Evidence of antigenic drift in the fusion machinery core of SARS-CoV-2 spike.

Antigenic drift of SARS-CoV-2 is typically defined by mutations in the N-terminal domain and receptor binding domain of spike protein. In contrast, whether antigenic drift occurs in the S2 domain remains largely elusive. Here, we perform a deep mutational scanning experiment to identify S2 mutations that affect binding of SARS-CoV-2 spike to three S2 apex public antibodies. Our results indicate that spatially diverse mutations, including D950N and Q954H, which are observed in Delta and Omicron variants, respectively, weaken the binding of spike to these antibodies. Although S2 apex antibodies are known to be nonneutralizing, we show that they confer protection in vivo through Fc-mediated effector functions. Overall, this study indicates that the S2 domain of SARS-CoV-2 spike can undergo antigenic drift, which represents a potential challenge for the development of more universal coronavirus vaccines.

A T cell-based SARS-CoV-2 spike protein vaccine provides protection without antibodies.

SARS-CoV-2 spike-based vaccines are used to control the COVID-19 pandemic. However, emerging variants have become resistant to antibody neutralization and further mutations may lead to full resistance. We tested whether T cells alone could provide protection without antibodies. We designed a T cell-based vaccine in which SARS-CoV-2 spike sequences were rearranged and attached to ubiquitin. Immunization of mice with the vaccine induced no specific antibodies, but strong specific T cell responses. We challenged mice with SARS-CoV-2 wild-type strain or an Omicron variant after the immunization and monitored survival or viral titers in the lungs. The mice were significantly protected against death and weight loss caused by the SARS-CoV-2 wild-type strain, and the viral titers in the lungs of mice challenged with the SARS-CoV-2 wild-type strain or the Omicron variant were significantly reduced. Importantly, depletion of CD4+ or CD8+ T cells led to significant loss of the protection. Our analyses of spike protein sequences of the variants indicated that fewer than one-third presented by dominant HLA alleles were mutated and that most of the mutated epitopes were in the subunit 1 region. As the subunit 2 region is conservative, the vaccines targeting spike protein are expected to protect against future variants due to the T cell responses.

Functional and antigenic characterization of SARS-CoV-2 spike fusion peptide by deep mutational scanning.

The fusion peptide of SARS-CoV-2 spike protein is functionally important for membrane fusion during virus entry and is part of a broadly neutralizing epitope. However, sequence determinants at the fusion peptide and its adjacent regions for pathogenicity and antigenicity remain elusive. In this study, we performed a series of deep mutational scanning (DMS) experiments on an S2 region spanning the fusion peptide of authentic SARS-CoV-2 in different cell lines and in the presence of broadly neutralizing antibodies. We identified mutations at residue 813 of the spike protein that reduced TMPRSS2-mediated entry with decreased virulence. In addition, we showed that an F823Y mutation, present in bat betacoronavirus HKU9 spike protein, confers resistance to broadly neutralizing antibodies. Our findings provide mechanistic insights into SARS-CoV-2 pathogenicity and also highlight a potential challenge in developing broadly protective S2-based coronavirus vaccines.

Contrasting roles of MERS-CoV and SARS-CoV-2 internal proteins in pathogenesis in mice.

Betacoronaviruses encode an internal (I) gene via an alternative reading frame within the nucleocapsid gene, called ORF8b for Middle-East respiratory syndrome coronavirus (MERS-CoV) and ORF9b for severe acute respiratory syndrome coronavirus (SARS-CoV) and SARS-CoV-2. Previous reports suggested that proteins 8b and 9b are involved in evading multiple innate immune signaling pathways. However, their roles in mediating pathogenesis in infected animals have not been determined. In this study, we abrogated the expression of protein 8b in MERS-CoV and protein 9b in SARS-CoV-2. Using mouse models of MERS-CoV and SARS-CoV-2 infection, we found that MERS-CoV lacking protein 8b expression was more virulent, while SARS-CoV-2 lacking protein 9b expression was attenuated compared with the respective wild-type viruses. Upon further analysis, we detected increased levels of type I interferon and enhanced infiltration of immune cells to the lungs of mice infected with MERS-CoV lacking protein 8b expression. These data suggest that the I protein of MERS-CoV plays a role in limiting pathogenesis while that of SARS-CoV-2 enhances disease severity. IMPORTANCE The function of betacoronavirus internal protein has been relatively understudied. The earliest report on the internal protein of mouse hepatitis virus suggested that the internal protein is a structural protein without significant functions in virus replication and virulence. However, the internal proteins of severe acute respiratory syndrome coronavirus (SARS-CoV), Middle-East respiratory syndrome coronavirus, and SARS-CoV-2 have been shown to evade immune responses. Despite the reported functions of the internal protein in these highly pathogenic human coronaviruses, its role in mediating pathogenesis in experimentally infected animals has not been characterized. Our data indicated that despite the similar genomic location and expression strategy of these internal proteins, their effects on virulence are vastly different and virus specific, highlighting the complexity between host-virus interaction and disease outcome.

