Vaccine-induced protection against SARS-CoV-2 requires IFN-γ-driven cellular immune response.

The overall success of worldwide mass vaccination in limiting the negative effect of the COVID-19 pandemics is inevitable, however, recent SARS-CoV-2 variants of concern, especially Omicron and its sub-lineages, efficiently evade humoral immunity mounted upon vaccination or previous infection. Thus, it is an important question whether these variants, or vaccines against them, induce anti-viral cellular immunity. Here we show that the mRNA vaccine BNT162b2 induces robust protective immunity in K18-hACE2 transgenic B-cell deficient (µMT) mice. We further demonstrate that the protection is attributed to cellular immunity depending on robust IFN-γ production. Viral challenge with SARS-CoV-2 Omicron BA.1 and BA.5.2 sub-variants induce boosted cellular responses in vaccinated µMT mice, which highlights the significance of cellular immunity against the ever-emerging SARS-CoV-2 variants evading antibody-mediated immunity. Our work, by providing evidence that BNT162b2 can induce significant protective immunity in mice that are unable to produce antibodies, thus highlights the importance of cellular immunity in the protection against SARS-CoV-2.

Intranasal infection by SARS-CoV-2 Omicron variants can induce inflammatory brain damage in newly weaned hamsters.

SUMMARY: Intranasal infection of newly-weaned Syrian hamsters by SARS-CoV-2 Omicron variants can lead to brain inflammation and neuron degeneration with detectable low level of viral load and sparse expression of viral nucleoprotein.

Human airway and nasal organoids reveal escalating replicative fitness of SARS-CoV-2 emerging variants.

The high transmissibility of SARS-CoV-2 Omicron subvariants was generally ascribed to immune escape. It remained unclear whether the emerging variants have gradually acquired replicative fitness in human respiratory epithelial cells. We sought to evaluate the replicative fitness of BA.5 and earlier variants in physiologically active respiratory organoids. BA.5 exhibited a dramatically increased replicative capacity and infectivity than B.1.1.529 and an ancestral strain wildtype (WT) in human nasal and airway organoids. BA.5 spike pseudovirus showed a significantly higher entry efficiency than that carrying WT or B.1.1.529 spike. Notably, we observed prominent syncytium formation in BA.5-infected nasal and airway organoids, albeit elusive in WT- and B.1.1.529-infected organoids. BA.5 spike-triggered syncytium formation was verified by lentiviral overexpression of spike in nasal organoids. Moreover, BA.5 replicated modestly in alveolar organoids, with a significantly lower titer than B.1.1.529 and WT. Collectively, the higher entry efficiency and fusogenic activity of BA.5 spike potentiated viral spread through syncytium formation in the human airway epithelium, leading to enhanced replicative fitness and immune evasion, whereas the attenuated replicative capacity of BA.5 in the alveolar organoids may account for its benign clinical manifestation.

A new generation Mpro inhibitor with potent activity against SARS-CoV-2 Omicron variants.

Emerging SARS-CoV-2 variants, particularly the Omicron variant and its sublineages, continually threaten the global public health. Small molecule antivirals are an effective treatment strategy to fight against the virus. However, the first-generation antivirals either show limited clinical efficacy and/or have some defects in pharmacokinetic (PK) properties. Moreover, with increased use of these drugs across the globe, they face great pressure of drug resistance. We herein present the discovery and characterization of a new generation antiviral drug candidate (SY110), which is a potent and selective inhibitor of SARS-CoV-2 main protease (Mpro). This compound displayed potent in vitro antiviral activity against not only the predominant SARS-CoV-2 Omicron sublineage BA.5, but also other highly pathogenic human coronaviruses including SARS-CoV-1 and MERS-CoV. In the Omicron-infected K18-hACE2 mouse model, oral treatment with SY110 significantly lowered the viral burdens in lung and alleviated the virus-induced pathology. Importantly, SY110 possesses favorable PK properties with high oral drug exposure and oral bioavailability, and also an outstanding safety profile. Furthermore, SY110 exhibited sensitivity to several drug-resistance Mpro mutations. Collectively, this investigation provides a promising new drug candidate against Omicron and other variants of SARS-CoV-2.

