Cross-neutralizing Anti-hemagglutinin Antibodies Isolated from Patients Infected with Avian Influenza A(H5N1) Virus

Objective To recover broad-neutralizing monoclonal antibodies(Bn Abs)from avian influenza A(H5N1)virus infection cases and investigate their genetic and functional features.Methods We screened the Abs repertoires of expanded B cells circulating in the peripheral blood of H5N1 patients.The genetic basis,biological functions,and epitopes of the obtained Bn Abs were assessed and modeled.Results Two Bn Abs,2-12 D5,and 3-37 G7.1,were respectively obtained from two human H5N1 cases on days 12 and 21 after disease onset.Both Abs demonstrated cross-neutralizing and Ab-dependent cellular cytotoxicity(ADCC)activity.Albeit derived from distinct Ab lineages,i.e.,V^H1-69-D2-15-JH^4(2-12D5)and V^H1-2-D3-9-JH^5(3-32 G7.1),the Bn Abs were directed toward CR6261-like epitopes in the HA stem,and HA2 I45 in the hydrophobic pocket was the critical residue for their binding.Signature motifs for binding with the HA stem,namely,IFY in VH1-69-encoded Abs and LXYFXW in D3-9-encoded Abs,were also observed in 2-12D5 and 3-32 G7.1,respectively.Conclusions Cross-reactive B cells of different germline origins could be activated and re-circulated by avian influenza virus.The HA stem epitopes targeted by the Bn Abs,and the two Ab-encoding genes usage implied the VH1-69 and D3-9 are the ideal candidates triggered by influenza virus for vaccine development.

Serological Investigation into the Infected Genotypes of Patients with Japanese Encephalitis in the Coastal Provinces of China

Objective Genotypes(G)1,3,and 5 of the Japanese encephalitis virus(JEV)have been isolated in China,but the dominant genotype circulating in Chinese coastal areas remains unknown.We searched for G5 JEV-infected cases and attempted to elucidate which JEV genotype was most closely related to human Japanese encephalitis(JE)in the coastal provinces of China.Methods In this study,we collected serum specimens from patients with JE in three coastal provinces of China(Guangdong,Zhejiang,and Shandong)from 2018 to 2020 and conducted JEV cross-neutralization tests against G1,G3,and G5.Results Acute serum specimens from clinically reported JE cases were obtained for laboratory confirmation from hospitals in Shandong(92 patients),Zhejiang(192 patients),and Guangdong(77 patients),China,from 2018 to 2020.Seventy of the 361 serum specimens were laboratory-confirmed to be infected with JEV.Two cases were confirmed to be infected with G1 JEV,32 with G3 JEV,and two with G5 JEV.Conclusion G3 was the primary infection genotype among JE cases with a definite infection genotype,and the infection caused by G5 JEV was confirmed serologically in China.

Sequentially immune-induced antibodies could cross-neutralize SARS-CoV-2 variants

Background:The Omicron(B.1.1.529)SARS-COV-2 variant has raised serious concerns because of its unprecedented rapid rate of spreading and the fact that there are 36 mutations in the spike protein.Since the vaccine-induced neutralizing antibody targets are the spike protein,this may lead to the possibility of vaccine-induced hu-moral immunity escape.Methods:We measured the neutralizing activity in vitro for Omicron and compared this with wild type(WH-09)and Delta variants in human and monkey sera from different types of immunity.The monkey sera samples were collected at 1 and 3 months post three-dose inactivated(PiCoVacc)and recombinant protein(ZF2001)vaccination.Human sera were collected from 1 month post three-dose inactivated vaccination.Results:In inactivated vaccine sera,at 1/3 months post three-dose,geometric mean titers(GMTs)of neutralization antibody(NAb)against the Omicron variant were 4.9/5.2-fold lower than those of the wild type.In recombinant protein vaccine sera,GMTs of NAb against Omicron were 15.7/8.9-fold lower than those of the wild type.In human sera,at 1 month post three-dose inactivated vaccination,GMTs of NAb against Omicron were 3.1-fold lower than those of the wild type.Conclusion:This study demonstrated that despite a reduction in neutralization titers,cross-neutralizing activity against Omicron and Delta variants was still observed after three doses of inactivated and recombinant protein vaccination.