Omicron subvariant BA.5 efficiently infects lung cells.

The SARS-CoV-2 Omicron subvariants BA.1 and BA.2 exhibit reduced lung cell infection relative to previously circulating SARS-CoV-2 variants, which may account for their reduced pathogenicity. However, it is unclear whether lung cell infection by BA.5, which displaced these variants, remains attenuated. Here, we show that the spike (S) protein of BA.5 exhibits increased cleavage at the S1/S2 site and drives cell-cell fusion and lung cell entry with higher efficiency than its counterparts from BA.1 and BA.2. Increased lung cell entry depends on mutation H69Δ/V70Δ and is associated with efficient replication of BA.5 in cultured lung cells. Further, BA.5 replicates in the lungs of female Balb/c mice and the nasal cavity of female ferrets with much higher efficiency than BA.1. These results suggest that BA.5 has acquired the ability to efficiently infect lung cells, a prerequisite for causing severe disease, suggesting that evolution of Omicron subvariants can result in partial loss of attenuation.

SARS-CoV-2-specific immunoglobulin Y antibodies are protective in infected mice.

Safe, passive immunization methods are required against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and its variants. Immunization of chickens with antigen is known to induce specific IgY antibodies concentrated in the egg yolk and has a good safety profile, high yield of IgY per egg, can be topically applied, not requiring parenteral delivery. Our data provide the first evidence of the prophylactic efficacy of Immunoglobulin Y antibodies against SARS-CoV-2 in mice. Lohmann hens were injected with recombinant SARS-CoV-2 RBD protein; IgY-Abs were extracted from the eggs and characterized using SDS-PAGE. Antiviral activity was evaluated using plaque reduction neutralization tests. In additional experiments, IgY-RBD efficacy was examined in mice sensitized to SARS-CoV-2 infection by transduction with Ad5-hACE2 (mild disease) or by using mouse-adapted virus (severe disease). In both cases, prophylactic intranasal administration of IgY-Abs reduced SARS-CoV-2 replication, and reduced morbidity, inflammatory cell infiltration, hemorrhage, and edema in the lungs and increased survival compared to control groups that received non-specific IgY-Abs. These results indicate that further evaluation of IgY-RBD antibodies in humans is warranted.

A Glycosylated RBD Protein Induces Enhanced Neutralizing Antibodies against Omicron and Other Variants with Improved Protection against SARS-CoV-2 Infection.

SARS-CoV-2 has mutated frequently since its first emergence in 2019. Numerous variants, including the currently emerging Omicron variant, have demonstrated high transmissibility or increased disease severity, posing serious threats to global public health. This study describes the identification of an immunodominant non-neutralizing epitope on SARS-CoV-2 receptor-binding domain (RBD). A subunit vaccine against this mutant RBD, constructed by masking this epitope with a glycan probe, did not significantly affect RBD's receptor-binding affinity or antibody-binding affinity, or its ability to induce antibody production. However, this vaccine enhanced the neutralizing activity of this RBD and its protective efficacy in immunized mice. Specifically, this vaccine elicited significantly higher-titer neutralizing antibodies than the prototypic RBD protein against Alpha (B.1.1.7 lineage), Beta (B.1.351 lineage), Gamma (P.1 lineage), and Epsilon (B.1.427 or B.1.429 lineage) variant pseudoviruses containing single or combined mutations in the spike (S) protein, albeit the neutralizing antibody titers against some variants were slightly lower than against original SARS-CoV-2. This vaccine also significantly improved the neutralizing activity of the prototypic RBD against pseudotyped and authentic Delta (B.1.617.2 lineage) and Omicron (B.1.1.529 lineage) variants, although the neutralizing antibody titers were lower than against original SARS-CoV-2. In contrast to the prototypic RBD, the mutant RBD completely protected human ACE2 (hACE2)-transgenic mice from lethal challenge with a prototype SARS-CoV-2 strain and a Delta variant without weight loss. Overall, these findings indicate that this RBD vaccine has broad-spectrum activity against multiple SARS-CoV-2 variants, as well as the potential to be effective and have improved efficacy against Omicron and other pandemic variants. IMPORTANCE Several SARS-CoV-2 variants have shown increased transmissibility, calling for a need to develop effective vaccines with broadly neutralizing activity against multiple variants. This study identified a non-neutralizing epitope on the receptor-binding domain (RBD) of SARS-CoV-2 spike protein, and further shielded it with a glycan probe. A subunit vaccine based on this mutant RBD significantly enhanced the ability of prototypic RBD against multiple SARS-CoV-2 variants, including the Delta and Omicron strains, although the neutralizing antibody titers against some of these variants were lower than those against original SARS-CoV-2. This mutant vaccine also enhanced the protective efficacy of the prototypic RBD vaccine against SARS-CoV-2 infection in immunized animals. In conclusion, this study identified an engineered RBD vaccine against Omicron and other SARS-CoV-2 variants that induced stronger neutralizing antibodies and protection than the original RBD vaccine. It also highlights the need to improve the effectiveness of current COVID-19 vaccines to prevent pandemic SARS-CoV-2 variants.