COVID-19 mRNA vaccine protects against SARS-CoV-2 Omicron BA.1 infection in diet-induced obese mice through boosting host innate antiviral responses.

BACKGROUND: Obesity is a worldwide epidemic and is considered a risk factor of severe manifestation of Coronavirus Disease 2019 (COVID-19). The pathogenicity of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and host responses to infection, re-infection, and vaccination in individuals with obesity remain incompletely understood. METHODS: Using the diet-induced obese (DIO) mouse model, we studied SARS-CoV-2 Alpha- and Omicron BA.1-induced disease manifestations and host immune responses to infection, re-infection, and COVID-19 mRNA vaccination. FINDINGS: Unlike in lean mice, Omicron BA.1 and Alpha replicated to comparable levels in the lungs of DIO mice and resulted in similar degree of tissue damages. Importantly, both T cell and B cell mediated adaptive immune responses to SARS-CoV-2 infection or COVID-19 mRNA vaccination are impaired in DIO mice, leading to higher propensity of re-infection and lower vaccine efficacy. However, despite the absence of neutralizing antibody, vaccinated DIO mice are protected from lung damage upon Omicron challenge, accompanied with significantly more IFN-α and IFN-ß production in the lung tissue. Lung RNAseq and subsequent experiments indicated that COVID-19 mRNA vaccination in DIO mice boosted antiviral innate immune response, including the expression of IFN-α, when compared to the nonvaccinated controls. INTERPRETATION: Our findings suggested that COVID-19 mRNA vaccination enhances host innate antiviral responses in obesity which protect the DIO mice to a certain degree when adaptive immunity is suboptimal. FUNDING: A full list of funding bodies that contributed to this study can be found in the Acknowledgements section.

Intranasal Boosting with Spike Fc-RBD of Wild-Type SARS-CoV-2 Induces Neutralizing Antibodies against Omicron Subvariants and Reduces Viral Load in the Nasal Turbinate of Mice.

The emergence of new immune-evasive severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants and subvariants outpaces the development of vaccines specific against the dominant circulating strains. In terms of the only accepted immune correlate of protection, the inactivated whole-virion vaccine using wild-type SARS-CoV-2 spike induces a much lower serum neutralizing antibody titre against the Omicron subvariants. Since the inactivated vaccine given intramuscularly is one of the most commonly used coronavirus disease 2019 (COVID-19) vaccines in developing regions, we tested the hypothesis that intranasal boosting after intramuscular priming would provide a broader level of protection. Here, we showed that one or two intranasal boosts with the Fc-linked trimeric spike receptor-binding domain from wild-type SARS-CoV-2 can induce significantly higher serum neutralizing antibodies against wild-type SARS-CoV-2 and the Omicron subvariants, including BA.5.2 and XBB.1, with a lower titre in the bronchoalveolar lavage of vaccinated Balb/c mice than vaccination with four intramuscular doses of inactivated whole virion vaccine. The intranasally vaccinated K18-hACE2-transgenic mice also had a significantly lower nasal turbinate viral load, suggesting a better protection of the upper airway, which is the predilected site of infection by Omicron subvariants. This intramuscular priming and intranasal boosting approach that achieves broader cross-protection against Omicron variants and subvariants may lengthen the interval required for changing the vaccine immunogen from months to years.

Virological features and pathogenicity of SARS-CoV-2 Omicron BA.2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron BA.2 was a dominant circulating SARS-CoV-2 variant worldwide. Recent reports hint that BA.2 is similarly potent regarding antibody evasion but may be more transmissible than BA.1. The pathogenicity of BA.2 remains unclear and is of critical public health significance. Here we investigated the virological features and pathogenicity of BA.2 with in vitro and in vivo models. We show that BA.2 is less dependent on transmembrane protease serine 2 (TMPRSS2) for virus entry in comparison with BA.1 in vitro. In K18-hACE2 mice, BA.2 replicates more efficiently than BA.1 in the nasal turbinates and replicates marginally less efficiently in the lungs, leading to decreased body weight loss and improved survival. Our study indicates that BA.2 is similarly attenuated in lungs compared with BA.1 but is potentially more transmissible because of its better replication at the nasal turbinates.

