Identification Of Key Epitopes Of Potent Neutralizing Antibody Against Human Respiratory Syncytial Virus

Background and Objective:Human respiratory syncytial virus(RSV)causes acute lower respiratory infection that is one of the leading causes of infant mortality,especially in developing countries.RSV epidemic usually occurs in late fall till next early spring,and near every infant is at risk of infection,especially those who are premature,immunocompromised or at high risk of congenital heart and lung diseases.In spite of more than 60 years after its discovery,RSV still poses a huge disease burden each year as no safe and effective vaccine has yet been developed.Currently,the only specific prophylactic treatment for RSV is Synagis that is humanized mouse monoclonal antibody(m Ab)to RSV F protein,which is used in the high-risk infants and young children.However,due to its low potency and short half-life,it is often needed to receive 5-6 injections for population at risk to cover an epidemic season resulting in high medical cost burden.There is an urgent clinical need to develop a better prophylactic treatment for RSV infection in the world,especially in China.It has been found that major portion of neutralizing antibodies against RSV in human after RSV infection were directed to the pre-fusion conformation of RSV F protein(Pre-F).Discovery of RSV Pre-F has been regarded as one of scientific breakthrough discoveries in the year of 2013.Ever since,efforts have been made to use RSV Pre-F as immunogen for vaccine development and as a target for therapeutic antibodies by numbers of research institutions and drug companies with a goal to substitute Synagis for prophylactic treatment against RSV infection.Among those,potent RSV neutralizing antibodies to pre-F have been discovered,which include MEDI8897 being developed by a global drug company Astra Zeneca and having been recently confirmed to have achieved the clinical goal in phase III trials and another antibody TRN1021 that has been developed by Zhuhai Trinomab.It has been demonstrated that TRN1021 has more than 30-fold higher RSV neutralization titers in vitro as well as in vivo protective activity against RSV challenge in cotton rats than does Synagis and has at least three folds higher protective activity in animals than does MEDI8897.However,the key epitopes targeted by TRN1021 remains to be elucidated.Understanding and elucidating the key targets by potent RSV neutralizing m Ab will provide insightful information for design and development of effective RSV vaccines and pave the foundation for development of TRN1021 antibody as a therapeutic for the prevention and treatment of RSV infection.Based on the initial work on TRN1021 by Zhuhai Trinomab,sequence analysis of drug resistant escaping virus mutants induced by m Ab TRN1021 identified amino acid residues of RSV F protein associated with the drug resistance.Computer model molecular docking was carried out to simulate the interactions of m Ab TRN1021 with RSV Pre-F protein and to predicate TRN1021 epitope on RSV Pre-F protein.Computational alanine scanning mutations were performed to evaluate the effect of mutations of the predicated key amino acid residues on the binding of TRN1021.Furthermore,recombinant RSV Pre-F mutant proteins were produced,purified and used to verify the findings from computer simulation.This study has demonstrated that the amino acid residues valine at position 76(V76),lysine at position 209(K209E)on RSVpre F protein identified through drug-resistant mutants as well as the amino acid residues cysteine at position 69 and lysine at position 203 identified through the computer molecular simulation predication and confirmed by using RSV-Pre-F mutants on RSV-Pre-F are critical epitopes recognized by targeted TRN1021.Sequence analysis of total of more than 1500 different RSV subtypes A and B F protein sequences collected in NCBI database indicates that these critical neutralizing epitopes are highly conserved in clinical RSV isolates.Taken together,this study identified key neutralization epitope on RSV Pre-F protein which is insightful and critically important information for structure-based RSV vaccine design and development,provided experimental base for elaborating the mechanism of action of RSV neutralizing antibody TRN1021 and indicated that m Ab TRN1021 can be a potent broadly neutralizing antibody for the prevention and treatment of RSV infection.Methods:1.Computer model molecular docking was carried out to simulate the interactions of m Ab TRN1021 with RSV Pre-F protein and to predicate key amino acid residues of TRN1021 epitope on RSV Pre-F protein.2.Electronic alanine scanning mutations were performed to evaluate the effect of mutations of the predicated key amino acid residues on the binding of TRN1021.3.Mammalian expression constructs for production of RSV Pre-F mutants were produced by site-directed mutagenesis using the wild-type RSV Pre-F gene construct as the template.4.Recombinant wild-type and mutant RSV Pre-F proteins as well as recombinant TRN1021 antibody were produced in 293 F cells by transient transfection and purified by using nickel columns and/or protein A columns.5.Binding and affinity of TRN1021 antibody to the purified recombinant mutant RSV PreF proteins and the wild-type and mutant RSV Pre-F protein were determined and compared in ELISA and SPR assays.6.Sequences of RSV F proteins collected in NCBI database were downloaded and analyzed for variations of the amino acid residues in RSV F protein at the positions identified in this study.Conclusion:1.Computer model molecular docking simulation predicated the binding epitopes of TRN1021 antibody are amino acid residues at positions of 66,68-73,76,200,204,205,208-213.2.Amino acid residues cysteine at position 69(C69)and lysine in RSV-Pre F at position 203(L203)were identified through computer molecular simulation predication and confirmed by using RSV pre-F mutants as the critical epitopes recognized by TRN1021.3.Analysis of F protein sequences of different RSV subtypes A(n=961)and B(n=610)collected in NCBI database indicates that critical amino acid residues V76,K209,C69 and L 203 of epitope targeted by TRN1021 neutralizing antibody are highly conserved in circulating clinical RSV isolates.Taken together,this study identified key neutralization epitope on RSV Pre-F protein which is insightful and critically important information for better design and development of RSV vaccine,provided experimental base for elaborating the mechanism of action of RSV neutralizing antibody TRN1021 and indicated that m Ab TRN1021 as potent broadly neutralizing antibody can be potentially developed for the prevention and treatment of RSV infection.