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.

Antibody-independent protection against heterologous SARS-CoV-2 challenge conferred by prior infection or vaccination.

Vaccines have reduced severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) morbidity and mortality, yet emerging variants challenge their effectiveness. The prevailing approach to updating vaccines targets the antibody response, operating under the presumption that it is the primary defense mechanism following vaccination or infection. This perspective, however, can overlook the role of T cells, particularly when antibody levels are low or absent. Here we show, through studies in mouse models lacking antibodies but maintaining functional B cells and lymphoid organs, that immunity conferred by prior infection or mRNA vaccination can protect against SARS-CoV-2 challenge independently of antibodies. Our findings, using three distinct models inclusive of a novel human/mouse ACE2 hybrid, highlight that CD8+ T cells are essential for combating severe infections, whereas CD4+ T cells contribute to managing milder cases, with interferon-γ having an important function in this antibody-independent defense. These findings highlight the importance of T cell responses in vaccine development, urging a broader perspective on protective immunity beyond just antibodies.

Adaptation of SARS-CoV-2 to ACE2H353K mice reveals new spike residues that drive mouse infection.

The Coronavirus Disease 2019 (COVID-19) pandemic continues to cause extraordinary loss of life and economic damage. Animal models of severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) infection are needed to better understand disease pathogenesis and evaluate preventive measures and therapies. While mice are widely used to model human disease, mouse angiotensin converting enzyme 2 (ACE2) does not bind the ancestral SARS-CoV-2 spike protein to mediate viral entry. To overcome this limitation, we "humanized" mouse Ace2 using CRISPR gene editing to introduce a single amino acid substitution, H353K, predicted to facilitate S protein binding. While H353K knockin Ace2 (mACE2H353K) mice supported SARS-CoV-2 infection and replication, they exhibited minimal disease manifestations. Following 30 serial passages of ancestral SARS-CoV-2 in mACE2H353K mice, we generated and cloned a more virulent virus. A single isolate (SARS2MA-H353K) was prepared for detailed studies. In 7-11-month-old mACE2H353K mice, a 104 PFU inocula resulted in diffuse alveolar disease manifested as edema, hyaline membrane formation, and interstitial cellular infiltration/thickening. Unexpectedly, the mouse-adapted virus also infected standard BALB/c and C57BL/6 mice and caused severe disease. The mouse-adapted virus acquired five new missense mutations including two in spike (K417E, Q493K), one each in nsp4, nsp9, and M and a single nucleotide change in the 5' untranslated region. The Q493K spike mutation arose early in serial passage and is predicted to provide affinity-enhancing molecular interactions with mACE2 and further increase the stability and affinity to the receptor. This new model and mouse-adapted virus will be useful to evaluate COVID-19 disease and prophylactic and therapeutic interventions.IMPORTANCEWe developed a new mouse model with a humanized angiotensin converting enzyme 2 (ACE2) locus that preserves native regulatory elements. A single point mutation in mouse ACE2 (H353K) was sufficient to confer in vivo infection with ancestral severe acute respiratory syndrome-coronavirus-2 virus. Through in vivo serial passage, a virulent mouse-adapted strain was obtained. In aged mACE2H353K mice, the mouse-adapted strain caused diffuse alveolar disease. The mouse-adapted virus also infected standard BALB/c and C57BL/6 mice, causing severe disease. The mouse-adapted virus acquired five new missense mutations including two in spike (K417E, Q493K), one each in nsp4, nsp9, and M and a single nucleotide change in the 5' untranslated region. The Q493K spike mutation arose early in serial passage and is predicted to provide affinity-enhancing molecular interactions with mACE2 and further increase the stability and affinity to the receptor. This new model and mouse-adapted virus will be useful to evaluate COVID-19 disease and prophylactic and therapeutic interventions.

Potent 3CLpro inhibitors effective against SARS-CoV-2 and MERS-CoV in animal models by therapeutic treatment.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and Middle East respiratory syndrome coronavirus (MERS-CoV) are zoonotic betacoronaviruses that continue to have a significant impact on public health. Timely development and introduction of vaccines and antivirals against SARS-CoV-2 into the clinic have substantially mitigated the burden of COVID-19. However, a limited or lacking therapeutic arsenal for SARS-CoV-2 and MERS-CoV infections, respectively, calls for an expanded and diversified portfolio of antivirals against these coronavirus infections. In this report, we examined the efficacy of two potent 3CLpro inhibitors, 5d and 11d, in fatal animal models of SARS-CoV-2 and MERS-CoV to demonstrate their broad-spectrum activity against both viral infections. These compounds significantly increased the survival of mice in both models when treatment started 1 day post infection compared to no treatment which led to 100% fatality. Especially, the treatment with compound 11d resulted in 80% and 90% survival in SARS-CoV-2 and MERS-CoV-infected mice, respectively. Amelioration of lung viral load and histopathological changes in treated mice correlated well with improved survival in both infection models. Furthermore, compound 11d exhibited significant antiviral activities in K18-hACE2 mice infected with SARS-CoV-2 Omicron subvariant XBB.1.16. The results suggest that these are promising candidates for further development as broad-spectrum direct-acting antivirals against highly virulent human coronaviruses.IMPORTANCEHuman coronaviruses such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and Middle East respiratory syndrome coronavirus (MERS-CoV) continue to have a significant impact on public health. A limited or lacking therapeutic arsenal for SARS-CoV-2 and MERS-CoV infections calls for an expanded and diversified portfolio of antivirals against these coronavirus infections. We have previously reported a series of small-molecule 3C-like protease (3CLpro) inhibitors against human coronaviruses. In this report, we demonstrated the in vivo efficacy of 3CLpro inhibitors for their broad-spectrum activity against both SARS-CoV-2 and MERS-CoV infections using the fatal animal models. The results suggest that these are promising candidates for further development as broad-spectrum direct-acting antivirals against highly virulent human coronaviruses.