Altered host protease determinants for SARS-CoV-2 Omicron.

Successful severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection requires proteolytic cleavage of the viral spike protein. While the role of the host transmembrane protease serine 2 in SARS-CoV-2 infection is widely recognized, the involvement of other proteases capable of facilitating SARS-CoV-2 entry remains incompletely explored. Here, we show that multiple members from the membrane-type matrix metalloproteinase (MT-MMP) and a disintegrin and metalloproteinase families can mediate SARS-CoV-2 entry. Inhibition of MT-MMPs significantly reduces SARS-CoV-2 replication in vitro and in vivo. Mechanistically, we show that MT-MMPs can cleave SARS-CoV-2 spike and angiotensin-converting enzyme 2 and facilitate spike-mediated fusion. We further demonstrate that Omicron BA.1 has an increased efficiency on MT-MMP usage, while an altered efficiency on transmembrane serine protease usage for virus entry compared with that of ancestral SARS-CoV-2. These results reveal additional protease determinants for SARS-CoV-2 infection and enhance our understanding on the biology of coronavirus entry.

Comparative analysis of SARS-CoV-2 Omicron BA.2.12.1 and BA.5.2 variants.

The initial severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron subvariants, BA.1 and BA.2, are being progressively displaced by BA.5 in many countries. To provide insight on the replacement of BA.2 by BA.5 as the dominant SARS-CoV-2 variant, we performed a comparative analysis of Omicron BA.2.12.1 and BA.5.2 variants in cell culture and hamster models. We found that BA.5.2 exhibited enhanced replicative kinetics over BA.2.12.1 in vitro and in vivo, which is evidenced by the dominant BA.5.2 viral genome detected at different time points, regardless of immune selection pressure with vaccine-induced serum antibodies. Utilizing reverse genetics, we constructed a mutant SARS-CoV-2 carrying spike F486V substitution, which is an uncharacterized mutation that concurrently discriminates Omicron BA.5.2 from BA.2.12.1 variant. We noticed that the 486th residue does not confer viral replication advantage to the virus. We also found that 486V displayed generally reduced immune evasion capacity when compared with its predecessor, 486F. However, the surge of fitness in BA.5.2 over BA.2.12.1 was not due to stand-alone F486V substitution but as a result of the combination of multiple mutations. Our study upholds the urgency for continuous monitoring of SARS-CoV-2 Omicron variants with enhanced replication fitness.

Analogous comparison unravels heightened antiviral defense and boosted viral infection upon immunosuppression in bat organoids.

Horseshoe bats host numerous SARS-related coronaviruses without overt disease signs. Bat intestinal organoids, a unique model of bat intestinal epithelium, allow direct comparison with human intestinal organoids. We sought to unravel the cellular mechanism(s) underlying bat tolerance of coronaviruses by comparing the innate immunity in bat and human organoids. We optimized the culture medium, which enabled a consecutive passage of bat intestinal organoids for over one year. Basal expression levels of IFNs and IFN-stimulated genes were higher in bat organoids than in their human counterparts. Notably, bat organoids mounted a more rapid, robust and prolonged antiviral defense than human organoids upon Poly(I:C) stimulation. TLR3 and RLR might be the conserved pathways mediating antiviral response in bat and human intestinal organoids. The susceptibility of bat organoids to a bat coronavirus CoV-HKU4, but resistance to EV-71, an enterovirus of exclusive human origin, indicated that bat organoids adequately recapitulated the authentic susceptibility of bats to certain viruses. Importantly, TLR3/RLR inhibition in bat organoids significantly boosted viral growth in the early phase after SARS-CoV-2 or CoV-HKU4 infection. Collectively, the higher basal expression of antiviral genes, especially more rapid and robust induction of innate immune response, empowered bat cells to curtail virus propagation in the early phase of infection.