RBD-mRNA vaccine induces broadly neutralizing antibodies against Omicron and multiple other variants and protects mice from SARS-CoV-2 challenge.

Multiple SARS-CoV-2 variants are identified with higher rates of transmissibility or greater disease severity. Particularly, recent emergence of Omicron variant with rapid human-to-human transmission posts new challenges to the current prevention strategies. In this study, following vaccination with an mRNA vaccine encoding SARS-CoV-2 receptor-binding domain (RBD-mRNA), we detected serum antibodies that neutralized pseudoviruses expressing spike (S) protein harboring single or multiple mutations, as well as authentic SARS-CoV-2 variants, and evaluated its protection against SARS-CoV-2 infection. The vaccine induced durable antibodies that potently neutralized prototypic strain and B.1.1.7 lineage variant pseudoviruses containing N501Y or D614G mutations alone or in combination with a N439K mutation (B.1.258 lineage), with a L452R mutation (B.1.427 or B.1.429 lineage), or a L452R-E484Q double mutation (B.1.617.1 variant), although neutralizing activity against B.1.1.7 lineage variant containing 10 amino acid changes in the S protein was slightly reduced. The RBD-mRNA-induced antibodies exerted moderate neutralization against authentic B.1.617.2 and B.1.1.529 variants, and pseudotyped B.1.351 and P.1 lineage variants containing K417N/T, E484K, and N501Y mutations, the B.1.617.2 lineage variant harboring L452R, T478K, and P681R mutations, and the B.1.1.529 lineage variant containing 38 mutations in the S protein. Particularly, RBD-mRNA vaccine completely protected mice from challenge with a virulent mouse-adapted SARS-CoV-2 variant. Among these lineages, B.1.1.7, B.1.351, P.1, B.1.617.2, and B.1.1.529 belong to Alpha, Beta, Gamma, Delta, and Omicron variants, respectively. Our observations reveal that RBD-mRNA vaccine is promising and highlights the need to design novel vaccines with improved neutralization against current and future pandemic SARS-CoV-2 variants.

SARS-CoV-2 Omicron virus causes attenuated disease in mice and hamsters.

The recent emergence of B.1.1.529, the Omicron variant1,2, has raised concerns of escape from protection by vaccines and therapeutic antibodies. A key test for potential countermeasures against B.1.1.529 is their activity in preclinical rodent models of respiratory tract disease. Here, using the collaborative network of the SARS-CoV-2 Assessment of Viral Evolution (SAVE) programme of the National Institute of Allergy and Infectious Diseases (NIAID), we evaluated the ability of several B.1.1.529 isolates to cause infection and disease in immunocompetent and human ACE2 (hACE2)-expressing mice and hamsters. Despite modelling data indicating that B.1.1.529 spike can bind more avidly to mouse ACE2 (refs. 3,4), we observed less infection by B.1.1.529 in 129, C57BL/6, BALB/c and K18-hACE2 transgenic mice than by previous SARS-CoV-2 variants, with limited weight loss and lower viral burden in the upper and lower respiratory tracts. In wild-type and hACE2 transgenic hamsters, lung infection, clinical disease and pathology with B.1.1.529 were also milder than with historical isolates or other SARS-CoV-2 variants of concern. Overall, experiments from the SAVE/NIAID network with several B.1.1.529 isolates demonstrate attenuated lung disease in rodents, which parallels preliminary human clinical data.

The development of Nanosota-1 as anti-SARS-CoV-2 nanobody drug candidates.

Combating the COVID-19 pandemic requires potent and low-cost therapeutics. We identified a series of single-domain antibodies (i.e., nanobody), Nanosota-1, from a camelid nanobody phage display library. Structural data showed that Nanosota-1 bound to the oft-hidden receptor-binding domain (RBD) of SARS-CoV-2 spike protein, blocking viral receptor angiotensin-converting enzyme 2 (ACE2). The lead drug candidate possessing an Fc tag (Nanosota-1C-Fc) bound to SARS-CoV-2 RBD ~3000 times more tightly than ACE2 did and inhibited SARS-CoV-2 pseudovirus ~160 times more efficiently than ACE2 did. Administered at a single dose, Nanosota-1C-Fc demonstrated preventive and therapeutic efficacy against live SARS-CoV-2 infection in both hamster and mouse models. Unlike conventional antibodies, Nanosota-1C-Fc was produced at high yields in bacteria and had exceptional thermostability. Pharmacokinetic analysis of Nanosota-1C-Fc documented an excellent in vivo stability and a high tissue bioavailability. As effective and inexpensive drug candidates, Nanosota-1 may contribute to the battle against COVID-19.

