Bispecific antibodies targeting two glycoproteins on SFTSV exhibit synergistic neutralization and protection in a mouse model.

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease with a high fatality rate of up to 30% caused by SFTS virus (SFTSV). However, no specific vaccine or antiviral therapy has been approved for clinical use. To develop an effective treatment, we isolated a panel of human monoclonal antibodies (mAbs). SF5 and SF83 are two neutralizing mAbs that recognize two viral glycoproteins (Gn and Gc), respectively. We found that their epitopes are closely located, and we then engineered them as several bispecific antibodies (bsAbs). Neutralization and animal experiments indicated that bsAbs display more potent protective effects than the parental mAbs, and the cryoelectron microscopy structure of a bsAb3 Fab-Gn-Gc complex elucidated the mechanism of protection. In vivo virus passage in the presence of antibodies indicated that two bsAbs resulted in less selective pressure and could efficiently bind to all single parental mAb-escape mutants. Furthermore, epitope analysis of the protective mAbs against SFTSV and RVFV indicated that they are all located on the Gn subdomain I, where may be the hot spots in the phleboviruses. Collectively, these data provide potential therapeutic agents and molecular basis for the rational design of vaccines against SFTSV infection.

Causal association between air pollution and frailty: a Mendelian randomization study.

Backgrounds: Frailty is a significant problem for older persons since it is linked to a number of unfavorable consequences. According to observational researches, air pollution may raise the risk of frailty. We investigated the causal association between frailty and air pollution (including PM2.5, PM2.5-10, PM10, nitrogen dioxide, and nitrogen oxides) using Mendelian randomization approach. Methods: We conducted MR analysis using extensive publically accessible GWAS (genome-wide association studies) summary data. The inverse variance weighted (IVW) method was employed as the primary analysis method. The weighted median model, MR-Egger, simple model, and weighted model approaches were chosen for quality control. The Cochran's Q test was utilized to evaluate heterogeneity. Pleiotropy is found using the MR-Egger regression test. The MR-PRESSO method was used to recognize outliers. The leave-one-out strategy was used to conduct the sensitivity analysis. Results: MR results suggested that PM2.5 was statistically significantly associated with frailty [odds ratio (OR) = 1.33; 95%confidence interval (CI) = 1.12-1.58, p = 0.001] in IVW method. We observed no statistical association between PM2.5-10(OR = 1.00, 95% CI = 0.79-1.28, p = 0.979), PM10(OR = 0.91, 95% CI = 0.75-1.11, p = 0.364), nitrogen dioxide (OR = 0.98, 95% CI = 0.85-1.12, p = 0.730), nitrogen oxides (OR = 1.15, 95% CI = 0.98-1.36, p = 0.086) and frailty. There was no pleiotropy in the results. The sensitivity analysis based on the leave-one-out method showed that the individual single nucleotide polymorphisms (SNPs) did not affect the robustness of the results. Conclusion: The current MR investigation shows a causal association between PM2.5 and frailty. Frailty's detrimental progression may be slowed down with the help of air pollution prevention and control.

Canine interleukin-31 binds directly to OSMRß with higher binding affinity than to IL-31RA.

Interleukin-31 (IL-31) is a pro-inflammatory cytokine involved in skin inflammation and tumor progression. The IL-31 signaling cascade is initiated by its binding to two receptors, IL-31 receptor alpha (IL-31RA) and oncostatin M receptor subunit beta (OSMRß). The previous study suggested that human IL-31 (hIL-31) directly interacts with IL-31RA and OSMRß, independently, but the binding ability of hIL-31 to IL-31RA is stronger than to OSMRß. In different to its human ortholog, feline IL-31 (fIL-31) has a higher binding affinity for feline OSMRß. However, the binding pattern of canine IL-31 to its receptors remains to be elucidated. In this study, we purified the recombinant canine IL-31 (rcIL-31) protein and revealed its secondary structure to be mainly composed of alpha-helices. Moreover, in vitro studies show that rcIL-31 has the ability to induce the phosphorylation of signal transducer activator of transcription 3 (STAT3) and STAT5 in DH-82 cells. In the following, the binding efficacies of bioactive rcIL-31 for its individual receptor components have been measured using a flow cytometry assay. The result demonstrates that correctly refolded rcIL-31 binds independently with cIL-31RA and cOSMRß which were expressed on the cell surface. Of note, rcIL-31 has a greater than tenfold higher affinity to OSMRß than to IL-31RA. Additionally, we demonstrated that D1-D4, especially D4 of cOSMRß, is crucial for its binding to cIL-31. Furthermore, this study proved that rcIL-31 has a high binding affinity to the soluble cOSMRß with a KD value of 3.59 × 10-8 M. The results presented in the current study will have a significant implication in the development of drugs or antibodies against diseases induced by cIL-31 signaling.

