Search

The recurrent outbreak of coronaviruses and variants underscores the need for broadly reactive antivirals and vaccines. Here, a novel broad-spectrum human antibody named 76E1 was isolated from a COVID-19 convalescent patient and showed broad neutralization activity against multiple α- and β-coronaviruses, including the SARS-CoV-2 variants and also exhibited the binding breath to peptides containing the epitope from γ- and δ- coronaviruses. 76E1 cross-protects mice from SARS-CoV-2 and HCoV-OC43 infection in both prophylactic and treatment models. The epitope including the fusion peptide and S2’ cleavage site recognized by 76E1 was significantly conserved among α-, β-, γ- and δ- coronaviruses. We uncovered a novel mechanism of antibody neutralization that the epitope of 76E1 was proportionally less exposed in the prefusion trimeric structure of spike protein but could be unmasked by binding to the receptor ACE2. Once the epitope exposed, 76E1 inhibited S2’ cleavage, thus blocked the membrane fusion process. Our data demonstrate a key epitope targeted by broadly-neutralizing antibodies and will guide next-generation epitope-based pan-coronavirus vaccine design.

Background The receptor-binding domain (RBD) variants of SARS-CoV-2 could impair antibody-mediated neutralization of the virus by host immunity; thus, prospective surveillance of antibody escape mutants and understanding the evolution of RBD are urgently needed. Methods Using the single B cell cloning technology, we isolated and characterized 93 RBD-specific antibodies from the memory B cells of four COVID-19 convalescent individuals in the early stage of the pandemic. Then, global RBD alanine scanning with a panel of 19 selected neutralizing antibodies (NAbs), including several broadly reactive NAbs, was performed. Furthermore, we assessed the impact of single natural mutation or co-mutations of concern at key positions of RBD on the neutralization escape and ACE2 binding function by recombinant proteins and pseudoviruses. Results Thirty-three amino acid positions within four independent antigenic sites (1 to 4) of RBD were identified as valuable indicators of antigenic changes in the RBD. The comprehensive escape mutation map not only confirms the widely circulating strains carrying important immune escape RBD mutations such as K417N, E484K, and L452R, but also facilitates the discovery of new immune escape-enabling mutations such as F486L, N450K, F490S, and R346S. Of note, these escape mutations could not affect the ACE2 binding affinity of RBD, among which L452R even enhanced binding. Furthermore, we showed that RBD co-mutations K417N, E484K, and N501Y present in B.1.351 appear more resistant to NAbs and human convalescent plasma from the early stage of the pandemic, possibly due to an additive effect. Conversely, double mutations E484Q and L452R present in B.1.617.1 variant show partial antibody evasion with no evidence for an additive effect. Conclusions Our study provides a global view of the determinants for neutralizing antibody recognition, antigenic conservation, and RBD conformation. The in-depth escape maps may have value for prospective surveillance of SARS-CoV-2 immune escape variants. Special attention should be paid to the accumulation of co-mutations at distinct major antigenic sites. Finally, the new broadly reactive NAbs described here represent new potential opportunities for the prevention and treatment of COVID-19.

Background The immune protective mechanisms during severe acute respiratory syndrome coronavirus (SARS-CoV-2) infection remain to be deciphered for the development of an effective intervention approach. Methods We examined early responses of interleukin 37 (IL-37), a powerful anti-inflammatory cytokine, in 254 SARS-CoV-2–infected patients before any clinical intervention and determined its correlation with clinical prognosis. Results Our results demonstrated that SARS-CoV-2 infection causes elevation of plasma IL-37. Higher early IL-37 responses were correlated with earlier viral RNA negative conversion, chest computed tomographic improvement, and cough relief, consequently resulted in earlier hospital discharge. Further assays showed that higher IL-37 was associated with lower interleukin 6 and interleukin 8 (IL-8) and higher interferon α responses and facilitated biochemical homeostasis. Low IL-37 responses predicted severe clinical prognosis in combination with IL-8 and C-reactive protein. In addition, we observed that IL-37 administration was able to attenuate lung inflammation and alleviate respiratory tissue damage in human angiotensin-converting enzyme 2–transgenic mice infected with SARS-CoV-2. Conclusions Overall, we found that IL-37 plays a protective role by antagonizing inflammatory responses while retaining type I interferon, thereby maintaining the functionalities of vital organs. IL-37, IL-8, and C-reactive protein might be formulated as a precise prediction model for screening severe clinical cases and have good value in clinical practice.