Air and environmental sampling for SARS-CoV-2 around hospitalized patients with coronavirus disease 2019 (COVID-19).

BACKGROUND: The role of severe respiratory coronavirus virus 2 (SARS-CoV-2)-laden aerosols in the transmission of coronavirus disease 2019 (COVID-19) remains uncertain. Discordant findings of SARS-CoV-2 RNA in air samples were noted in early reports. METHODS: Sampling of air close to 6 asymptomatic and symptomatic COVID-19 patients with and without surgical masks was performed with sampling devices using sterile gelatin filters. Frequently touched environmental surfaces near 21 patients were swabbed before daily environmental disinfection. The correlation between the viral loads of patients' clinical samples and environmental samples was analyzed. RESULTS: All air samples were negative for SARS-CoV-2 RNA in the 6 patients singly isolated inside airborne infection isolation rooms (AIIRs) with 12 air changes per hour. Of 377 environmental samples near 21 patients, 19 (5.0%) were positive by reverse-transcription polymerase chain reaction (RT-PCR) assay, with a median viral load of 9.2 × 102 copies/mL (range, 1.1 × 102 to 9.4 × 104 copies/mL). The contamination rate was highest on patients' mobile phones (6 of 77, 7.8%), followed by bed rails (4 of 74, 5.4%) and toilet door handles (4 of 76, 5.3%). We detected a significant correlation between viral load ranges in clinical samples and positivity rate of environmental samples (P < .001). CONCLUSION: SARS-CoV-2 RNA was not detectable by air samplers, which suggests that the airborne route is not the predominant mode of transmission of SARS-CoV-2. Wearing a surgical mask, appropriate hand hygiene, and thorough environmental disinfection are sufficient infection control measures for COVID-19 patients isolated singly in AIIRs. However, this conclusion may not apply during aerosol-generating procedures or in cohort wards with large numbers of COVID-19 patients.

A molecularly engineered, broad-spectrum anti-coronavirus lectin inhibits SARS-CoV-2 and MERS-CoV infection in vivo.

"Pan-coronavirus" antivirals targeting conserved viral components can be designed. Here, we show that the rationally engineered H84T-banana lectin (H84T-BanLec), which specifically recognizes high mannose found on viral proteins but seldom on healthy human cells, potently inhibits Middle East respiratory syndrome coronavirus (MERS-CoV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (including Omicron), and other human-pathogenic coronaviruses at nanomolar concentrations. H84T-BanLec protects against MERS-CoV and SARS-CoV-2 infection in vivo. Importantly, intranasally and intraperitoneally administered H84T-BanLec are comparably effective. Mechanistic assays show that H84T-BanLec targets virus entry. High-speed atomic force microscopy depicts real-time multimolecular associations of H84T-BanLec dimers with the SARS-CoV-2 spike trimer. Single-molecule force spectroscopy demonstrates binding of H84T-BanLec to multiple SARS-CoV-2 spike mannose sites with high affinity and that H84T-BanLec competes with SARS-CoV-2 spike for binding to cellular ACE2. Modeling experiments identify distinct high-mannose glycans in spike recognized by H84T-BanLec. The multiple H84T-BanLec binding sites on spike likely account for the drug compound's broad-spectrum antiviral activity and the lack of resistant mutants.

Dual‐Functional Anti‐Pathogen Coatings

A dual-functional anti-pathogenic coating with controllable repelling and capturing/inactivation of pathogens, which make it capable of eliminating a broad range of pathogenic bacteria and viruses, including SARS-CoV-2, is reported. The reversible switch between repelling and capturing/inactivation is readily made via its CO2-responsive wettability. In its superhydrophobic state, the coating enables a SARS-CoV-2 repellent efficacy of 99.9997%. In its superhydrophilic state, the coating has a virucidal efficacy of up to 99.9897%, which is comparable to the inactivation rate of chemical disinfectants. The coating is highly flexible with anti-corrosive and anti-frosting properties, which works for repelling essentially all pathogens and potentially could be applied into various daily used products and materials. The coating has the potential to set a new prevention standard in fighting the current pandemic and preventing future ones via especially intercepting the transmission of pathogens through contaminated surfaces.