Expression, purification, and biological characterization of recombinant human interleukin-31 protein.

Interleukin-31 (IL-31), belonging to the IL-6 cytokine family, is involved in skin inflammation and pruritus, as well as some tumors' progression. Here, we reported the expression and purification of recombinant human IL-31 (rhIL-31) using a prokaryotic system. This recombinant protein was expressed in the form of inclusion bodies, refolded and purified by size-exclusion chromatography. Circular dichroism analysis revealed that the secondary structure of rhIL-31 was mainly composed of alpha-helix, which is in consistence with the 3D model structure built by AlphaFold server. In vitro studies showed that rhIL-31 exhibited a good binding ability to the recombinant hIL-31 receptor alpha fused with human Fc fragment (rhIL-31RA-hFc) with EC50 value of 16.36 µg/mL in ELISA assay. Meanwhile, flow cytometry demonstrated that rhIL-31 was able to bind to hIL-31RA or hOSMRß expressed on the cell surface, independently. Furthermore, rhIL-31 could induce the phosphorylation of STAT3 in A549 cells. In conclusion, the prepared rhIL-31 in this study possesses the binding ability to its receptors, and can activate the signal pathway of JAK/STAT. Thus, it can be applied in further studies, including investigation of hIL-31-related diseases, structural analysis, and development of therapeutic drugs, and monoclonal antibodies targeting hIL-31.

Study on lactation performance and development of KASP marker for milk traits in Xinjiang donkey (Equus asinus).

Donkey milk has high nutritional and medicinal value, but there are few researches in donkey milk traits, especially on genome. The whole lactation of 89 donkeys was recorded and it was found that Xinjiang donkey had good lactation performance while great differences among individuals. In our previous study, four genes including LGALS2, NUMB, ADCY8 and CA8 were identified as milk-associated with Chinese Kazakh house, based on Equine 670k Chip genomic analysis. And then 15 SNPs of the four key genes were conducted for genotyping in Xinjiang donkey in this study, one of Chinese indigenous breed, 14 SNPs were successful classified. And those SNPs were correlation analysis with milk yield of Xinjiang donkeys. The results showed that NUMB g.46709914T > G was significantly correlated with daily milk yield of Xinjiang donkey in the early, middle, and late periods, while ADCY8 g.48366302T > C, CA8 g.89567442T > G and CA8 g.89598328T > A were significantly correlated with lactation in the late periods. These results indicate that NUMB g.46709914T > G can be as markers of candidate genes for lactating traits in donkeys, SNPs of ADCY8 and CA8 as potential. Our findings will not only help confirm key genes for donkey milk traits, but also provide future for genomic selection in donkeys.

Structural and inhibitor sensitivity analysis of influenza B-like viral neuraminidases derived from Asiatic toad and spiny eel.

