nAb responses compared to SARS-CoV-2 infection or vaccination alone 10-13. Here, we utilized a targeted donor selection strategy to isolate a large panel of broadly neutralizing antibodies (bnAbs) to sarbecoviruses from two such donors. Many of the bnAbs are remarkably effective in neutralization against sarbecoviruses that use ACE2 for viral entry and a substantial fraction also show notable binding to non-ACE2-using sarbecoviruses. The bnAbs are equally effective against most SARS-CoV-2 VOCs and many neutralize the Omicron variant. Neutralization breadth is achieved by bnAb binding to epitopes on a relatively conserved face of the receptor binding domain (RBD) as opposed to strain-specific nAbs to the receptor binding site that are commonly elicited in SARS-CoV-2 infection and vaccination 14-18. Consistent with targeting of conserved sites, select RBD bnAbs exhibited in vivo protective efficacy against diverse SARS-like coronaviruses in a prophylaxis challenge model. The generation of a large panel of potent bnAbs provides new opportunities and choices for next-generation antibody prophylactic and therapeutic applications and, importantly, provides a molecular basis for effective design of pan-sarbecovirus vaccines.

Antibodies with potent and broad neutralizing activity against antigenically diverse and highly transmissible SARS-CoV-2 variants

The emergence of highly transmissible SARS-CoV-2 variants of concern (VOC) that are resistant to therapeutic antibodies highlights the need for continuing discovery of broadly reactive antibodies. We identify four receptor-binding domain targeting antibodies from three early-outbreak convalescent donors with potent neutralizing activity against 12 variants including the B.1.1.7 and B.1.351 VOCs. Two of them are ultrapotent, with sub-nanomolar neutralization titers (IC50 <0.0006 to 0.0102 g/mL; IC80 < 0.0006 to 0.0251 g/mL). We define the structural and functional determinants of binding for all four VOC-targeting antibodies, and show that combinations of two antibodies decrease the in vitro generation of escape mutants, suggesting potential means to mitigate resistance development. These results define the basis of therapeutic cocktails against VOCs and suggest that targeted boosting of existing immunity may increase vaccine breadth against VOCs. One Sentence SummaryUltrapotent antibodies from convalescent donors neutralize and mitigate resistance of SARS-CoV-2 variants of concern.

SARS-CoV-2 vaccination induces neutralizing antibodies against pandemic and pre-emergent SARS-related coronaviruses in monkeys

Betacoronaviruses (betaCoVs) caused the severe acute respiratory syndrome (SARS) and Middle East Respiratory Syndrome (MERS) outbreaks, and now the SARS-CoV-2 pandemic. Vaccines that elicit protective immune responses against SARS-CoV-2 and betaCoVs circulating in animals have the potential to prevent future betaCoV pandemics. Here, we show that immunization of macaques with a multimeric SARS-CoV-2 receptor binding domain (RBD) nanoparticle adjuvanted with 3M-052-Alum elicited cross-neutralizing antibody responses against SARS-CoV-1, SARS-CoV-2, batCoVs and the UK B.1.1.7 SARS-CoV-2 mutant virus. Nanoparticle vaccination resulted in a SARS-CoV-2 reciprocal geometric mean neutralization titer of 47,216, and robust protection against SARS-CoV-2 in macaque upper and lower respiratory tracts. Importantly, nucleoside-modified mRNA encoding a stabilized transmembrane spike or monomeric RBD protein also induced SARS-CoV-1 and batCoV cross-neutralizing antibodies, albeit at lower titers. These results demonstrate current mRNA vaccines may provide some protection from future zoonotic betaCoV outbreaks, and provide a platform for further development of pan-betaCoV nanoparticle vaccines.