Spike mutations contributing to the altered entry preference of SARS-CoV-2 omicron BA.1 and BA.2.

SARS-CoV-2 B.1.1.529.1 (Omicron BA.1) emerged in November 2021 and quickly became the predominant circulating SARS-CoV-2 variant globally. Omicron BA.1 contains more than 30 mutations in the spike protein, which contribute to its altered virological features when compared to the ancestral SARS-CoV-2 or previous SARS-CoV-2 variants. Recent studies by us and others demonstrated that Omicron BA.1 is less dependent on transmembrane serine protease 2 (TMPRSS2), less efficient in spike cleavage, less fusogenic, and adopts an altered propensity to utilize the plasma membrane and endosomal pathways for virus entry. Ongoing studies suggest that these virological features of Omicron BA.1 are in part retained by the subsequent Omicron sublineages. However, the exact spike determinants that contribute to these altered features of Omicron remain incompletely understood. In this study, we investigated the spike determinants for the observed virological characteristics of Omicron. By screening for the individual changes on Omicron BA.1 and BA.2 spike, we identify that 69-70 deletion, E484A, and H655Y contribute to the reduced TMPRSS2 usage while 25-27 deletion, S375F, and T376A result in less efficient spike cleavage. Among the shared spike mutations of BA.1 and BA.2, S375F and H655Y reduce spike-mediated fusogenicity. Interestingly, the H655Y change consistently reduces serine protease usage while increases the use of endosomal proteases. In keeping with these findings, the H655Y substitution alone reduces plasma membrane entry and facilitates endosomal entry when compared to SARS-CoV-2 WT. Overall, our study identifies key changes in Omicron spike that contributes to our understanding on the virological determinant and pathogenicity of Omicron.

Human Nasal Organoids Model SARS-CoV-2 Upper Respiratory Infection and Recapitulate the Differential Infectivity of Emerging Variants.

The human upper respiratory tract, specifically the nasopharyngeal epithelium, is the entry portal and primary infection site of respiratory viruses. Productive infection of SARS-CoV-2 in the nasal epithelium constitutes the cellular basis of viral pathogenesis and transmissibility. Yet a robust and well-characterized in vitro model of the nasal epithelium remained elusive. Here we report an organoid culture system of the nasal epithelium. We derived nasal organoids from easily accessible nasal epithelial cells with a perfect establishment rate. The derived nasal organoids were consecutively passaged for over 6 months. We then established differentiation protocols to generate 3-dimensional differentiated nasal organoids and organoid monolayers of 2-dimensional format that faithfully simulate the nasal epithelium. Moreover, when differentiated under a slightly acidic pH, the nasal organoid monolayers represented the optimal correlate of the native nasal epithelium for modeling the high infectivity of SARS-CoV-2, superior to all existing organoid models. Notably, the differentiated nasal organoid monolayers accurately recapitulated higher infectivity and replicative fitness of the Omicron variant than the prior variants. SARS-CoV-2, especially the more transmissible Delta and Omicron variants, destroyed ciliated cells and disassembled tight junctions, thereby facilitating virus spread and transmission. In conclusion, we establish a robust organoid culture system of the human nasal epithelium for modeling upper respiratory infections and provide a physiologically-relevant model for assessing the infectivity of SARS-CoV-2 emerging variants. IMPORTANCE An in vitro model of the nasal epithelium is imperative for understanding cell biology and virus-host interaction in the human upper respiratory tract. Here we report an organoid culture system of the nasal epithelium. Nasal organoids were derived from readily accessible nasal epithelial cells with perfect efficiency and stably expanded for more than 6 months. The long-term expandable nasal organoids were induced maturation into differentiated nasal organoids that morphologically and functionally simulate the nasal epithelium. The differentiated nasal organoids adequately recapitulated the higher infectivity and replicative fitness of SARS-CoV-2 emerging variants than the ancestral strain and revealed viral pathogenesis such as ciliary damage and tight junction disruption. Overall, we established a human nasal organoid culture system that enables a highly efficient reconstruction and stable expansion of the human nasal epithelium in culture plates, thus providing a facile and robust tool in the toolbox of microbiologists.