Mosaic RBD Nanoparticles Elicit Protective Immunity Against Multiple Human Coronaviruses in Animal Models.

To combat SARS-CoV-2 variants and MERS-CoV, as well as the potential re-emergence of SARS-CoV and spillovers of sarbecoviruses, which pose a significant threat to global public health, vaccines that can confer broad-spectrum protection against betacoronaviruses (ß-CoVs) are urgently needed. A mosaic ferritin nanoparticle vaccine is developed that co-displays the spike receptor-binding domains of SARS-CoV, MERS-CoV, and SARS-CoV-2 Wild-type (WT) strain and evaluated its immunogenicity and protective efficacy in mice and nonhuman primates. A low dose of 10 µg administered at a 21-day interval induced a Th1-biased immune response in mice and elicited robust cross-reactive neutralizing antibody responses against a variety of ß-CoVs, including a series of SARS-CoV-2 variants. It is also able to effectively protect against challenges of SARS-CoV, MERS-CoV, and SARS-CoV-2 variants in not only young mice but also the more vulnerable mice through induction of long-lived immunity. Together, these results suggest that this mosaic 3-RBD nanoparticle has the potential to be developed as a pan-ß-CoV vaccine.

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.

Discovery of Nanosota-2, -3, and -4 as super potent and broad-spectrum therapeutic nanobody candidates against COVID-19.

IMPORTANCE: The COVID-19 pandemic exposed limitations of conventional antibodies as therapeutics, including high cost, limited potency, ineffectiveness against new viral variants, and primary reliance on injection-only delivery. Nanobodies are single-domain antibodies with therapeutic potentials. We discovered three anti-SARS-CoV-2 nanobodies, named Nanosota-2, -3, and -4, from an immunized alpaca. Nanosota-2 is super potent against prototypic SARS-CoV-2, Nanosota-3 is highly potent against the omicron variant, and Nanosota-4 is effective against both SARS-CoV-1 and SARS-CoV-2. In addition to their super potency and combined broad antiviral spectrum, these nanobodies are cost-effective, can be easily adapted to new viral variants through phage display, and can potentially be administered as inhalers. The Nanosota series are powerful therapeutic candidates to combat circulating SARS-CoV-2 and prepare for possible future coronavirus pandemics.

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.

Glycosylated Delta-receptor-binding domain mucosal vaccine elicits broadly neutralizing antibodies with protection against SARS-CoV-2 challenge.

Mucosal COVID-19 vaccines are needed to block SARS-CoV-2 infection at the mucosal site. Intranasal delivery of a glycosylated Delta variant receptor-binding domain (Delta-RBD) mucosal vaccine elicited potent and balanced systemic antibody titers comparable to those induced by the intramuscular injection of the same vaccine or Omicron-S subunit vaccine, as well as high mucosal IgA antibody responses. It elicited broadly neutralizing antibodies against the original SARS-CoV-2 strain, Delta and Omicron BA1/BA2 variants, completely protecting transgenic mice from lethal challenge with a Delta variant, including complete absence of weight loss. Of note, intramuscular priming with the Omicron-S protein followed by intranasal boosting with the Delta-RBD protein improved the vaccine's ability to generate broad-spectrum neutralizing antibodies against recent BA5 and XBB Omicron variants. Overall, this vaccine has the potential to prevent the SARS-CoV-2 infection of the respiratory mucosa, while the i.m. priming and i.n. boosting vaccination strategy may offer protection against known and emerging SARS-CoV-2 variants.

Monovalent SARS-CoV-2 mRNA vaccine does not boost Omicron-specific immune response in diabetic and control pediatric patients.

While the immunogenicity of SARS-CoV-2 vaccines has been well described in adults, pediatric populations have been less studied. In particular, children with type 1 diabetes are generally at elevated risk for more severe disease after infections, but are understudied in terms of COVID-19 and SARS-CoV-2 vaccine responses. We investigated the immunogenicity of COVID-19 mRNA vaccinations in 35 children with type 1 diabetes (T1D) and 23 controls and found that these children develop levels of SARS-CoV-2 neutralizing antibody titers and spike protein-specific T cells comparable to non-diabetic children. However, in comparing the neutralizing antibody responses in children who received two doses of mRNA vaccines (24 T1D; 14 controls) with those who received a third, booster dose (11 T1D; 9 controls), we found that the booster dose increased neutralizing antibody titers against ancestral SARS-CoV-2 strains but, unexpectedly, not omicron lineage variants. In contrast, boosting enhanced omicron variant neutralizing antibody titers in adults.

