A strategy to produce monoclonal antibodies against gp96 by prime-boost regimen using endogenous protein and E.coli heterologously-expressed fragment

Gp96, a member of HSP90 family, is a versatile molecular chaperone with various newly-discovered functions, for example to serve as a low affinity, high capacity calcium binding protein, a natural adjuvant for therapeutic cancer vaccines, a tumor rejection antigen, an immune regulator to pathological cell death. Its multi-functional and structural characteristics make it also an interesting target to develop antibody-based therapeutics. However, its low immunogenicity to mice, because of its high-sequence similarity among different species, is an obstacle to obtain valuable monoclonal antibodies (MAbs). This is a common problem for any low immunogenic proteins, whose sequences share close identity between mice and other species. Here, a new strategy of priming was employed by swine endogenous full-length gp96 and then boosting by E. coli-system heterologously expressed gp96 N-terminal fragment (N-355) to generate MAbs. Twelve different highly-specific MAbs against swine/human endogenous gp96 were successfully obtained. The binding activities of these MAbs were confirmed by enzyme-linked immunosorbent assay (ELISA), Western blot (WB), immunofluorescence and flow cytometry analysis. This provides some important reagents for further research and potential therapeutics. The methods employed can be used for MAb production of any sequence-highly-conserved proteins between mice and swine/human (or any other species).

Structure and receptor-binding properties of an airborne transmissible avian infl uenza A virus hemagglutinin H5(VN1203mut)

Avian infl uenza A virus continues to pose a global threat with occasional H5N1 human infections,which is em-phasized by a recent severe human infection caused by avian-origin H7N9 in China.Luckily these viruses do not transmit effi ciently in human populations.With a few ami-no acid substitutions of the hemagglutinin H5 protein in the laboratory,two H5 mutants have been shown to obtain an air-borne transmission in a mammalian ferret model.Here in this study one of the mutant H5 proteins devel-oped by Kawaoka’s group(VN1203mut)was expressed in a baculovirus system and its receptor-binding properties were assessed.We herein show that the VN1203mut had a dramatically reduced binding affi nity for the avianα2,3-linkage receptor compared to wild type but showed no detectable increase in affi nity for the humanα2,6-linkage receptor,using Surface Plasmon Resonance techonology.Further,the crystal structures of the VN1203mut and its complexes with either human or avian receptors demon-strate that the VN1203mut binds the human receptor in the same binding manner(cis conformation)as seen for the HAs of previously reported 1957 and 1968 pandemic influenza viruses.Our receptor binding and crystallo-graphic data shown here further confi rm that the ability to bind the avian receptor has to decrease for a higher hu-man receptor binding affi nity.As the Q226L substitution is shown important for obtaining human receptor binding,we suspect that the newly emerged H7N9 binds human receptor as H7 has a Q226L substitution.

Characterization of human αβTCR repertoire and discovery of D-D fusion in TCRβ chains

The characterization of the human T-cell receptor （TCR） repertoire has made remarkable progress, with most of the work focusing on the TCRβ chains. Here, we ana- lyzed the diversity and complexity of both the TCRa and TCRβ repertoires of three healthy donors. We found that the diversity of the TCRα repertoire is higher than that of the TCRβ repertoire, whereas the usages of the V and J genes tended to be preferential with similar TRAV and TRAJ patterns in all three donors. The V-J pairings, like the V and J gene usages, were slightly preferential. We also found that the TRDV1 gene rearranges with the majority of TRAJ genes, suggesting that TRDV1 is a shared TRAV/DV gene （TRAV42/DV1）. Moreover, we uncovered the presence of tandem TRBD （TRB D gene） usage in -2% of the productive human TCRβ CDR3 sequences.

Linking innate and adaptive immunity

The evolution of mechanisms to fight pathogens in plants and animals is reflected in the complexity of the components of their so-called immune systems. Higher vertebrates have

Structural characterization of SARS-CoV-2 dimeric ORF9b reveals potential fold-switching trigger mechanism

The constant emergence of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)variants indicates the evolution and adaptation of the virus.Enhanced innate immune evasion through increased expression of viral antagonist proteins,including ORF9b,contributes to the improved transmission of the Alpha variant;hence,more attention should be paid to these viral proteins.ORF9b is an accessory protein that suppresses innate immunity via a monomer conformation by binding to Tom70.Here,we solved the dimeric structure of SARS-CoV-2 ORF9b with a long hydrophobic tunnel containing a lipid molecule that is crucial for the dimeric conformation and determined the specific lipid ligands as monoglycerides by conducting a liquid chromatography with tandem mass spectrometry analysis,suggesting an important role in the viral life cycle.Notably,a long intertwined loop accessible for host factor binding was observed in the structure.Eight phosphorylated residues in ORF9b were identified,and residues S50 and S53 were found to contribute to the stabilization of dimeric ORF9b.Additionally,we proposed a model of multifunctional ORF9b with a distinct conformation,suggesting that ORF9b is a fold-switching protein,while both lipids and phosphorylation contribute to the switching.Specifically,the ORF9b monomer interacts with Tom70 to suppress the innate immune response,whereas the ORF9b dimer binds to the membrane involving mature virion assembly.Our results provide a better understanding of the multiple functions of ORF9b.