Sex disparities and neutralizing antibody durability to SARS-CoV-2 infection in convalescent individuals

The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome-related coronavirus-2 (SARS-CoV-2) has now caused over 2 million deaths worldwide and continues to expand. Currently, much is unknown about functionally neutralizing human antibody responses and durability to SARS-CoV-2. Using convalescent sera collected from 101 COVID-19 recovered individuals 21-212 days after symptom onset with forty-eight additional longitudinal samples, we measured functionality and durability of serum antibodies. We also evaluated associations between individual demographic and clinical parameters with functional neutralizing antibody responses to COVID-19. We found robust antibody durability out to six months, as well as significant positive associations with the magnitude of the neutralizing antibody response and male sex. We also show that SARS-CoV-2 convalescent neutralizing antibodies are higher in individuals with cardio-metabolic comorbidities. SignificanceIn this study we found that neutralizing antibody responses in COVID-19 convalescent individuals vary in magnitude but are durable and correlate well with RBD Ig binding antibody levels compared to other SARS-CoV-2 antigen responses. In our cohort, higher neutralizing antibody titers are independently and significantly associated with male sex compared to female sex. We also show for the first time, that higher convalescent antibody titers in male donors are associated with increased age and symptom grade. Furthermore, cardio-metabolic co-morbidities are associated with higher antibody titers independently of sex. Here, we present an in-depth evaluation of serologic, demographic, and clinical correlates of functional antibody responses and durability to SARS-CoV-2.

The functions of SARS-CoV-2 neutralizing and infection-enhancing antibodies in vitro and in mice and nonhuman primates

SARS-CoV-2 neutralizing antibodies (NAbs) protect against COVID-19. A concern regarding SARS-CoV-2 antibodies is whether they mediate disease enhancement. Here, we isolated NAbs against the receptor-binding domain (RBD) and the N-terminal domain (NTD) of SARS-CoV-2 spike from individuals with acute or convalescent SARS-CoV-2 or a history of SARS-CoV-1 infection. Cryo-electron microscopy of RBD and NTD antibodies demonstrated function-specific modes of binding. Select RBD NAbs also demonstrated Fc receptor-{gamma} (Fc{gamma}R)-mediated enhancement of virus infection in vitro, while five non-neutralizing NTD antibodies mediated Fc{gamma}R-independent in vitro infection enhancement. However, both types of infection-enhancing antibodies protected from SARS-CoV-2 replication in monkeys and mice. Nonetheless, three of 31 monkeys infused with enhancing antibodies had higher lung inflammation scores compared to controls. One monkey had alveolar edema and elevated bronchoalveolar lavage inflammatory cytokines. Thus, while in vitro antibody-enhanced infection does not necessarily herald enhanced infection in vivo, increased lung inflammation can occur in SARS-CoV-2 antibody-infused macaques.

LY-CoV555, a rapidly isolated potent neutralizing antibody, provides protection in a non-human primate model of SARS-CoV-2 infection

SARS-CoV-2 poses a public health threat for which therapeutic agents are urgently needed. Herein, we report that high-throughput microfluidic screening of antigen-specific B-cells led to the identification of LY-CoV555, a potent anti-spike neutralizing antibody from a convalescent COVID-19 patient. Biochemical, structural, and functional characterization revealed high-affinity binding to the receptor-binding domain, ACE2 binding inhibition, and potent neutralizing activity. In a rhesus macaque challenge model, prophylaxis doses as low as 2.5 mg/kg reduced viral replication in the upper and lower respiratory tract. These data demonstrate that high-throughput screening can lead to the identification of a potent antiviral antibody that protects against SARS-CoV-2 infection. One Sentence SummaryLY-CoV555, an anti-spike antibody derived from a convalescent COVID-19 patient, potently neutralizes SARS-CoV-2 and protects the upper and lower airways of non-human primates against SARS-CoV-2 infection.

SARS-CoV-2 mRNA Vaccine Development Enabled by Prototype Pathogen Preparedness

A SARS-CoV-2 vaccine is needed to control the global COVID-19 public health crisis. Atomic-level structures directed the application of prefusion-stabilizing mutations that improved expression and immunogenicity of betacoronavirus spike proteins. Using this established immunogen design, the release of SARS-CoV-2 sequences triggered immediate rapid manufacturing of an mRNA vaccine expressing the prefusion-stabilized SARS-CoV-2 spike trimer (mRNA-1273). Here, we show that mRNA-1273 induces both potent neutralizing antibody and CD8 T cell responses and protects against SARS-CoV-2 infection in lungs and noses of mice without evidence of immunopathology. mRNA-1273 is currently in a Phase 2 clinical trial with a trajectory towards Phase 3 efficacy evaluation.

