An RBD bispecific antibody effectively neutralizes a SARS-CoV-2 Omicron variant

Potent neutralizing antibodies (nAbs) against SARS-CoV-2 are a promising therapeutic against the ongoing COVID-19 pandemic. However, the continuous emergence of neutralizing antibody escape variants makes it challenging for antibody therapeutics based on monospecific nAbs. Here, we generated an IgG-like bispecific antibody (bsAb), Bi-Nab, based on a pair of human neutralizing antibodies targeting multiple and invariant sites of the spike receptor binding domain (RBD): 35B5 and 32C7. We demonstrated that Bi-Nab exhibited higher binding affinity to the Delta spike protein than its parental antibodies and presented an extended inhibition breadth of preventing RBD binding to angiotensin-converting enzyme 2 (ACE2), the cellular receptor of SARS-CoV-2. In addition, pseudovirus neutralization results showed that Bi-Nab improved the neutralization potency and breadth with a lower half maximum inhibitory concentration (IC50) against wild-type SARS-CoV-2, variants being monitored (VBMs) and variants of concern (VOCs). Notably, the IgG-like Bi-Nab enhanced the neutralizing activity against Omicron variants with potent capabilities for transmission and immune evasion in comparison with its parental monoclonal antibody (mAb) 32C7 and a cocktail (with the lowest IC50 values of 31.6 ng/mL against the Omicron BA.1 and 399.2 ng/mL against the Omicron BA.2), showing evidence of synergistic neutralization potency of Bi-Nab against the Omicron variants. Thus, Bi-Nab represents a feasible and effective strategy against SARS-CoV-2 variants of concern.

Gasdermin D Inhibits Coronavirus Infection by Promoting the Noncanonical Secretion of Beta Interferon.

Pyroptosis, a programmed cell death, functions as an innate immune effector mechanism and plays a crucial role against microbial invasion. Gasdermin D (GSDMD), as the main pyroptosis effector, mediates pyroptosis and promotes releasing proinflammatory molecules into the extracellular environment through pore-forming activity, modifying inflammation and immune responses. While the substantial importance of GSDMD in microbial infection and cancer has been widely investigated, the role of GSDMD in virus infection, including coronaviruses, remains unclear. Enteric coronavirus transmissible gastroenteritis virus (TGEV) and porcine deltacoronavirus (PDCoV) are the major agents for lethal watery diarrhea in neonatal pigs and pose the potential for spillover from pigs to humans. In this study, we found that alphacoronavirus TGEV upregulated and activated GSDMD, resulting in pyroptosis after infection. Furthermore, the fragment of swine GSDMD from amino acids 242 to 279 (242-279 fragment) was required to induce pyroptosis. Notably, GSDMD strongly inhibited both TGEV and PDCoV infection. Mechanistically, the antiviral activity of GSDMD was mediated through promoting the nonclassical release of antiviral beta interferon (IFN-ß) and then enhancing the interferon-stimulated gene (ISG) responses. These findings showed that GSDMD dampens coronavirus infection by an uncovered GSDMD-mediated IFN secretion, which may present a novel target of coronavirus antiviral therapeutics. IMPORTANCE Coronaviruses, primarily targeting respiratory and gastrointestinal epithelia in vivo, have a serious impact on humans and animals. GSDMD, a main executioner of pyroptosis, is highly expressed in epithelial cells and involves viral infection pathogenesis. While the functions and importance of GSDMD as a critical regulator of inflammasome activities in response to intracellular bacterial infection have been extensively investigated, the roles of GSDMD during coronavirus infection remain unclear. We here show that alphacoronavirus TGEV triggered pyroptosis and upregulated GSDMD expression, while GSDMD broadly suppressed the infection of enteric coronavirus TGEV and PDCoV by its pore-forming activity via promoting unconventional release of IFN-ß. Our study highlights the importance of GSDMD as a regulator of innate immunity and may open new avenues for treating coronavirus infection.

Different Mechanisms Are Utilized by Coronavirus Transmissible Gastroenteritis Virus To Regulate Interferon Lambda 1 and Interferon Lambda 3 Production.