Influenza virus neuraminidase (NA) is an important target for antiviral development because it plays a crucial role in releasing newly assembled viruses. Two unique influenza-like virus genomes were recently reported in the Wuhan Asiatic toad and Wuhan spiny eel. Their NA genes appear to be highly divergent from all known influenza NAs, raising key questions as to whether the Asiatic toad influenza-like virus NA (tNA) and spiny eel NA (eNA) have canonical NA activities and structures and whether they show sensitivity to NA inhibitors (NAIs). Here, we found that both tNA and eNA have neuraminidase activities. A detailed structural analysis revealed that tNA and eNA present similar overall structures to currently known NAs, with a conserved calcium binding site. Inhibition assays indicated that tNA is resistant to NAIs, while eNA is still sensitive to NAIs. E119 is conserved in canonical NAs. The P119E substitution in tNA can restore sensitivity to NAIs, and, in contrast, the E119P substitution in eNA decreased its sensitivity to NAIs. The structures of NA-inhibitor complexes further provide a detailed insight into NA-inhibitor interactions at the atomic level. Moreover, tNA and eNA have unique N-glycosylation sites compared with canonical NAs. Collectively, the structural features, NA activities, and sensitivities to NAIs suggest that fish- and amphibian-derived influenza-like viruses may circulate in these vertebrates. More attention should be paid to these influenza-like viruses because their NA molecules may play roles in the emergence of NAI resistance.

Development and characterization of a novel mouse anti-canine oncostatin M receptor beta monoclonal antibody.

Oncostatin M receptor beta (OSMRß) mediates signaling of Oncostatin M (OSM) and interleukine-31 (IL-31), two key cytokines involved in many important biological processes including inflammation and cancer progression. More importantly, OSMRß might be a potential biomarker and therapeutic target for some diseases, such as inflammatory bowel disease, pruritus and ovarian cancer. In this study, soluble recombinant canine OSMRß (cOSMRß) was experimentally expressed as a native antigen to develop an effective cOSMRß-specific monoclonal antibody (mAb), 2O2, using hybridoma technology. It was demonstrated that 2O2 is able to detect OSMRß expressed on cell surface using immunofluorescence assay (IFA) and flow cytometry (FACS). This mAb exhibits very high binding affinity to cOSMRß with the KD and half-maximal effective concentration (EC50) values of 2.49 nM and 96.96 ng/ml, respectively. Meanwhile, it didn't show any cross-relativities with feline OSMRß (fOSMRß) and human OSMRß (hOSMRß). Moreover, we determined the binding epitope of 2O2, which localizes in the domain VI (DVI, amino acids 623-734) of cOSMRß. In conclusion, this novel mAb, 2O2, can be used in immunoassays, including IFA, FACS and enzyme-linked immunosorbent assay (ELISA) to facilitate studies in dogs.

[Expression of influenza B virus hemagglutinin and its immunogenicity determination].

Influenza B virus is one of the causes for seasonal influenza, which can account for serious illness or even death in some cases. We tested the expression of extracellular domain of hemagglutinin (HA-ecto) of influenza B viruses in mammalian cells, and then determined the immunogenicity of HA-ecto in mice. The gene sequence encoding influenza B virus HA-ecto, foldon sequence, and HIS tag was optimized and inserted into pCAGGS vector. The opening reading frame (ORF) of neuraminidase was also cloned into pCAGGS. The pCAGGS-HA-ecto and pCAGGS-NA were co-transfected into 293T cells using linear polyethylenimine. Cell supernatant after transfection was collected after 96 h, and the secreted trimmeric HA-ecto protein was purified by nickel ion affinity chromatography and size exclusion chromatography. Subsequently, the mice were immunized with HA-ecto protein, and the corresponding antibody titers were detected by ELISA and hemagglutination inhibition (HAI) assays. The results showed that soluble trimeric HA-ecto protein could be obtained using mammalian cell expression system. Moreover, trimeric HA-ecto protein, in combination with the adjuvant, induced high levels of ELISA and HAI antibodies against homogenous and heterologous antigens in mice. Thus, the soluble HA-ecto protein expressed in mammalian cells could be used as a recombinant subunit vaccine candidate for influenza B virus.

The molecular basis for SARS-CoV-2 binding to dog ACE2.

SARS-CoV-2 can infect many domestic animals, including dogs. Herein, we show that dog angiotensin-converting enzyme 2 (dACE2) can bind to the SARS-CoV-2 spike (S) protein receptor binding domain (RBD), and that both pseudotyped and authentic SARS-CoV-2 can infect dACE2-expressing cells. We solved the crystal structure of RBD in complex with dACE2 and found that the total number of contact residues, contact atoms, hydrogen bonds and salt bridges at the binding interface in this complex are slightly fewer than those in the complex of the RBD and human ACE2 (hACE2). This result is consistent with the fact that the binding affinity of RBD to dACE2 is lower than that of hACE2. We further show that a few important mutations in the RBD binding interface play a pivotal role in the binding affinity of RBD to both dACE2 and hACE2. Our work reveals a molecular basis for cross-species transmission and potential animal spread of SARS-CoV-2, and provides new clues to block the potential transmission chains of this virus.

