Preparation, Characterization And Immune Protective Role Of A Monoclonal Antibody Against Human Respiratory Syncytial Virus

Objective:The worldwide spread human respiratory syncytial virus (RSV) is the most important etiological cause of viral lower respiratory tract illness in infants and children. So far no safe and effective vaccines are available. In this research, we used hybridoma technology to preparare neutralizing monoclonal anbibody (McAb) against RSV, which is of great significance in immunotherapy on RSV infection.Methods: The RSV was proliferated on HEp-2 cells and the resulting culture supernant was used to vaccinate BALB/c mice intranasally. The Spleen cells from the mouse with the highest titer serum was fused with myeloma cells. Hybridomas were screened by indirect ELISA after subcloning by the limiting dilution technique. The McAb was purified through protein G affinity chromatography, whose specificity and affinity were analyzed by indirect ELISA, indirect immunofluoresecnce (IIF) and Western blot. Then, the indirect ELISA, non-competitive ELISA and competitive ELISA were employed to identify biological characteristics of this McAb including subtype, affinity and the recognized epitope. Through the immunoenzyme method, established by our group, we investigated the neutralizing activity and fusion inhibition capacity of McAb F8 in vitro. Later, total RNA was extracted from the hybridoma cells. The general primes specific for VH and VL of mouse antibody were used to amplify VH and VL genes of this McAb. The PCR products were inserted into pGEM-T vector, sequenced and compared with the reported McAb against RSV. Animal model of RSV infection was established and applied to identify the neutralizing activity of this selected McAb in vivo.Result: A strain of McAb F8 against RSV fusion protein (F) was obtained. The purified McAb F8 attained 95% purity and the affinity constant (Ka) and the isotype of McAb F8 was 6.8×108 L/mol and IgG1, respectively. The rescognized protein and epitope of McAb F8 were F1 fragment of RSV F protein and amino acids 205-222. McAb F8 had the neutralizing activity and fusion inhibition capacity in vitro. The VH gene contained 371 bp and encoded 124 amino acid residues; the VL gene contained 323 bp and encoded 107 amino acid residues. They were homologous with the sequences of variable region of mouse IgG, which were published in GeneBank. Compared further with RSV murine McAb against F protein from the U.S. Patents, the VH and VL homologies were 75.4% and 95.37% respectively. The in vivo immunotherapy experiment displayed that McAb F8 was also able to reduce the lung virus titer in RSV infected mice.Conclusion: The McAb F8 specific for RSV F had been obtained and purified. Some biological features of McAb F8 were identified. The sequence of variable region of McAb F8 had been successfully cloned, and found highly homologous with the reported McAb against RSV F. Additionally, McAb F8 displayed neutralizing activity and fusion inhibition capacity both in vitro and in vivo. All of the results were benefitial to its immunotherapy role on RSV infection.