抗禽流感病毒多表位DNA疫苗的构建及其免疫效力研究

多表位DNA疫苗是建立在常规DNA疫苗基础上的一种新型疫苗。它是用表位作免疫原，这样就比较容易在一个表达载体上克隆病原体的多个抗原基因中具有免疫活性的部分。本试验以H5N1亚型禽流感病毒的HA和NP基因及其表位为基础构建了4个重组质粒：1 pIRES/HA（表达全长的HA基因）；2 pIRES/tHA（只表达HA基因的主要抗原表位区）；3 pIRES/tHANpep（融合表达HA基因的抗原表位区和NP基因的3个CTL表位）；4 pIRES/tHANpep-IFN-γ（用鸡的IFN-γ基因取代质粒pIRES/tHANpep中的neo基因）。分别用这4个重组质粒和空载体质粒pIRES1neo肌注免疫30日龄SPF鸡。免疫3次，间隔为2周，每次每只鸡的剂量为200μg。第3次免疫后两周以高致病性禽流感病毒H5N1强毒攻击，免疫及攻毒前后均采血检测HI抗体效价和外周血CD4⁺、CD8⁺T细胞的变化。结果发现，攻毒前各质粒免疫组均检测不到HI抗体，攻毒后1周存活鸡HI抗体效价迅速升高到64～256。流式细胞仪检测显示外周血CD4⁺、CD8⁺T细胞在疫苗免疫后都有不同程度的升高。空载体质粒对照组鸡（10只）在攻毒后3～8 d内全部死亡，其他各重组质粒免疫组鸡都获得了部分保护，保护率分别是：pIRES/HA组为545%（6/11），pIRES/tHA组为30%（3/10），pIRES/tHANPep组为36.3%（4/11）， pIRES/tHANPepIFNγ组为50%（5/10）。这些结果表明我们构建的多表位DNA疫苗能够诱导机体产生特异性免疫应答，并在同型禽流感强毒攻击时对鸡只提供了一定的保护。

Multi-epitope DNA Vaccines Against Avian Influenza in Chickens

Multiple epitopes from one or more viruses can be lined up and co-expressed in one vector to generate multi-epitopes DNA vaccines. In the study, four recombinant plasmids were constructed based on HA and NP gene of avian influenza virus (AIV) (H5N1): (1) pIRES/HA, carrying the complete HA gene; (2) pIRES/tHA, carrying a truncated HA gene fragment of major neutralizing antigenic epitopes; (3) pIRES/tHA-NPep, in which three CTL epitopes of NP gene of AIV were fused to the truncated HA from the C-terminal; and (4) pIRES/tHA-NPep-IFN-gamma, which was constructed by replacing neo gene in pIRES/ tHA-NPep with IFN-y of chicken. Fifty five SPF chickens were randomly divided into five groups and immunized with the above four constructs and control plasmid. Each chicken was intramuscally immunized with 200 microg plasmid DNA three times in a two-week interval. Two weeks after the third immunization, chickens were injected with H5N1 subtype avian influenza virus. Before the virus loading no detectable antibodies to HA were found in the chicken serum; but high levels of HI antibodies were detected in the serum of the survived chickens. The percentages of CD4+ and CD8+ T lymphocyte in peripheral blood of immunized chickens increased steadily after the vaccination. After virus loading all chickens in the control group died within three to eight days, and the survival rates of the four DNA vaccine groups were as follows: pIRES/HA, 54.5%; pIRES/tHA, 30%, pIRES/ tHA-NPep, 36.3%, pIRES/tHA-NPep-IFN-gamma, 50%. These results indicated that multi-epitopes DNA immunization can induce immune response and protect chickens from homologous virus loading.