Avian influenza A (H7N9) virus: from low pathogenic to highly pathogenic.

The avian influenza A (H7N9) virus is a zoonotic virus that is closely associated with live poultry markets. It has caused infections in humans in China since 2013. Five waves of the H7N9 influenza epidemic occurred in China between March 2013 and September 2017. H7N9 with low-pathogenicity dominated in the first four waves, whereas highly pathogenic H7N9 influenza emerged in poultry and spread to humans during the fifth wave, causing wide concern. Specialists and officials from China and other countries responded quickly, controlled the epidemic well thus far, and characterized the virus by using new technologies and surveillance tools that were made possible by their preparedness efforts. Here, we review the characteristics of the H7N9 viruses that were identified while controlling the spread of the disease. It was summarized and discussed from the perspectives of molecular epidemiology, clinical features, virulence and pathogenesis, receptor binding, T-cell responses, monoclonal antibody development, vaccine development, and disease burden. These data provide tools for minimizing the future threat of H7N9 and other emerging and re-emerging viruses, such as SARS-CoV-2.

A noncompeting pair of human neutralizing antibodies block COVID-19 virus binding to its receptor ACE2.

Neutralizing antibodies could potentially be used as antivirals against the coronavirus disease 2019 (COVID-19) pandemic. Here, we report isolation of four human-origin monoclonal antibodies from a convalescent patient, all of which display neutralization abilities. The antibodies B38 and H4 block binding between the spike glycoprotein receptor binding domain (RBD) of the virus and the cellular receptor angiotensin-converting enzyme 2 (ACE2). A competition assay indicated different epitopes on the RBD for these two antibodies, making them a potentially promising virus-targeting monoclonal antibody pair for avoiding immune escape in future clinical applications. Moreover, a therapeutic study in a mouse model validated that these antibodies can reduce virus titers in infected lungs. The RBD-B38 complex structure revealed that most residues on the epitope overlap with the RBD-ACE2 binding interface, explaining the blocking effect and neutralizing capacity. Our results highlight the promise of antibody-based therapeutics and provide a structural basis for rational vaccine design.

Treatment of 5 Critically Ill Patients With COVID-19 With Convalescent Plasma.

Importance: Coronavirus disease 2019 (COVID-19) is a pandemic with no specific therapeutic agents and substantial mortality. It is critical to find new treatments. Objective: To determine whether convalescent plasma transfusion may be beneficial in the treatment of critically ill patients with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Design, Setting, and Participants: Case series of 5 critically ill patients with laboratory-confirmed COVID-19 and acute respiratory distress syndrome (ARDS) who met the following criteria: severe pneumonia with rapid progression and continuously high viral load despite antiviral treatment; Pao2/Fio2 <300; and mechanical ventilation. All 5 were treated with convalescent plasma transfusion. The study was conducted at the infectious disease department, Shenzhen Third People's Hospital in Shenzhen, China, from January 20, 2020, to March 25, 2020; final date of follow-up was March 25, 2020. Clinical outcomes were compared before and after convalescent plasma transfusion. Exposures: Patients received transfusion with convalescent plasma with a SARS-CoV-2-specific antibody (IgG) binding titer greater than 1:1000 (end point dilution titer, by enzyme-linked immunosorbent assay [ELISA]) and a neutralization titer greater than 40 (end point dilution titer) that had been obtained from 5 patients who recovered from COVID-19. Convalescent plasma was administered between 10 and 22 days after admission. Main Outcomes and Measures: Changes of body temperature, Sequential Organ Failure Assessment (SOFA) score (range 0-24, with higher scores indicating more severe illness), Pao2/Fio2, viral load, serum antibody titer, routine blood biochemical index, ARDS, and ventilatory and extracorporeal membrane oxygenation (ECMO) supports before and after convalescent plasma transfusion. Results: All 5 patients (age range, 36-65 years; 2 women) were receiving mechanical ventilation at the time of treatment and all had received antiviral agents and methylprednisolone. Following plasma transfusion, body temperature normalized within 3 days in 4 of 5 patients, the SOFA score decreased, and Pao2/Fio2 increased within 12 days (range, 172-276 before and 284-366 after). Viral loads also decreased and became negative within 12 days after the transfusion, and SARS-CoV-2-specific ELISA and neutralizing antibody titers increased following the transfusion (range, 40-60 before and 80-320 on day 7). ARDS resolved in 4 patients at 12 days after transfusion, and 3 patients were weaned from mechanical ventilation within 2 weeks of treatment. Of the 5 patients, 3 have been discharged from the hospital (length of stay: 53, 51, and 55 days), and 2 are in stable condition at 37 days after transfusion. Conclusions and Relevance: In this preliminary uncontrolled case series of 5 critically ill patients with COVID-19 and ARDS, administration of convalescent plasma containing neutralizing antibody was followed by improvement in their clinical status. The limited sample size and study design preclude a definitive statement about the potential effectiveness of this treatment, and these observations require evaluation in clinical trials.