Structural basis of Semliki Forest virus entry using the very-low-density lipoprotein receptor

Alphaviruses are a group of important viruses that cause significant diseases in humans.Among them,Semliki Forest vi-rus(SFV)not only causes symptoms such as joint pain but also infects neuron cells and induces encephalitis in rodents.Recently,the very-low-density lipoprotein receptor(VLDLR)was identified as the cellular receptor for SFV entry.In this study,we present the cryo-electron microscopy structure of SFV bound to human VLDLR.VLDLR targets E1-DIII region of SFV using its membrane-distal LDLR class A(LA)repeats.Structural and functional analyses emphasize the synergistic role of multiple VLDLR repeats in the SFV entry.Remarkably,VLDLR’s binding mode to SFV closely mirrors that of minor group human rhinoviruses but differs significantly from other alphaviruses’interactions with receptors in the canyon re-gion of the E protein.We also assessed SFV binding to VLDLR or apolipoprotein E receptor 2(ApoER2)proteins in horses and mosquitoes and revealed their use of multiple but different LA repeats for binding.Our findings illuminate SFV’s cross-species infectivity,offering insights into potential antiviral strategies against alphavirus infections.

Tyrosine metabolic enzymes from insects and mammals：A comparative perspective

Differences in the metabolism of tyrosine between insects and mammals present an interesting example of molecular evolution. Both insects and mammals possess finetuned systems of enzymes to meet their specific demands for tyrosine metabolites; however, more homologous enzymes involved in tyrosine metabolism have emerged in many insect species. Without knowledge of modem genomics, one might suppose that mammals, which are generally more complex than insects and require tyrosine as a precur sor for important catecholamine neurotransmitters and for melanin, should possess more enzymes to control tyrosine metabolism. Therefore, the question of why insects actually possess more tyrosine metabolic enzymes is quite interesting. It has long been known that insects rely heavily on tyrosine metabolism for cuticle hardening and for innate immune responses, and these evolutionary constraints are likely the key answers to this question. In terms of melanogenesis, mammals also possess a high level of regulation; yet mam malian systems possess more mechanisms for detoxification whereas insects accelerate pathways like melanogenesis and therefore must bear increased oxidative pressure. Our research group has had the opportunity to characterize the structure and function of many key proteins involved in tyrosine metabolism from both insects and mammals. In this mini review we will give a brief overview of our research on tyrosine metabolic enzymes in the scope of an evolutionary perspective of mammals in comparison to insects.

An octamer of enolase from Streptococcus suis

Enolase is a conserved cytoplasmic metalloenzyme existing universally in both eukaryotic and prokaryotic cells.The enzyme can also locate on the cell surface and bind to plasminogen,via which contributing to the mucosal surface localization of the bacterial pathogens and assisting the invasion into the host cells.The functions of the eukaryotic enzymes on the cell surface expression(including T cells,B cells,neutrophils,monocytoes,neuronal cells and epithelial cells)are not known.Streptococcus suis serotype 2(S.suis 2,SS2)is an important zoonotic pathogen which has recently caused two large-scale outbreaks in southern China with severe streptococcal toxic shock syndrome(STSS)never seen before in human sufferers.We recently identified the SS2 enolase as an important protective antigen which could protect mice from fatal S.suis 2 infection.In this study,a 2.4-angstrom structure of the SS2 enolase is solved,revealing an octameric arrangement in the crystal.We further demonstrated that the enzyme exists exclusively as an octamer in solution via a sedimentation assay.These results indicate that the octamer is the biological unit of SS2 enolase at least in vitro and most likely in vivo as well.This is,to our knowledge,the first comprehensive characterization of the SS2 enolase octamer both structurally and biophysically,and the second octamer enolase structure in addition to that of Streptococcus pneumoniae.We also investigated the plasminogen binding property of the SS2 enzyme.