Type III interferons (IFN-λ) are shown to be preferentially produced by epithelial cells, which provide front-line protection at barrier surfaces. Transmissible gastroenteritis virus (TGEV), belonging to the genus Alphacoronavirus of the family Coronaviridae, can cause severe intestinal injuries in porcine, resulting in enormous economic losses for the swine industry, worldwide. Here, we demonstrated that although IFN-λ1 had a higher basal expression, TGEV infection induced more intense IFN-λ3 production in vitro and in vivo than did IFN-λ1. We explored the underlying mechanism of IFN-λ induction by TGEV and found a distinct regulation mechanism of IFN-λ1 and IFN-λ3. The classical RIG-I-like receptor (RLR) pathway is involved in IFN-λ3 but not IFN-λ1 production. Except for the signaling pathways mediated by RIG-I and MDA5, TGEV nsp1 induces IFN-λ1 and IFN-λ3 by activating NF-κB via the unfolded protein responses (UPR) PERK-eIF2α pathway. Furthermore, functional domain analysis indicated that the induction of IFN-λ by the TGEV nsp1 protein was located at amino acids 85 to 102 and was dependent on the phosphorylation of eIF2α and the nuclear translocation of NF-κB. Moreover, the recombinant TGEV with the altered amino acid motif of nsp1 85-102 was constructed, and the nsp1 (85-102sg) mutant virus significantly reduced the production of IFN-λ, compared with the wild strain. Compared to the antiviral activities of IFN-λ1, the administration of IFN-λ3 showed greater antiviral activity against TGEV infections in IPEC-J2 cells. In summary, our data point to the significant role of IFN-λ in the host innate antiviral responses to coronavirus infections within mucosal organs and in the distinct mechanisms of IFN-λ1 and IFN-λ3 regulation. IMPORTANCE Coronaviruses cause infectious diseases in various mammals and birds and exhibit an epithelial cell tropism in enteric and respiratory tracts. It is critical to explore how coronavirus infections modulate IFN-λ, a key innate cytokine against mucosal viral infection. Our results uncovered the different processes of IFN-λ1 and IFN-λ3 production that are involved in the classical RLR pathway and determined that TGEV nsp1 induces IFN-λ1 and IFN-λ3 production by activating NF-κB via the PERK-eIF2α pathway in UPR. These studies highlight the unique regulation of antiviral defense in the intestine during TGEV infection. We also demonstrated that IFN-λ3 induced greater antiviral activity against TGEV replication than did IFN-λ1 in IPEC-J2 cells, which is helpful in finding a novel strategy for the treatment of coronavirus infections.

A Bispecific Antibody Targeting RBD and S2 Potently Neutralizes SARS-CoV-2 Omicron and Other Variants of Concern.

Emerging severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) variants, especially the Omicron variant, have impaired the efficacy of existing vaccines and most therapeutic antibodies, highlighting the need for additional antibody-based tools that can efficiently neutralize emerging SARS-CoV-2 variants. The use of a "single" agent to simultaneously target multiple distinct epitopes on the spike is desirable in overcoming the neutralizing escape of SARS-CoV-2 variants. Herein, we generated a human-derived IgG-like bispecific antibody (bsAb), Bi-Nab35B5-47D10, which successfully retained parental specificity and simultaneously bound to the two distinct epitopes on receptor-binding domain (RBD) and S2. Bi-Nab35B5-47D10 showed improved spike binding breadth among wild-type (WT) SARS-CoV-2, variants of concern (VOCs), and variants being monitored (VBMs) compared with its parental monoclonal antibodies (MAbs). Furthermore, pseudotyped virus neutralization demonstrated that Bi-Nab35B5-47D10 can efficiently neutralize VBMs, including Alpha (B.1.1.7), Beta (B.1.351), and Kappa (B.1.617.1), as well as VOCs, including Delta (B.1.617.2), Omicron BA.1, and Omicron BA.2. Crucially, Bi-Nab35B5-47D10 substantially improved neutralizing activity against Omicron BA.1 (IC50 = 0.15 nM) and Omicron BA.2 (IC50 = 0.67 nM) compared with its parental MAbs. Therefore, Bi-Nab35B5-47D10 represents a potential effective countermeasure against SARS-CoV-2 Omicron and other variants of concern. IMPORTANCE The new, highly contagious SARS-CoV-2 Omicron variant caused substantial breakthrough infections and has become the dominant strain in countries across the world. Omicron variants usually bear high mutations in the spike protein and exhibit considerable escape of most potent neutralization monoclonal antibodies and reduced efficacy of current COVID-19 vaccines. The development of neutralizing antibodies with potent efficacy against the Omicron variant is still an urgent priority. Here, we generated a bsAb, Bi-Nab35B5-47D10, which simultaneously targets SARS-CoV-2 RBD and S2 and improves the neutralizing potency and breadth against SARS-CoV-2 WT and the tested variants compared with their parental antibodies. Notably, Bi-Nab35B5-47D10 has more potent neutralizing activity against the VOC Omicron pseudotyped virus. Therefore, Bi-Nab35B5-47D10 is a feasible and potentially effective strategy by which to treat and prevent COVID-19.