Nsp3-N interactions are critical for SARS-CoV-2 fitness and virulence.

SARS-CoV-2, the causative agent of COVID-19 encodes at least 16 nonstructural proteins of variably understood function. Nsp3, the largest nonstructural protein contains several domains, including a SARS-unique domain (SUD), which occurs only in Sarbecovirus. The SUD has a role in preferentially enhancing viral translation. During isolation of mouse-adapted SARS-CoV-2, we isolated an attenuated virus that contained a single mutation in a linker region of nsp3 (nsp3-S676T). The S676T mutation decreased virus replication in cultured cells and primary human cells and in mice. Nsp3-S676T alleviated the SUD translational enhancing ability by decreasing the interaction between two translation factors, Paip1 and PABP1. We also identified a compensatory mutation in the nucleocapsid (N) protein (N-S194L) that restored the virulent phenotype, without directly binding to SUD. Together, these results reveal an aspect of nsp3-N interactions, which impact both SARS-CoV-2 replication and, consequently, pathogenesis.

MERS-CoV RBD-mRNA vaccine induces potent and broadly neutralizing antibodies with protection against MERS-CoV infection.

Middle East respiratory syndrome coronavirus (MERS-CoV), a highly pathogenic coronavirus in the same Betacoronavirus genus and Coronaviridae family as SARS-CoV-2, continues to post a threat to human health. Mortality remains high; therefore, there is a need to develop effective vaccines to prevent MERS-CoV infection. The receptor-binding domain (RBD) within the MERS-CoV spike (S) protein is a critical vaccine target. The latest mRNA technology has enabled rapid development of much-needed vaccines with high efficiency and scalable manufacturing capacity. Here, we designed a mRNA vaccine encoding the RBD of MERS-CoV S protein (RBD-mRNA) and evaluated its immunogenicity and protective efficacy in a mouse model. The data showed that nucleoside-modified RBD-mRNA, but not RBD-mRNA lacking the nucleoside modification, was stable and elicited broadly and durable neutralizing antibody and cellular immune responses, which neutralized the original strain and multiple MERS-CoV variants. Among all immunization routes tested, the intradermal route was appropriate for this RBD-mRNA to induce strong B-cell responses and the highest neutralizing antibody titers. Importantly, injection of nucleoside-modified RBD-mRNA through the intradermal route protected immunized mice against challenge with MERS-CoV. This protection correlated with serum neutralizing antibody titers. Overall, we have developed an effective MERS-CoV RBD-based mRNA vaccine (with potential for further development) that prevents infection by divergent strains of MERS-CoV.

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.

What's what in a pandemic? Virus, disease, and societal disaster must be differentiated.

Viruses, the diseases they can trigger, and the possible associated societal disaster represent different entities. To engage with the complexities of viral pandemics, we need to recognize each entity by using a distinctive name.

ICTV Virus Taxonomy Profile: Coronaviridae 2023.

The family Coronaviridae includes viruses with positive-sense RNA genomes of 22-36 kb that are expressed through a nested set of 3' co-terminal subgenomic mRNAs. Members of the subfamily Orthocoronavirinae are characterized by 80-160 nm diameter, enveloped virions with spike projections. The orthocoronaviruses, severe acute respiratory syndrome coronavirus and Middle East respiratory syndrome-related coronavirus are extremely pathogenic for humans and in the last two decades have been responsible for the SARS and MERS epidemics. Another orthocoronavirus, severe acute respiratory syndrome coronavirus 2, was responsible for the recent global COVID-19 pandemic. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Coronaviridae which is available at www.ictv.global/report/coronaviridae.

Nirmatrelvir treatment of SARS-CoV-2-infected mice blunts antiviral adaptive immune responses.

Alongside vaccines, antiviral drugs are becoming an integral part of our response to the SARS-CoV-2 pandemic. Nirmatrelvir-an orally available inhibitor of the 3-chymotrypsin-like cysteine protease-has been shown to reduce the risk of progression to severe COVID-19. However, the impact of nirmatrelvir treatment on the development of SARS-CoV-2-specific adaptive immune responses is unknown. Here, by using mouse models of SARS-CoV-2 infection, we show that nirmatrelvir administration blunts the development of SARS-CoV-2-specific antibody and T cell responses. Accordingly, upon secondary challenge, nirmatrelvir-treated mice recruited significantly fewer memory T and B cells to the infected lungs and mediastinal lymph nodes, respectively. Together, the data highlight a potential negative impact of nirmatrelvir treatment with important implications for clinical management and might help explain the virological and/or symptomatic relapse after treatment completion reported in some individuals.