Potently neutralizing human antibodies that block SARS-CoV-2 receptor binding and protect animals

The COVID-19 pandemic is a major threat to global health for which there are only limited medical countermeasures, and we lack a thorough understanding of mechanisms of humoral immunity1,2. From a panel of monoclonal antibodies (mAbs) targeting the spike (S) glycoprotein isolated from the B cells of infected subjects, we identified several mAbs that exhibited potent neutralizing activity with IC50 values as low as 0.9 or 15 ng/mL in pseudovirus or wild-type (wt) SARS-CoV-2 neutralization tests, respectively. The most potent mAbs fully block the receptor-binding domain of S (SRBD) from interacting with human ACE2. Competition-binding, structural, and functional studies allowed clustering of the mAbs into defined classes recognizing distinct epitopes within major antigenic sites on the SRBD. Electron microscopy studies revealed that these mAbs recognize distinct conformational states of trimeric S protein. Potent neutralizing mAbs recognizing unique sites, COV2-2196 and COV2-2130, bound simultaneously to S and synergistically neutralized authentic SARS-CoV-2 virus. In two murine models of SARS-CoV-2 infection, passive transfer of either COV2-2916 or COV2-2130 alone or a combination of both mAbs protected mice from severe weight loss and reduced viral burden and inflammation in the lung. These results identify protective epitopes on the SRBD and provide a structure-based framework for rational vaccine design and the selection of robust immunotherapeutic cocktails.

Potent neutralization of SARS-CoV-2 in vitro and in an animal model by a human monoclonal antibody

Effective therapies are urgently needed for the SARS-CoV-2/COVID19 pandemic. We identified panels of fully human monoclonal antibodies (mAbs) from eight large phage-displayed Fab, scFv and VH libraries by panning against the receptor binding domain (RBD) of the SARS-CoV-2 spike (S) glycoprotein. One high affinity mAb, IgG1 ab1, specifically neutralized replication competent SARS-CoV-2 with exceptional potency as measured by two different assays. There was no enhancement of pseudovirus infection in cells expressing Fc{gamma} receptors at any concentration. It competed with human angiotensin-converting enzyme 2 (hACE2) for binding to RBD suggesting a competitive mechanism of virus neutralization. IgG1 ab1 potently neutralized mouse ACE2 adapted SARS-CoV-2 in wild type BALB/c mice and native virus in hACE2 expressing transgenic mice. The ab1 sequence has relatively low number of somatic mutations indicating that ab1-like antibodies could be quickly elicited during natural SARS-CoV-2 infection or by RBD-based vaccines. IgG1 ab1 does not have developability liabilities, and thus has potential for therapy and prophylaxis of SARS-CoV-2 infections. The rapid identification (within 6 days) of potent mAbs shows the value of large antibody libraries for response to public health threats from emerging microbes.

Rapid isolation and profiling of a diverse panel of human monoclonal antibodies targeting the SARS-CoV-2 spike protein

Antibodies are a principal determinant of immunity for most RNA viruses and have promise to reduce infection or disease during major epidemics. The novel coronavirus SARS-CoV-2 has caused a global pandemic with millions of infections and hundreds of thousands of deaths to date1,2. In response, we used a rapid antibody discovery platform to isolate hundreds of human monoclonal antibodies (mAbs) against the SARS-CoV-2 spike (S) protein. We stratify these mAbs into five major classes based on their reactivity to subdomains of S protein as well as their cross-reactivity to SARS-CoV. Many of these mAbs inhibit infection of authentic SARS-CoV-2 virus, with most neutralizing mAbs recognizing the receptor-binding domain (RBD) of S. This work defines sites of vulnerability on SARS-CoV-2 S and demonstrates the speed and robustness of new antibody discovery methodologies.

