Mimotope Of Hemagglutinin-neuraminidase From Newcastle Disease Virus And Its Immunological Properties

Newcastle disease, which is listed as class A diseases of poultry by OIE, is a kind of avian acute infectious disease caused by Newcastle disease virus (NDV). Up to now, many hemagglutinin-neuraminidase (HN) nucleotide sequences of NDV strains were obtained, but there is still few reports about its mimotope.In this study, monoclonal antibody (McAb) M22, specific for HN of NDV, was labeled with biotin and used as a molecular probe to screen its recognized mimotopes by biopanning a phage-display 12-mer peptide library. Through three rounds of affinity screening, washing and amplification, the results of enrichment experiment and the ratio of yield (input/output) assay showed that the positive clones were concentrated obviously. 20 single clones were selected randomly from the third round of biopanning elute, and four of them showed strong binding reactivity to McAb M22 in sandwich ELISA. It was further demonstrated that NDV could inhibit one of the clones, CLONE 20 to bind with McAb M22 in competitive ELISA, with the inhibition rate up to 67.3%. It suggested that CLONE 20 share HN mimicry molecules. Based on the sequence of CLONE 20 ssDNA, the deduced epitope sequence was SWFHHHQARAPM.Comparing the displayed-peptide of CLONE 20 and HN sequences of NDV strains such as F48E8 and La Sota, it was inferred that WF and QAR may play a major role in the antigenicity of HN. It was shown that displayed-peptide sequences were located within the predicted epitopes.The immunogenicity of CLONE 20 was evaluated in both chicken and mice. It could induce SPF chicken and BALB/c mice to generate specific antibodies against HN of NDV. As for the SPF chicken intramuscularly injected with CLONE 20 emulsified with Freund's adjuvant, after the third immunization, the geometric mean (GM) titers of HI was 1:5.1, and GM of ELISA titers was 1:78.2. As for the SPF chicken injected CLONE 20without adjuvant, after the third dose, GM of HI titers was 1:3.3, and that of ELISA titers was 1:26.4. In contrast, the negative control group immunized with negative clones and healthy control group' had no specific antibodies. With the statistic analysis, HI titers and ELISA titers were significantly increased in the CLONE 20 immunized groups compared with those of negative control and healthy control groups. Meanwhile there was no significant difference between CLONE 20 immunized groups with or without Freund's adjuvant.As for the BALB/c mice intramuscularly injected with CLONE 20 emulsified with Freund's adjuvant, after the third immunization, the GM titers of HI was 1:3.7, and GM of ELISA titers was 1:59.7. As for the BALB/c mice injected CLONE 20 without adjuvant, after the third administration, the GM titers of HI was 1:2.7, and that of ELISA titers was 1:52.0. In contrast, the negative control group immunized with negative clones and healthy control group had no specific antibodies. With the statistic analysis, HI titers and ELISA titers were significantly increased in the CLONE 20 immunized groups compared with those of negative control and healthy control groups. Meanwhile there was no significant difference between CLONE 20 immunized groups with or without Freund's adjuvant.In summary, the selected peptide sequence of SWFHHHQARAPM was the mimotope recognized by McAb M22. It showed good immunogenicity in both chicken and mice. It would be a new tool to further define antigen structure of HN of NDV and to study the mechanism of its immune response. These data also indicate that this mimotope might be potential components of peptide vaccines against NDV.