Summary COVID-19 has become a worldwide threat to humans, and neutralizing antibodies have therapeutic potential. We have purified more than one thousand memory B cells specific to SARS-CoV-2 S1 or RBD (receptor binding domain), and obtain 729 paired heavy and light chain fragments. Among these, 178 antibodies test positive for antigen binding, and the majority of the top 17 binders with EC50 below 1 nM are RBD binders. Furthermore, we identify 11 neutralizing antibodies, 8 of which show an IC50 within 10 nM, and the best one, 414-1, with IC50 of 1.75 nM. Through epitope mapping, we find 3 main epitopes in RBD recognized by these antibodies, and epitope B antibody 553-15 could substantially enhance the neutralizing abilities of most of the other antibodies. We also find that 515-5 could cross-neutralize the SARS-CoV pseudovirus. Altogether, our study provides 11 potent human neutralizing antibodies for COVID-19 as therapeutic candidates., Graphical Abstract, Highlights 11 neutralizing antibodies against SARS-CoV-2 targeting 3 main epitopes on RBD Epitope A antibody 414-1 shows neutralizing IC50 at 1.75 nM Epitope B antibody 553-15 can enhance the neutralizing abilities of other antibodies One neutralizing antibody, 515-5, can cross-neutralize SARS-CoV pseudovirus, Wan et al. identify 11 potent neutralizing antibodies against COVID-19 from 11 convalescent patients. These human neutralizing antibodies target 3 separate epitopes on the receptor binding domain (RBD) of the SARS-CoV-2 spike protein and epitope B antibody (553-15) can enhance the neutralizing abilities of most other antibodies.

The coronavirus induced disease 19 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become a worldwide threat to human lives, and neutralizing antibodies present a great therapeutic potential in curing affected patients. We purified more than one thousand memory B cells specific to SARS-CoV-2 S1 or RBD (receptor binding domain) antigens from 11 convalescent COVID-19 patients, and a total of 729 naturally paired heavy and light chain fragments were obtained by single B cell cloning technology. Among these, 178 recombinant monoclonal antibodies were tested positive for antigen binding, and the top 13 binders with Kd below 0.5 nM are all RBD binders. Importantly, all these 13 antibodies could block pseudoviral entry into HEK293T cells overexpressing ACE2, with the best ones showing IC50s around 2-3 nM. We further identified 8 neutralizing antibodies against authentic virus with IC50s within 10 nM. Among these, 414-1 blocked authentic viral entry at IC50 of 1.75 nM and in combination with 105-38 could achieve IC50 as low as 0.45 nM. Meanwhile, we also found that 3 antibodies could cross-react with the SARS-CoV spike protein. Altogether, our study provided a panel of potent human neutralizing antibodies for COVID19 as therapeutics candidates for further development.

Type I interferons (IFNs) are the first line of defense against viral infection. Using a mouse model of influenza A virus infection, we found that IFN-κ was one of the earliest responding type I IFNs after infection with H9N2, a low-pathogenic avian influenza A virus, whereas this early induction did not occur upon infection with the epidemic-causing H7N9 virus. IFN-κ efficiently suppressed the replication of various influenza viruses in cultured human lung cells, and chromodomain helicase DNA binding protein 6 (CHD6) was the major effector for the antiviral activity of IFN-κ, but not for that of IFN-α or IFN-β. The induction of CHD6 required both of the type I IFN receptor subunits IFNAR1 and IFNAR2, the mitogen-activated protein kinase (MAPK) p38, and the transcription factor c-Fos but was independent of signal transducer and activator of transcription 1 (STAT1) activity. In addition, we showed that pretreatment with IFN-κ protected mice from lethal influenza viral challenge. Together, our findings identify an IFN-κ-specific pathway that constrains influenza A virus and provide evidence that IFN-κ may have potential as a preventative and therapeutic agent against influenza A virus.