Comparison of the replication and neutralization of different SARS-CoV-2 Omicron subvariants in vitro

Background:New Omicron subvariants are emerging rapidly from BA.1 to BA.4 and BA.5.Their pathogenicity has changed from that of wild-type(WH-09)and Omicron variants have over time become globally dominant.The spike proteins of BA.4 and BA.5 that serve as the target for vaccine-induced neutralizing antibodies have also changed compared to the previous subvariants,which is likely to cause immune es-cape and the reduction of the protective effect of the vaccine.Our study addresses the above issues and provides a basis for formulating relevant prevention and control strategies.Methods:We collected cellular supernatant and cell lysates and measured the viral titers,viral RNA loads,and E subgenomic RNA(E sgRNA)loads in different Omicron subvariants grown in Vero E6 cells,using WH-09 and Delta variants as a reference.Additionally,we evaluated the in vitro neutralizing activity of different Omicron sub-variants and compared it to the WH-09 and Delta variants using macaque sera with different types of immunity.Results:As the SARS-CoV-2 evolved into Omicron BA.1,the replication ability in vitro began to decrease.Then with the emergence of new subvariants,the replication ability gradually recovered and became stable in the BA.4 and BA.5 subvariants.In WH-09-inactivated vaccine sera,geometric mean titers of neutralization antibodies against different Omicron subvariants declined by 3.7~15.4-fold compared to those against WH-09.In Delta-inactivated vaccine sera,geometric mean titers of neutrali-zation antibodies against Omicron subvariants declined by 3.1~7.4-fold compared to those against Delta.Conclusion:According to the findings of this research,the replication efficiency of all Omicron subvariants declined compared with WH-09 and Delta variants,and was lower in BA.1 than in other Omicron subvariants.After two doses of inactivated(WH-09 or Delta)vaccine,cross-neutralizing activities against various Omicron subvariants were seen despite a decline in neutralizing titers.

Genetic Variation of the VP1 Gene of the Virulent Duck Hepatitis A Virus Type 1(DHAV-1) Isolates in Shandong Province of China

To investigate the relationship of the variation of virulence and the external capsid proteins of the pandemic duck hepatitis A virus type 1(DHAV-1) isolates,the virulence,cross neutralization assays and the complete sequence of the virion protein 1(VP1) gene of nine virulent DHAV-1 strains,which were isolated from infected ducklings with clinical symptoms in Shandong province of China in 2007-2008,were tested.The fifth generation duck embryo allantoic liquids of the 9 isolates were tested on 12-day-old duck embryos and on 7-day-old ducklings for the median embryonal lethal doses(ELD 50 s) and the median lethal doses(LD 50 s),respectively.The results showed that the ELD 50 s of embryonic duck eggs of the 9 DHAV-1 isolates were between 1.9 × 10 6 /mL to 1.44 × 10 7 /mL,while the LD 50 s were 2.39 × 10 5 /mL to 6.15 × 10 6 /mL.Cross-neutralization tests revealed that the 9 DHAV-1 isolates were completely neutralized by the standard serum and the hyperimmune sera against the 9 DHAV-1 isolates,respectively.Compared with other virulent,moderate virulent,attenuated vaccine and mild strains,the VP1 genes of the 9 strains shared 89.8%-99.7% similarity at the nucleotide level and 92.4%-99.6% at amino acid level with other DHAV-1 strains.There were three hypervariable regions at the C-terminus(aa 158-160,180-193 and 205-219) and other variable points in VP1 protein,but which didn't cause virulence of DHAV-1 change.