Long-Term Expanding Porcine Airway Organoids Provide Insights into the Pathogenesis and Innate Immunity of Porcine Respiratory Coronavirus Infection.

Respiratory coronaviruses cause serious health threats to humans and animals. Porcine respiratory coronavirus (PRCoV), a natural transmissible gastroenteritis virus (TGEV) mutant with partial spike deletion, causes mild respiratory disease and is an interesting animal respiratory coronavirus model for human respiratory coronaviruses. However, the absence of robust ex vivo models of porcine airway epithelium hinders an understanding of the pathogenesis of PRCoV infection. Here, we generated long-term porcine airway organoids (AOs) derived from basal epithelial cells, which recapitulate the in vivo airway complicated epithelial cellularity. Both 3D and 2D AOs are permissive for PRCoV infection. Unlike TGEV, which established successful infection in both AOs and intestinal organoids, PRCoV was strongly amplified only in AOs, not intestinal organoids. Furthermore, PRCoV infection in AOs mounted vigorous early type I and III interferon (IFN) responses and upregulated the expression of overzealous inflammatory genes, including pattern recognition receptors (PRRs) and proinflammatory cytokines. Collectively, these data demonstrate that stem-derived porcine AOs can serve as a promising disease model for PRCoV infection and provide a valuable tool to study porcine respiratory infection. IMPORTANCE Porcine respiratory CoV (PRCoV), a natural mutant of TGEV, shows striking pathogenetic similarities to human respiratory CoV infection and provides an interesting animal model for human respiratory CoVs, including SARS-CoV-2. The lack of an in vitro model recapitulating the complicated cellularity and structure of the porcine respiratory tract is a major roadblock for the study of PRCoV infection. Here, we developed long-term 3D airway organoids (AOs) and further established 2D AO monolayer cultures. The resultant 3D and 2D AOs are permissive for PRCoV infection. Notably, PRCoV mediated pronounced IFN and inflammatory responses in AOs, which recapitulated the inflammatory responses associated with PRCoV in vivo infection. Therefore, porcine AOs can be utilized to characterize the pathogenesis of PRCoV and, more broadly, can serve as a universal platform for porcine respiratory infection.

Swine Enteric Coronaviruses (PEDV, TGEV, and PDCoV) Induce Divergent Interferon-Stimulated Gene Responses and Antigen Presentation in Porcine Intestinal Enteroids.

Swine enteric coronaviruses (SECoVs) including porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), and porcine deltacoronavirus (PDCoV), account for the majority of lethal watery diarrhea in neonatal pigs and pose significant economic and public health burdens in the world. While the three SECoVs primarily infect intestinal epithelia in vivo and cause similar clinical signs, there are evident discrepancies in their cellular tropism and pathogenicity. However, the underlying mechanisms to cause the differences remain unclear. Herein, we employed porcine enteroids that are a physiologically relevant model of the intestine to assess the host epithelial responses following infection with the three SECoVs (PEDV, TGEV, and PDCoV). Although SECoVs replicated similarly in jejunal enteroids, a parallel comparison of transcriptomics datasets uncovered that PEDV and TGEV infection induced similar transcriptional profiles and exhibited a more pronounced response with more differentially expressed genes (DEGs) in jejunal enteroids compared with PDCoV infection. Notably, TGEV and PDCoV induced high levels of type I and III IFNs and IFN-stimulated gene (ISG) responses, while PEDV displayed a delayed peak and elicited a much lesser extent of IFN responses. Furthermore, TGEV and PDCoV instead of PEDV elicited a substantial upregulation of antigen-presentation genes and T cell-recruiting chemokines in enteroids. Mechanistically, we demonstrated that IFNs treatment markedly elevated the expression of NOD-like receptor (NLR) family NLRC5 and major histocompatibility complex class I (MHC-I) molecules. Together, our results indicate unique and common viral strategies for manipulating the global IFN responses and antigen presentation utilized by SECoVs, which help us a better understanding of host-SECoVs interactions.

