T Cell Epitope Region In Neucleoprotein Of H5N1 Avian Influenza Virus

Avian influenza is an infectious disease of birds caused by influenza A virus. High pathogenic avian influenza virus (HPAIV) can cause birds, especially poultry acute disease and mass mortality, which brings poultry industry great financial loses. Since the first documented outbreak of human infections with H5N1 AIV, which occurred in Hong Kong in 1997, more and more attentions were paid to this virus in related fields and governments. Currently, the main methods to prevent and control the spread of this virus are derived from the generation of neutralizing antibody induced by virus surface antigens hemagglutinin (HA) and neuraminidase (NA). However, few studies related to the cell-mediated immunity caused by the internal proteins such as nucleoprotein (NP) were reported so far.To understand the cell-mediated immunity caused by AIV NP and determine whether or not any T cell epitope is located in this protein, the fowlpox virus (FPV) expression system was engaged and cloned into the full length or segments of NP from H5N1 AIV A/Goose/Guangdong/1/96(H5N1) (GD1/96) in this study. The full length of NP was divided into 6 overlapped segments, around 70 amino acids in each segment, with different primer pairs by PCR amplification. There were 30 to 35 amino acids overlaps between two consecutive segments. The full length and 6 segments of NP were cloned into plasmid pSY538, and LacZ expression unit was ligated to pSY538 downstream of the target genes for recombinant fowl virus screen. Then the target genes and LacZ expression unit were cut from pSY538 vector and sub-cloned into plasmid pSY638, and recombinant plasmid pSY-NPn-LacZ (n=1, 2, 3, 4, 5, 6) were obtained finally. To get the recombinant fowl viruses that express these target proteins, the primary chicken embryo fibroblast (CEF) cells were infected with FPV S-FPV-017 and transfected with pSY-NPn-LacZ successively. The transfected cells and supernatant were harvested after 3-5 days until 80-100% cytopathic effect (CPE) came out. After 3 cycles of freeze-thaw, the recombinant FPVs harbors different segments and full length of NP were purified and tittered with plaque assay on CEF cells. As a result, five rFPVs, named rFPV-NP1,rFPV-NP3,rFPV-NP4 rFPV-NP5 and rFPV-NP, were obtained and all the titers were around 2×106 pfu/mL.In order to induce the memory T cell specific to AIV NP in chicken, NP gene which was cut from recombinant plasmid pVAX-NP was sub-cloned into the downstream of an avian-derived promoter in the eukaryotic expression vector pCAGGS. The recombinant plasmid pCAGGS-NP was gained and verified by PCR identification and DNA sequencing. The positive pCAGGS-NP was subjected to transfect primary CEF and indirect florescent assay (IFA) was engaged to test the transient expression of NP. Then SPF chickens were immunized with 100μg recombinant plasmid pCAGG-NP and vector control pCAGGS intramuscularly or mock immunized. The immunized chickens were boosted with same amount of pCAGG-NP three weeks later. Another three weeks after the second immunization, chickens immunized with pCAGG-NP were challenged with AIV A/Chicken/Heilongjiang/35/00(H9N2) to be a secondary booster. Serum samples were collected every week post immunization and challenge and analyzed for the virus-specific IgG to NP by ELISA. The results showed the serum NP antibody was induced after immunization with pCAGG-NP and dramatically boosted after being challenged. According to the principle of immunological effect of DNA vaccination, the relative cell mediated immunity including memory T cells was generated in vivo.To establish an effective method to evaluate the T cell responses generated by activated chicken lymphocytes after the stimulation of different NP segments, the real-time quantitative RT-PCR assay was developed to detect the chicken interferon gamma (ChIFN-γ) secreted by lymphocytes. Briefly, ChIFN-γand chicken 28S rRNA (Ch28S) genes were amplified from the total RNA of ConA-stimulated spleen lymphocytes by RT-PCR with specific primers respectively and cloned into the pET-28a(+) vector. Using linear recombinant plasmids as the templates, the purified RNAs of ChIFN-γand Ch28S were obtained from in vitro transcription system and diluted gradient by 10-fold. The ChIFN-γin the diluted standard samples was detected by the one-step real-time quantitative RT-PCR with their specific primers and Taqman probe while the Ch28S rRNA served as an internal control. The results showed that the Ct of ChIFN-γor Ch28S have a good linear relationship (R2﹥0.99) with the standard samples from 1×102 to 1×107 copies/μL. Therefore, the method of real-time quantitative RT-PCR was established successfully to measure the ChIFN-γin this study.All the immunized chickens and mock immunized chickens were sacrificed 4 days after AIV H9N2 boost, and the spleens were isolated for the preparation of lymphocytes. All the lymphocytes from each chicken were stimulated with rFPVs containing different fragments of NP respectively. After 24 hours stimulation, the media and cells were collected and separated by centrifuge. IFN-γproduction in the culture supernatant was measured by ELISA kit. The cell pellets were divided into two portions, one of which were used to detect ChIFN-γgene transcription with real-time RT-PCR, the other part were subjected to flow cytometry analysis for CD8+ T cell proliferation. All these data indicated that the fragment 5 (NP277-421) induced a stronger T cell responses for the spleen lymphocytes isolated from pCAGG-NP immunized chickens, which meant T cell epitope(s) was/were located in this fragment.