表位结构指导免疫优势转移的HPV交叉疫苗的理性设计

In vaccine development,broadly or cross-type neutralizing antibodies(bnAbs or cnAbs)are frequently targeted to enhance protection.Utilizing immunodominant antibodies could help fine-tune vaccine immunogenicity and augment the precision of immunization strategies.However,the methodologies to capitalize on the attributes of bnAbs in vaccine design have not been clearly elucidated.In this study,we discovered a cross-type neutralizing monoclonal antibody,13H5,against human papillomavirus 6(HPV6)and HPV11.This nAb exhibited a marked preference for HPV6,demonstrating superior binding activity to virus-like particles(VLPs)and significantly higher prevalence in anti-HPV6 human serum as compared to HPV11 antiserum(90%vs.31%).Through co-crystal structural analysis of the HPV6 L1 pentamer:13H5 complex,we delineated the epitope as spanning four segments of amino acids(Phe42-Ala47,Gly172-Asp173,Glu255-Val275,and Val337-Tyr351)on the L1 surface loops.Further interaction analysis and site-directed mutagenesis revealed that the Ser341 residue in the HPV6 HI loop plays a critical role in the interaction between 13H5 and L1.Substituting Ser341 with alanine,which is the residue type present in HPV11 L1,almost completely abolished binding activity to 13H5.By swapping amino acids in the HPV11 HI loop with corresponding residues in HPV6 L1(Ser341,Thr338,and Thr339),we engineered chimeric HPV11-6HI VLPs.Remarkably,the chimeric HPV11-6HI VLPs shifted the high immunodominance of 13H5 from HPV6 to the engineered VLPs and yielded comparable neutralization titers for both HPV6 and HPV11 in mice and non-human primates.This approach paves the way for the design of broadly protective vaccines from antibodies within the main immunization reservoir.

Cross-neutralization of SARS coronavirus-specific antibodies against bat SARS-like coronaviruses

Dear Editor,The 2002–2003 global pandemic caused by severe acute respiratory syndrome coronavirus（SARS-CoV）infected around 8,000 people with 10%mortality（http：//www.who.int/csr/sars/en/）.

Key residues of the receptor binding motif in the spike protein of SARS-CoV-2 that interact with ACE2 and neutralizing antibodies

Coronavirus disease 2019(COVID-19),caused by the novel human coronavirus SARS-CoV-2,is currently a major threat to public health worldwide.The viral spike protein binds the host receptor angiotensin-converting enzyme 2(ACE2)via the receptor-binding domain(RBD),and thus is believed to be a major target to block viral entry.Both SARS-CoV-2 and SARS-CoV share this mechanism.Here we functionally analyzed the key amino acid residues located within receptor binding motif of RBD that may interact with human ACE2 and available neutralizing antibodies.The in vivo experiments showed that immunization with either the SARS-CoV RBD or SARS-CoV-2 RBD was able to induce strong clade-specific neutralizing antibodies in mice;however,the cross-neutralizing activity was much weaker,indicating that there are distinct antigenic features in the RBDs of the two viruses.This finding was confirmed with the available neutralizing monoclonal antibodies against SARS-CoV or SARS-CoV-2.It is worth noting that a newly developed SARS-CoV-2 human antibody,HA001,was able to neutralize SARS-CoV-2,but failed to recognize SARS-CoV.Moreover,the potential epitope residues of HA001 were identified as A475 and F486 in the SARS-CoV-2 RBD,representing new binding sites for neutralizing antibodies.Overall,our study has revealed the presence of different key epitopes between SARS-CoV and SARSCoV-2,which indicates the necessity to develop new prophylactic vaccine and antibody drugs for specific control of the COVID-19 pandemic although the available agents obtained from the SARS-CoV study are unneglectable.

Characterization of the receptor-binding domain(RBD)of 2019 novel coronavirus:implication for development of RBD protein as a viral attachment inhibitor and vaccine

The outbreak of Coronavirus Disease 2019(COVID-19)has posed a serious threat to global public health,calling for the development of safe and effective prophylactics and therapeutics against infection of its causative agent,severe acute respiratory syndrome coronavirus 2(SARS-CoV-2),also known as 2019 novel coronavirus(2019-nCoV).The CoV spike(S)protein plays the most important roles in viral attachment,fusion and entry,and serves as a target for development of antibodies,entry inhibitors and vaccines.Here,we identified the receptor-binding domain(RBD)in SARS-CoV-2 S protein and found that the RBD protein bound strongly to human and bat angiotensin-converting enzyme 2(ACE2)receptors.SARS-CoV-2 RBD exhibited significantly higher binding affinity to ACE2 receptor than SARS-CoV RBD and could block the binding and,hence,attachment of SARS-CoV-2 RBD and SARS-CoV RBD to ACE2-expressing cells,thus inhibiting their infection to host cells.SARS-CoV RBD-specific antibodies could crossreact with SARS-CoV-2 RBD protein,and SARS-CoV RBD-induced antisera could cross-neutralize SARS-CoV-2,suggesting the potential to develop SARS-CoV RBD-based vaccines for prevention of SARS-CoV-2 and SARS-CoV infection.