Coronavirus transmissible gastroenteritis virus antagonizes the antiviral effect of the microRNA miR-27b via the IRE1 pathway.

Although the functional parameters of microRNAs (miRNAs) have been explored to some extent, the roles of these molecules in coronavirus infection and the regulatory mechanism of miRNAs in virus infection are still unclear. Transmissible gastroenteritis virus (TGEV) is an enteropathgenic coronavirus and causes high morbidity and mortality in suckling piglets. Here, we demonstrated that microRNA-27b-3p (miR-27b-3p) suppressed TGEV replication by directly targeting porcine suppressor of cytokine signaling 6 (SOCS6), while TGEV infection downregulated miR-27b-3p expression in swine testicular (ST) cells and in piglets. Mechanistically, the decrease of miR-27b-3p expression during TGEV infection was mediated by the activated inositol-requiring enzyme 1 (IRE1) pathway of the endoplasmic reticulum (ER) stress. Further studies showed that when ER stress was induced by TGEV, IRE1 acted as an RNase activated by autophosphorylation and unconventionally spliced mRNA encoding a potent transcription factor, X-box-binding protein 1 (Xbp1s). Xbp1s inhibited the transcription of miR-27 and ultimately reduced the production of miR-27b-3p. Therefore, our findings indicate that TGEV inhibits the expression of an anti-coronavirus microRNA through the IRE1 pathway and suggest a novel way in which coronavirus regulates the host cell response to infection.

Sensitivity of SARS-CoV-2 Variants to Neutralization by Convalescent Sera and a VH3-30 Monoclonal Antibody.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global pandemic of novel coronavirus disease (COVID-19). Though vaccines and neutralizing monoclonal antibodies (mAbs) have been developed to fight COVID-19 in the past year, one major concern is the emergence of SARS-CoV-2 variants of concern (VOCs). Indeed, SARS-CoV-2 VOCs such as B.1.1.7 (UK), B.1.351 (South Africa), P.1 (Brazil), and B.1.617.1 (India) now dominate the pandemic. Herein, we found that binding activity and neutralizing capacity of sera collected from convalescent patients in early 2020 for SARS-CoV-2 VOCs, but not non-VOC variants, were severely blunted. Furthermore, we observed evasion of SARS-CoV-2 VOCs from a VH3-30 mAb 32D4, which was proved to exhibit highly potential neutralization against wild-type (WT) SARS-CoV-2. Thus, these results indicated that SARS-CoV-2 VOCs might be able to spread in convalescent patients and even harbor resistance to medical countermeasures. New interventions against these SARS-CoV-2 VOCs are urgently needed.

The dichotomous and incomplete adaptive immunity in COVID-19 patients with different disease severity.

The adaptive immunity that protects patients from coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is not well characterized. In particular, the asymptomatic patients have been found to induce weak and transient SARS-CoV-2 antibody responses, but the underlying mechanisms remain unknown; meanwhile, the protective immunity that guide the recovery of these asymptomatic patients is elusive. Here, we characterized SARS-CoV-2-specific B-cell and T-cell responses in 10 asymptomatic patients and 64 patients with other disease severity (mild, n = 10, moderate, n = 32, severe, n = 12) and found that asymptomatic or mild symptomatic patients failed to mount virus-specific germinal center (GC) B cell responses that result in robust and prolonged humoral immunity, assessed by GC response indicators including follicular helper T (TFH) cell and memory B cell responses as well as serum CXCL13 levels. Alternatively, these patients mounted potent virus-specific TH1 and CD8+ T cell responses. In sharp contrast, patients of moderate or severe disease induced vigorous virus-specific GC B cell responses and associated TFH responses; however, the virus-specific TH1 and CD8+ T cells were minimally induced in these patients. These results, therefore, uncovered the protective immunity in asymptomatic patients and also revealed the strikingly dichotomous and incomplete humoral and cellular immune responses in COVID-19 patients with different disease severity, providing important insights into rational design of effective COVID-19 vaccines.