The spike receptor-binding motif G496S substitution determines the replication fitness of SARS-CoV-2 Omicron sublineage.

The replication and pathogenicity of SARS-CoV-2 Omicron BA.2 are comparable to that of BA.1 in experimental animal models. However, BA.2 has rapidly emerged to overtake BA.1 to become the predominant circulating SARS-CoV-2 variant worldwide. Here, we compared the replication fitness of BA.1 and BA.2 in cell culture and in the Syrian hamster model of COVID-19. Using a reverse genetics approach, we found that the BA.1-specific spike mutation G496S compromises its replication fitness, which may contribute to BA.1 being outcompeted by BA.2 in the real world. Additionally, the BA.1-unique G496S substitution confers differentiated sensitivity to therapeutic monoclonal antibodies, which partially recapitulates the immunoevasive phenotype of BA.1 and BA.2. In summary, our study identified G496S as an important determinant during the evolutionary trajectory of SARS-CoV-2.

Coronaviruses exploit a host cysteine-aspartic protease for replication.

Highly pathogenic coronaviruses, including severe acute respiratory syndrome coronavirus 2 (refs. 1,2) (SARS-CoV-2), Middle East respiratory syndrome coronavirus3 (MERS-CoV) and SARS-CoV-1 (ref. 4), vary in their transmissibility and pathogenicity. However, infection by all three viruses results in substantial apoptosis in cell culture5-7 and in patient tissues8-10, suggesting a potential link between apoptosis and pathogenesis of coronaviruses. Here we show that caspase-6, a cysteine-aspartic protease of the apoptosis cascade, serves as an important host factor for efficient coronavirus replication. We demonstrate that caspase-6 cleaves coronavirus nucleocapsid proteins, generating fragments that serve as interferon antagonists, thus facilitating virus replication. Inhibition of caspase-6 substantially attenuates lung pathology and body weight loss in golden Syrian hamsters infected with SARS-CoV-2 and improves the survival of mice expressing human DPP4 that are infected with mouse-adapted MERS-CoV. Our study reveals how coronaviruses exploit a component of the host apoptosis cascade to facilitate virus replication.

A bipotential organoid model of respiratory epithelium recapitulates high infectivity of SARS-CoV-2 Omicron variant.

The airways and alveoli of the human respiratory tract are lined by two distinct types of epithelium, which are the primary targets of respiratory viruses. We previously established long-term expanding human lung epithelial organoids from lung tissues and developed a 'proximal' differentiation protocol to generate mucociliary airway organoids. However, a respiratory organoid system with bipotential of the airway and alveolar differentiation remains elusive. Here we defined a 'distal' differentiation approach to generate alveolar organoids from the same source for the derivation of airway organoids. The alveolar organoids consisting of type I and type II alveolar epithelial cells (AT1 and AT2, respectively) functionally simulate the alveolar epithelium. AT2 cells maintained in lung organoids serve as progenitor cells from which alveolar organoids derive. Moreover, alveolar organoids sustain a productive SARS-CoV-2 infection, albeit a lower replicative fitness was observed compared to that in airway organoids. We further optimized 2-dimensional (2D) airway organoids. Upon differentiation under a slightly acidic pH, the 2D airway organoids exhibit enhanced viral replication, representing an optimal in vitro correlate of respiratory epithelium for modeling the high infectivity of SARS-CoV-2. Notably, the higher infectivity and replicative fitness of the Omicron variant than an ancestral strain were accurately recapitulated in these optimized airway organoids. In conclusion, we have established a bipotential organoid culture system able to reproducibly expand the entire human respiratory epithelium in vitro for modeling respiratory diseases, including COVID-19.