Avian-to-Human Receptor-Binding Adaptation of Avian H7N9 Influenza Virus Hemagglutinin.

Since 2013, H7N9 avian influenza viruses (AIVs) have caused more than 1,600 human infections, posing a threat to public health. An emerging concern is whether H7N9 AIVs will cause pandemics among humans. Molecular analysis of hemagglutinin (HA), which is a critical determinant of interspecies transmission, shows that the current H7N9 AIVs are still dual-receptor tropic, indicating limited human-to-human transmission potency. Mutagenesis and structural studies reveal that a G186V substitution is sufficient for H7N9 AIVs to acquire human receptor-binding capacity, and a Q226L substitution would favor binding to both avian and human receptors only when paired with A138/V186/P221 hydrophobic residues. These data suggest a different evolutionary route of H7N9 viruses compared to other AIV-subtype HAs.

Light chain modulates heavy chain conformation to change protection profile of monoclonal antibodies against influenza A viruses.

The isolation of human monoclonal antibodies with broadly neutralizing breadth can provide a promising countermeasure for influenza A viruses infection. Most broadly neutralizing antibodies against influenza A viruses bind to the conserved stem region or the receptor-binding cavity of hemagglutinin and the interaction is dominated by the heavy chain. The light chain, however, contributes few or no direct contacts to the antigen. Here we report an H3-clade neutralizing human monoclonal antibody, AF4H1K1, which recognizes the hemagglutinin glycoproteins of all group 2 influenza A viruses. This human monoclonal antibody has been obtained through the screening by pairing different heavy and light chains from an H7N9-infected patient based on the next-generation sequencing technology. Further structural studies revealed that light chains modulate the neutralizing spectrum by affecting the local conformation of heavy chains, instead of direct interaction with the antigen. These findings provide important clues to understand the molecular basis of light chains in antigen recognition and to explore the strategies in particular of the use of light chain modification to develop broadly protective monoclonal antibodies against influenza A viruses and other emerging viruses.

Double Lock of a Human Neutralizing and Protective Monoclonal Antibody Targeting the Yellow Fever Virus Envelope.

Yellow fever virus (YFV), a deadly human pathogen, is the prototype of the genus Flavivirus. Recently, YFV re-emerged in Africa and Brazil, leading to hundreds of deaths, with some cases imported to China. Prophylactic or therapeutic countermeasures are urgently needed. Previously, several human monoclonal antibodies against YFV were screened out by phage display. Here, we find that one of them, 5A, exhibits high neutralizing potency and good protection. Crystallographic analysis of the YFV envelope (E) protein in its pre- and post-fusion states shows conformations similar to those observed in other E proteins of flaviviruses. Furthermore, the structures of 5A in complex with the E protein in both states are resolved, revealing an invariant recognition site. Structural analysis and functional data suggest that 5A has high neutralization potency because it interferes with virus entry by preventing both virus attachment and fusion. These findings will be instrumental for immunogen or inhibitor design.