Aminopeptidase N Expression, Not Interferon Responses, Determines the Intestinal Segmental Tropism of Porcine Deltacoronavirus.

Porcine deltacoronavirus (PDCoV) is an economically important enteropathogen of swine with worldwide distribution. PDCoV primarily infects the small intestine instead of the large intestine in vivo However, the underlying mechanism of PDCoV tropism to different intestinal segments remains poorly understood as a result of the lack of a suitable in vitro intestinal model that recapitulates the cellular diversity and complex functions of the gastrointestinal tract. Here, we established the PDCoV infection model of crypt-derived enteroids from different intestinal segments. Enteroids were susceptible to PDCoV, and multiple types of different functional intestinal epithelia were infected by PDCoV in vitro and in vivo We further found that PDCoV favorably infected the jejunum and ileum and restrictedly replicated in the duodenum and colon. Mechanistically, enteroids from different intestinal regions displayed a distinct gene expression profile, and the differential expression of primary viral receptor host aminopeptidase N (APN) instead of the interferon (IFN) responses determined the susceptibility of different intestinal segments to PDCoV, although PDCoV substantially elicited antiviral genes production in enteroids after infection. Additional studies showed that PDCoV infection significantly induced the expression of type I and III IFNs at the late stage of infection, and exogenous IFN inhibited PDCoV replication in enteroids. Hence, our results provide critical inputs to further dissect the molecular mechanisms of PDCoV-host interactions and pathogenesis.IMPORTANCE The zoonotic potential of the PDCoV, a coronavirus efficiently infecting cells from a broad range species, including porcine, chicken, and human, emphasizes the urgent need to further study the cell and tissue tropism of PDCoV in its natural host. Herein, we generated crypt stem cell-derived enteroids from porcine different intestinal regions, which well recapitulated the events in vivo of PDCoV infection that PDCoV targeted multiple types of intestinal epithelia and preferably infected the jejunum and ileum over the duodenum and colon. Mechanistically, we demonstrated that the expression of APN receptor rather than the IFN responses determined the susceptibility of different regions of the intestines to PDCoV infection, though PDCoV infection markedly elicited the IFN responses. Our findings provide important insights into how the distinct gene expression profiles of the intestinal segments determine the cell and tissue tropism of PDCoV.

The study of recombinant adenovirus and antiviral effect of porcineepidemic diarrhea virus (PEDV) neutralizing antibody PC10-IgG

To explore the PED treatment preparation that can directly be used in piglets, here we inserted the PEDV neutralizingantibody PC10-IgG gene sequence into the human adenovirus type V (Ad5) backbone plasmid to construct the recombinantadenovirus Ad5-PC10-IgG and analyzed its anti-PEDV effect. The supernatant of HEK293 cells infected with Ad5-PC10-IgG wasdetected by ELISA, and purified IgG from the supernatant was detected by SDS-PAGE and western blot. The results showed that HEK293 cells infected by Ad5-PC10-IgG were able to secrete and express PC10-IgG and the antibody titer was relatively high. Thesecreted PC10-IgG was also used to test the binding activity (by IFA) and neutralize activity (by PCR and IFA) against PEDV, andthe results showed that PC10-IgG expressed by Ad5-PC10-IgG in the supernatant could significantly inhibit PEDV infection in VeroE6 cells and had good binding activity. In order to verify whether Ad5-PC10-IgG can infect the pig's small intestine, Ad5-PC10-IgGwere used to infect porcine intestinal enteroids, and the supernatant after infection was detected by ELISA as well as the expressionof PC10-IgG by porcine intestinal enteroids were detected by IFA. The results showed that Ad5-PC10-IgG was able to infect porcineintestinal enteroids and the PC10-IgG was secreted by viral infected cells. In addition, mice were inoculated by Ad5-PC10-IgG, andthe serum and anal swabs were collected at different time points and detected by ELISA. After that, the binding activity andneutralizing activity of PC10-IgG in the serum against PEDV were detected through the virus binding experiment and the virusinhibition experiment. The results showed that on the 1, 3, 5 day after inoculation, the serum of the inoculated mice contained higherconcentrations of PC10-IgG instead of anal swabs, and the PC10-IgG in the serum had good binding activity and neutralizingactivity to PEDV. The above results indicate that Ad5-PC10-IgG can secrete and express the active neutralizing antibody PC10-IgGin vivo and in vitro. Therefore, this study lays the foundation for the further development of PEDV therapeutic antibody againstPEDV infection in the future.