The coronavirus induced disease 19 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become a worldwide threat to human lives, and neutralizing antibodies present a great therapeutic potential in curing affected patients. We purified more than one thousand memory B cells specific to SARS-CoV-2 S1 or RBD (receptor binding domain) antigens from 11 convalescent COVID-19 patients, and a total of 729 naturally paired heavy and light chain fragments were obtained by single B cell cloning technology. Among these, 178 recombinant monoclonal antibodies were tested positive for antigen binding, and the top 13 binders with Kd below 0.5 nM are all RBD binders. Interestingly, among these 13 antibodies and found 10 unique CDR3H sequences, while 505-3, 505-5 and 515-1 shared identical CDR3H. Importantly, all these 13 antibodies could block pseudoviral entry into HEK293T cells overexpressing ACE2, with the best ones showing IC50s around 2-3 nM. We further identified 8 neutralizing antibodies against authentic virus with IC50s within 10 nM. Among these, 414-1 blocked authentic viral entry at IC50 of 1.75 nM and in combination with 105-38 could achieve IC50 as low as 0.45 nM. Meanwhile, we also found that 3 antibodies could cross-react with the SARS-CoV spike protein. Altogether, our study provided a panel of potent human neutralizing antibodies for COVID19 as therapeutics candidates for further development. Funding: This work was supported by Zhejiang University special scientific research fund for COVID-19 prevention and control (2020XGZX023), National Key Research and Development program of China (2016YFA0101800 and 2018YFA0108700), the national Natural Science Foundation of China (31925010) and Shanghai Municipal Science and Technology Major Project (2017SHZDZX01). Conflict of Interest: Fei Lan is a scientific advisor of Active Motif China Inc. All other authors declare no conflicts of interest. Ethical Approval: The experiments involving authentic COVID-19 virus were performed in Fudan University biosafety level 3 (BSL-3) facility. The overall study was reviewed and approved by the SHAPHC Ethics Committee (approval no. 2020-Y008-01).

Given that continuing antigenic shift and drift of influenza A viruses result in the escape from previous vaccine-induced immune protection, a universal influenza vaccine has been actively sought. However, there were very few vaccines capable of eliciting cross-group ant-influenza immunity. Here, we designed two novel composite immunogens containing highly conserved T-cell epitopes of six influenza A virus internal antigens, and expressed them in DNA, recombinant adenovirus-based (AdC68) and recombinant vaccinia vectors, respectively, to formulate three vaccine forms. The introduction of the two immunogens via a DNA priming and viral vectored vaccine boosting modality afforded cross-group protection from both PR8 and H7N9 influenza virus challenges in mice. Both respiratory residential and systemic T cells contributed to the protective efficacy. Intranasal but not intramuscular administration of AdC68 based vaccine was capable of raising both T cell subpopulations to confer a full protection from lethal PR8 and H7N9 challenges, and blocking the lymphatic egress of T cells during challenges attenuated the protection. Thus, by targeting highly conserved internal viral epitopes to efficiently generate both respiratory and systemic memory T cells, the sequential vaccination strategy reported here represented a new promising candidate for the development of T-cell based universal influenza vaccines.

A multiplex reverse transcription-PCR (mRT-PCR) assay was developed for the rapid detection of influenza A viruses. The assay simultaneously differentiated H1, H3, H5 and H9 hemagglutinin subtypes in a single reaction mixture. Five sets of specific primers targeted to the M, H1, H3, H5 and H9 genes were used in this assay. The amplified products were visualized by agarose gel electrophoresis. The sizes of the PCR amplified fragments were 612 bp for H1, 187 bp for H3, 338 bp for H5, 289 bp for H9 and 239 bp for M. The detection limit of the viral RNA template was 1 ng for the H1, H3 and H5 subtypes and 0.1 ng for the H9 subtype. Nonspecific product bands from RNAs of other viral pathogens were not amplified. The sensitivity analysis demonstrated that the mRT-PCR assay is as sensitive as conventional RT-PCR and 10 times less sensitive than SYBR Green real-time RT-PCR. In conclusion, the mRT-PCR assay developed in this study was able to type influenza A viruses and simultaneously differentiate H1, H3, H5 and H9 subtypes in both human and avian clinical specimens, and thus, the mRT-PCR assay could be a rapid, convenient and relatively inexpensive molecular diagnostic tool for large-scale screening of clinical samples.

Genetically similar H5N1 viruses circulating in the avian reservoir exhibit different levels of pathogenicity in mice. In this study, we characterized two highly pathogenic H5N1 avian isolates--A/Hunan/316/2005 (HN05), which is highly pathogenic in mice, and A/Hubei/489/2004 (HB04), which is nonpathogenic. In mammalian cells, HN05 replicates more efficiently than HB04, although both viruses have similar growth kinetics in avian cells. We used reverse genetics to generate recombinant H5N1 strains containing genes from HN05 and HB04 and examined their virulence. HN05 genes encoding the polymerase complex determine pathogenicity and viral replication ability both in vitro and in vivo. The PB2 subunit plays an important role in enhancing viral replication, and the PB1 and PA subunits contribute mainly to pathogenicity in mice. These results can be used to elucidate host-range expansion and the molecular basis of the high virulence of H5N1 viruses in mammalian species.