Heterosubtypic Protections against Human-Infecting Avian Influenza Viruses Correlate to Biased Cross-T-Cell Responses.

Against a backdrop of seasonal influenza virus epidemics, emerging avian influenza viruses (AIVs) occasionally jump from birds to humans, posing a public health risk, especially with the recent sharp increase in H7N9 infections. Evaluations of cross-reactive T-cell immunity to seasonal influenza viruses and human-infecting AIVs have been reported previously. However, the roles of influenza A virus-derived epitopes in the cross-reactive T-cell responses and heterosubtypic protections are not well understood; understanding those roles is important for preventing and controlling new emerging AIVs. Here, among the members of a healthy population presumed to have previously been infected by pandemic H1N1 (pH1N1), we found that pH1N1-specific T cells showed cross- but biased reactivity to human-infecting AIVs, i.e., H5N1, H6N1, H7N9, and H9N2, which correlates with distinct protections. Through a T-cell epitope-based phylogenetic analysis, the cellular immunogenic clustering expanded the relevant conclusions to a broader range of virus strains. We defined the potential key conserved epitopes required for cross-protection and revealed the molecular basis for the immunogenic variations. Our study elucidated an overall profile of cross-reactivity to AIVs and provided useful recommendations for broad-spectrum vaccine development.IMPORTANCE We revealed preexisting but biased T-cell reactivity of pH1N1 influenza virus to human-infecting AIVs, which provided distinct protections. The cross-reactive T-cell recognition had a regular pattern that depended on the T-cell epitope matrix revealed via bioinformatics analysis. Our study elucidated an overall profile of cross-reactivity to AIVs and provided useful recommendations for broad-spectrum vaccine development.

CTL immunogenicity of Rv3615c antigen and diagnostic performances of an ESAT-6/CFP-10/Rv3615c antigen cocktail for Mycobacterium tuberculosis infection.

T cell immune responses have played pivotal roles in host immune protection against Mycobacterium tuberculosis (MTB) infection. MTB specific antigen, Rv3615c (EspC), was identified to be as immunodominant as the well-known ESAT-6 and CFP-10, and has brought promising expectations to more sensitive T-cell based diagnosis and vaccine development. However, limited knowledge about the immunogenicity and diagnostic values of this antigen has restricted its application in clinical practice. Herein, the Rv3615c antigen was identified as a robust CTL immunoantigen with broadly cross-human leucocyte antigen (HLA) allele recognized peptides which may contribute to the broad recognition of Rv3615c antigen among the population. A three-antigen-cocktail (3-Ag-cocktail) comprising of ESAT-6, CFP-10 and Rv3615c was investigated in a multicenter, randomized and double-blinded study to evaluate its clinical diagnostic performances. A significantly improved sensitivity was demonstrated against the 3-Ag-cocktail compared with that against ESAT-6 and CFP-10. Both responsive magnitude and sensitivity were significantly lower in patients concurrently suffering from cancer, indicating its restriction in diagnosis of immunocomprised patients. In conclusion, inclusion of the Rv3615c antigen with multiple HLA restricted CTL epitopes would benefit the T-cell based diagnosis of MTB infection.

Avian-to-Human Receptor-Binding Adaptation by Influenza A Virus Hemagglutinin H4.

Low-pathogenicity avian influenza viruses (LPAIVs) have caused a global concern to public health since the first novel LPAIV H7N9 outbreak occurred. The receptor-binding properties of the viral hemagglutinin are one key factor for efficient transmission and infection in humans. Recent evidence shows that H4 subtype viruses have been widely circulating in domestic poultry and human asymptomatic infections might have occurred. Here, we evaluated the receptor-binding properties of two representative isolates, avian H4N6 (containing Q226 and G228) and swine H4N6 (containing L226 and S228), and found that the avian isolate preferentially binds to avian receptors, whereas the swine isolate preferentially binds to human receptors. The Q226L and G228S substitutions are pivotal for the receptor-binding switch, which resulted in similar human receptor-binding features to the pandemic H2 and H3, implying that H4 has the potential to cause human infections. This early-warning study calls for future extensive surveillance.