Structural vaccinology: structure-based design of influenza A virus hemagglutinin subtype-specific subunit vaccines

There is a great need for new vaccine development against influenza A viruses due to the drawbacks of traditional vaccines that are mainly prepared using embryonated eggs.The main component of the current split influenza A virus vaccine is viral hemagglutinin(HA)which induces a strong antibody-mediated immune response.To develop a modern vaccine against influenza A viruses,the current research has been focused on the universal vaccines targeting viral M2,NP and HA proteins.Crystallographic studies have shown that HA forms a trimer embedded on the viral envelope surface,and each monomer consists of a globular head(HA1)and a“rod-like”stalk region(HA2),the latter being more conserved among different HA subtypes and being the primary target for universal vaccines.In this study,we rationally designed the HA head based on the crystal structure of the 2009-pandemic influenza A(H1N1)virus HA as a model,tested its immunogenicity in mice,solved its crystal structure and further examined its immunological characteristics.The results show that the HA globular head can be easily prepared by in vitro refolding in an E.coli expression system,which maintains its intact structure and allows for the stimulation of a strong immune response.Together with recent reports on some similar HA globular head preparations we conclude that structure-based rational design of the HA globular head can be used for subtype-specific vaccines against influenza viruses.

Genomic characterizations of H4 subtype avian influenza viruses from live poultry markets in Sichuan province of China, 2014–2015

Dear Editor,Avian influenza viruses （AIVs） have posed a serious threat to poultry production and public health. To date, more than fourteen AIV subtypes that are able to infect human beings have been documented. Also, it is suggested that new subtypes may be reported in the future, owing to the migration of wild birds and live poultry transportation （Gao, 2018）.Poultry may act as a potential incubator for novel subtypes of avian influenza virus （Bi et al., 2016a; Bi et al., 2016b; Liu et al., 2014a; Su et al., 2017）. Up to date.

Crystal structure of the C-terminal fragment of NS1 protein from yellow fever virus

Dear Editor,Yellow fever（YF）,a mosquito-borne flavivirus disease,is endemic in tropical areas of Africa and Central and South America.YF is transmitted via the bite of infected Aedes aegypti or Haemogogus mosquitoes and mainly affects humans and nonhuman primates.

Structural basis for the inhibition of the SARS-CoV-2 main protease by the anti-HCV drug narlaprevir

Dear Editor,The second wave of the coronavirus disease(COVID-19)pandemic has recently appeared in Europe.Most European countries,such as France,Germany,and Italy,have announced the implementation of a new round of epidemic prevention and control measures.However,no clinical drug or vaccine has been approved for the treatment of COVID-19.The interim results of the solidarity therapy trial coordinated by the World Health Organization(WHO)indicated that remdesivir,hydroxychloroquine,lopina-vir/ritonavir,and interferon appear to have little or no effect on the 28-day mortality of hospitalized patients or the hospitalization process of new COVID-19 patients.Therefore,there is an urgent need to develop new drugs against COVID-19.

The antigenicity of SARS-CoV-2 Delta variants aggregated 10 high-frequency mutations in RBD has not changed sufficiently to replace the current vaccine strain

Emerging SARS-CoV-2 variants are the most serious problem for COVID-19 prophylaxis and treatment.To determine whether the SARS-CoV-2 vaccine strain should be updated following variant emergence like seasonal flu vaccine,the changed degree on antigenicity of SARS-CoV-2 variants and H3N2 flu vaccine strains was compared.The neutralization activities of Alpha,Beta and Gamma variants’spike protein-immunized sera were analysed against the eight current epidemic variants and 20 possible variants combining the top 10 prevalent RBD mutations based on the Delta variant,which were constructed using pseudotyped viruses.Meanwhile,the neutralization activities of convalescent sera and current inactivated and recombinant protein vaccine-elicited sera were also examined against all possible Delta variants.Eight HA protein-expressing DNAs elicited-animal sera were also tested against eight pseudotyped viruses of H3N2 flu vaccine strains from 2011–2019.Our results indicate that the antigenicity changes of possible Delta variants were mostly within four folds,whereas the antigenicity changes among different H3N2 vaccine strains were approximately 10–100-fold.Structural analysis of the antigenic characterization of the SARS-CoV-2 and H3N2 mutations supports the neutralization results.This study indicates that the antigenicity changes of the current SARS-CoV-2 may not be sufficient to require replacement of the current vaccine strain.

A neutralizing-protective supersite of human monoclonal antibodies for yellow fever virus

The yellow fever virus(YFV)is a life-threatening human pathogen.Owing to the lack of available therapeutics,non-vaccinated individuals are at risk.Here,we isolated eight human monoclonal antibodies that neutralize YFV infection.Five recognized overlapping epitopes and exhibited potent neutralizing activity.Two(YD6 and YD73)were ultra-potent and conferred complete protection against the lethal challenge of YFV as both prophylactics and therapeutics in a mouse model.Crystal structures revealed that YD6 engaged the YFV envelope protein in both pre-and post-fusion states,suggesting viral inhibition by a“double-lock”mechanism.The recognition determinants for YD6 and YD73 are clustered at the premembrane(prM)-binding site.Notably,antibodies targeting this site were present in minute traces in YFV-infected individuals but contributed significantly to neutralization,suggesting a vulnerable supersite of YFV.We provide two promising candidates for immunotherapy against YFV,and the supersite represents an ideal target for epitope-based vaccine design.