Disease severity dictates SARS-CoV-2-specific neutralizing antibody responses in COVID-19.

COVID-19 patients exhibit differential disease severity after SARS-CoV-2 infection. It is currently unknown as to the correlation between the magnitude of neutralizing antibody (NAb) responses and the disease severity in COVID-19 patients. In a cohort of 59 recovered patients with disease severity including severe, moderate, mild, and asymptomatic, we observed the positive correlation between serum neutralizing capacity and disease severity, in particular, the highest NAb capacity in sera from the patients with severe disease, while a lack of ability of asymptomatic patients to mount competent NAbs. Furthermore, the compositions of NAb subtypes were also different between recovered patients with severe symptoms and with mild-to-moderate symptoms. These results reveal the tremendous heterogeneity of SARS-CoV-2-specific NAb responses and their correlations to disease severity, highlighting the needs of future vaccination in COVID-19 patients recovered from asymptomatic or mild illness.

The Coronavirus PEDV Evades Type III Interferon Response Through the miR-30c-5p/SOCS1 Axis.

Porcine epidemic diarrhea virus (PEDV) is an economically important pathogen that has evolved several mechanisms to evade type I IFN responses. Type III interferon (IFN-λ), an innate cytokine that primarily targets the mucosal epithelia, is critical in fighting mucosal infection in the host and has been reported to potently inhibit PEDV infection in vitro. However, how PEDV escapes IFN-λ antiviral response remains unclear. In this study, we found that PEDV infection induced significant IFN-λ expression in type I IFN-defective Vero E6 cells, but virus-induced endogenous IFN-λ did not reduce PEDV titers. Moreover, we demonstrated that PEDV escaped IFN-λ responses by substantially upregulating the suppressor of cytokine signaling protein 1 (SOCS1) expression, which impaired the induction of IFN-stimulated genes (ISGs) and dampened the IFN-λ antiviral response and facilitated PEDV replication in Vero E6 cells. We further showed that PEDV infection increased SOCS1 expression by decreasing host miR-30c-5p expression. MiR-30c-5p suppressed SOCS1 expression through targeting the 3' untranslated region (UTR) of SOCS1. The inhibition of IFN-λ elicited ISGs expression by SOCS1 was specifically rescued by overexpression of miR-30c-5p. Collectively, our findings identify a new strategy by PEDV to escape IFN-λ-mediated antiviral immune responses by engaging the SOCS1/miR-30c axis, thus improving our understanding of its pathogenesis.

Research progress in enteroids in porcine enteric coronavirus infection

Porcine enteric coronavirus, an important agent of diarrhea in piglets, primarily infects the villous epithelia of the small intestine, causing substantial economic losses to the pork industry. The lack of an in vitro model that can recapitulate the highly complex physiological properties of the gastrointestinal tract significantly limits the study of the interactions between porcine enteric coronavirus infection and host intestinal epithelium. With the rapid development of stem cell technology, an in vitro model that can mimic diverse cellular nature and complex structure of the intestine-enteroids, has attracted widespread attention. Compared with conventional cell lines, enteroids not only simulate the structure and function of the intestine, but also retain the genetic characteristics of the host. Enteroids would be expected to be an ideal model for studying the interactions of host-enteric pathogens. This article reviews the recent research progress of porcine enteric coronavirus and the applications of enteroids in enteric pathogens research, in order to provide new insights for the future fundamental research of porcine enteric coronavirus.