Exponential increase in neutralizing and spike specific antibodies following vaccination of COVID-19 convalescent plasma donors.

BACKGROUND: With the recent approval of COVID-19 vaccines, recovered COVID-19 subjects who are vaccinated may be ideal candidates to donate COVID-19 convalescent plasma (CCP). CASE SERIES: Eleven recovered COVID-19 patients were screened to donate CCP. All had molecularly confirmed COVID-19, and all but one were antibody positive by chemiluminescence immunoassay (DiaSorin) prior to vaccination. All were tested again for antibodies 11-21 days after they were vaccinated (Pfizer/Moderna). All showed dramatic increases (~50-fold) in spike-specific antibody levels and had at least a 20-fold increase in the IC50 neutralizing antibody titer based on plaque reduction neutralization testing (PRNT). The spike-specific antibody levels following vaccination were significantly higher than those seen in any non-vaccinated COVID-19 subjects tested to date at our facility. CONCLUSION: Spike-specific and neutralizing antibodies demonstrated dramatic increases following a single vaccination after COVID-19 infection, which significantly exceeded values seen with COVID-19 infection alone. Recovered COVID-19 subjects who are vaccinated may make ideal candidates for CCP donation.

The SARS-CoV-2 B.1.1.529 Omicron virus causes attenuated infection and disease in mice and hamsters.

Despite the development and deployment of antibody and vaccine countermeasures, rapidly-spreading SARS-CoV-2 variants with mutations at key antigenic sites in the spike protein jeopardize their efficacy. The recent emergence of B.1.1.529, the Omicron variant1,2, which has more than 30 mutations in the spike protein, has raised concerns for escape from protection by vaccines and therapeutic antibodies. A key test for potential countermeasures against B.1.1.529 is their activity in pre-clinical rodent models of respiratory tract disease. Here, using the collaborative network of the SARS-CoV-2 Assessment of Viral Evolution (SAVE) program of the National Institute of Allergy and Infectious Diseases (NIAID), we evaluated the ability of multiple B.1.1.529 Omicron isolates to cause infection and disease in immunocompetent and human ACE2 (hACE2) expressing mice and hamsters. Despite modeling and binding data suggesting that B.1.1.529 spike can bind more avidly to murine ACE2, we observed attenuation of infection in 129, C57BL/6, and BALB/c mice as compared with previous SARS-CoV-2 variants, with limited weight loss and lower viral burden in the upper and lower respiratory tracts. Although K18-hACE2 transgenic mice sustained infection in the lungs, these animals did not lose weight. In wild-type and hACE2 transgenic hamsters, lung infection, clinical disease, and pathology with B.1.1.529 also were milder compared to historical isolates or other SARS-CoV-2 variants of concern. Overall, experiments from multiple independent laboratories of the SAVE/NIAID network with several different B.1.1.529 isolates demonstrate attenuated lung disease in rodents, which parallels preliminary human clinical data.

The Development of a Novel Nanobody Therapeutic for SARS-CoV-2.

Combating the COVID-19 pandemic requires potent and low-cost therapeutics. We identified a novel series of single-domain antibodies (i.e., nanobody), Nanosota-1, from a camelid nanobody phage display library. Structural data showed that Nanosota-1 bound to the oft-hidden receptor-binding domain (RBD) of SARS-CoV-2 spike protein, blocking out viral receptor ACE2. The lead drug possessing an Fc tag ( Nanosota-1C-Fc ) bound to SARS-CoV-2 RBD with a K d of 15.7picomolar (∼3000 times more tightly than ACE2 did) and inhibited SARS-CoV-2 infection with an ND 50 of 0.16microgram/milliliter (∼6000 times more potently than ACE2 did). Administered at a single dose, Nanosota-1C-Fc demonstrated preventive and therapeutic efficacy in hamsters subjected to SARS-CoV-2 infection. Unlike conventional antibody drugs, Nanosota-1C-Fc was produced at high yields in bacteria and had exceptional thermostability. Pharmacokinetic analysis of Nanosota-1C-F c documented a greater than 10-day in vivo half-life efficacy and high tissue bioavailability. Nanosota-1C-Fc is a potentially effective and realistic solution to the COVID-19 pandemic. IMPACT STATEMENT: Potent and low-cost Nanosota-1 drugs block SARS-CoV-2 infections both in vitro and in vivo and act both preventively and therapeutically.