Haunted with and hunting for viruses

Early this year, a subtype influenza A virus （H7N9）, was found to cause severe human infections with a 29% case fatality rate （CFR）, making it a top-profile piece of news in the world （www.who.org）. The infection was shortly con- firmed to be related to live animal markets and poultry dealing , which led to a close-down of the markets and the outbreak has since been well controlled （www.chinacdc.cn）.

Natural killer cells are activated and play a protective role against ZIKA virus infection in mice

Zika virus(ZIKV)is a mosquito-borne Flavivirus that was responsible for an epidemic in the Americas during 2015–2016[1].ZIKV infections in adults have been linked to Guillain-Barrésyndrome[2,3],as well as abnormalities such as abortion,stillbirth,and microcephaly in some cases[4].In addition,we have reported that ZIKV infect testicular stem-like peritubular

Crystal structures of the two membrane-proximal Ig-like domains(D3D4)of LILRB1/B2:alternative models for their involvement in peptide-HLA binding

Leukocyte immunoglobulin-like receptors(LILRs),also called CD85s,ILTs,or LIRs,are important mediators of immune activation and tolerance that contain tandem immunoglobulin(Ig)-like folds.There are 11(in addition to two pseudogenes)LILRs in total,two with two Ig-like domains(D1D2)and the remaining nine with four Ig-like domains(D1D2D3D4).Thus far,the structural features of the D1D2 domains of LILR proteins are well defi ned,but no structures for the D3D4 domains have been reported.This is a very important fi eld to be studied as it relates to the unknown functions of the D3D4 domains,as well as their relative orientation to the D1D2 domains on the cell surface.Here,we report the crystal structures of the D3D4 domains of both LILRB1 and LILRB2.The two Ig-like domains of both LILRB1-D3D4 and LILRB2-D3D4 are arranged at an acute angle(~60°)to form a bent struc-ture,resembling the structures of natural killer inhibitory receptors.Based on these two D3D4 domain structures and previously reported D1D2/HLA I complex structures,two alternative models of full-length(four Ig-like domains)LILR molecules bound to HLA I are proposed.

Structural and functional insights into MCR-2 mediated colistin resistance

Dear Editor,The spread of antibiotic resistance genes among bacteria is a serious and growing threat to global health (Peleg and Hoopet,2010).One emerging case is transferable genes found in bacteria-encoding enzymes resistant to colistin.Colistin is a cationic polypeptide antibiotic with broadspectrum activity against Gram-negative bacteria through interactions with the lipid A moiety of bacterial lipopoly-saccharide(LPS),subsequently destroying the cell envelope.During late 2015,a plasmid-mediated gene,mcr-1,was first reported from Escherichia coli(E.coli)in China to confer resistance to colistin(Liu et a1.,2016),and detected world-wide soon afterwards(Gao et a1.,2016;Hu et a1.,2016).

Structural vaccinology: structure-based design of influenza A virus hemagglutinin subtype-specific subunit vaccines

The figure legends for Figure 4 and Figure 5 should be changed as follows:Figure 4.Hemagglutination inhibition(HAI)titers in mice.BALB/c mice were immunized intramuscularly with either 25μg or 50μg of HA57–264.Sera were collected before viral challenge and were assessed against A/California/07/2009.Bars indicate geometric mean titers(GMT).n=8 mice per group.

In vitro assembly of Ebola virus nucleocapsidlike complex expressed in E. coil

Ebola virus （EBOV） harbors an RNA genome encapsi- dated by nucleoprotein （NP） along with other viral pro- teins to form a nucleocapsid complex. Previous Cryo- eletron tomography and biochemical studies have shown the helical structure of EBOV nucleocapsid at nanometer resolution and the first 450 amino-acid of NP （NP△451-739） alone is capable of forming a helical nucleocapsid-Iike complex （NLC）. However, the struc- tural basis for NP-NP interaction and the dynamic pro- cedure of the nucleocapsid assembly is yet poorly understood. In this work, we, by using an E. coli expression system, captured a series of images of NP△451-739 conformers at different stages of NLC assembly by negative-stain electron microscopy, which allowed us to picture the dynamic procedure of EBOV nucleocapsid assembly. Along with further biochemical studies, we showed the assembly of NLC is salt-sensi- tive, and also established an indispensible role of RNA in this process. We propose the diverse modes of NLC elongation might be the key determinants shaping the plasticity of EBOV virions. Our findings provide a new model for characterizing the self-oligomerization of viral nucleoproteins and studying the dynamic assembly process of viral nucleocapsid in vitro.