A mouse-adapted SARS-CoV-2 model for the evaluation of COVID-19 medical countermeasures

Coronaviruses are prone to emergence into new host species most recently evidenced by SARS-CoV-2, the causative agent of the COVID-19 pandemic. Small animal models that recapitulate SARS-CoV-2 disease are desperately needed to rapidly evaluate medical countermeasures (MCMs). SARS-CoV-2 cannot infect wildtype laboratory mice due to inefficient interactions between the viral spike (S) protein and the murine ortholog of the human receptor, ACE2. We used reverse genetics to remodel the S and mACE2 binding interface resulting in a recombinant virus (SARS-CoV-2 MA) that could utilize mACE2 for entry. SARS-CoV-2 MA replicated in both the upper and lower airways of both young adult and aged BALB/c mice. Importantly, disease was more severe in aged mice, and showed more clinically relevant phenotypes than those seen in hACE2 transgenic mice. We then demonstrated the utility of this model through vaccine challenge studies in immune competent mice with native expression of mACE2. Lastly, we show that clinical candidate interferon (IFN) lambda-1a can potently inhibit SARS-CoV-2 replication in primary human airway epithelial cells in vitro, and both prophylactic and therapeutic administration diminished replication in mice. Our mouse-adapted SARS-CoV-2 model demonstrates age-related disease pathogenesis and supports the clinical use of IFN lambda-1a treatment in human COVID-19 infections.

The RBD Of The Spike Protein Of SARS-Group Coronaviruses Is A Highly Specific Target Of SARS-CoV-2 Antibodies But Not Other Pathogenic Human and Animal Coronavirus Antibodies

A new Severe Acute Respiratory Syndrome Coronavirus variant (SARS-CoV-2) that first emerged in late 2019 is responsible for a pandemic of severe respiratory illness. People infected with this highly contagious virus present with clinically inapparent, mild or severe disease. Currently, the presence of the virus in individual patients and at the population level is being monitored by testing symptomatic cases by PCR for the presence of viral RNA. There is an urgent need for SARS-CoV-2 serologic tests to identify all infected individuals, irrespective of clinical symptoms, to conduct surveillance and implement strategies to contain spread. As the receptor binding domain (RBD) of the viral spike (S) protein is poorly conserved between SARS-CoVs and other pathogenic human coronaviruses, the RBD represents a promising antigen for detecting CoV specific antibodies in people. Here we use a large panel of human sera (70 SARS-CoV-2 patients and 71 control subjects) and hyperimmune sera from animals exposed to zoonotic CoVs to evaluate the performance of the RBD as an antigen for accurate detection of SARS-CoV-2-specific antibodies. By day 9 after the onset of symptoms, the recombinant SARS-CoV-2 RBD antigen was highly sensitive (98%) and specific (100%) to antibodies induced by SARS-CoVs. We observed a robust correlation between levels of RBD binding antibodies and SARS-CoV-2 neutralizing antibodies in patients. Our results, which reveal the early kinetics of SARS-CoV-2 antibody responses, strongly support the use of RBD-based antibody assays for population-level surveillance and as a correlate of neutralizing antibody levels in people who have recovered from SARS-CoV-2 infections.

Remdesivir potently inhibits SARS-CoV-2 in human lung cells and chimeric SARS-CoV expressing the SARS-CoV-2 RNA polymerase in mice.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in 2019 as the causative agent of the novel pandemic viral disease COVID-19. With no approved therapies, this pandemic illustrates the urgent need for safe, broad-spectrum antiviral countermeasures against SARS-CoV-2 and future emerging CoVs. We report that remdesivir (RDV), a monophosphoramidate prodrug of an adenosine analog, potently inhibits SARS-CoV-2 replication in human lung cells and primary human airway epithelial cultures (EC50 = 0.01 M). Weaker activity was observed in Vero E6 cells (EC50 = 1.65 M) due to their low capacity to metabolize RDV. To rapidly evaluate in vivo efficacy, we engineered a chimeric SARS-CoV encoding the viral target of RDV, the RNA-dependent RNA polymerase, of SARS-CoV-2. In mice infected with chimeric virus, therapeutic RDV administration diminished lung viral load and improved pulmonary function as compared to vehicle treated animals. These data provide evidence that RDV is potently active against SARS-CoV-2 in vitro and in vivo, supporting its further clinical testing for treatment of COVID-19.