H5亚型2.3.4.4分支禽流感病毒HA基因重组质粒的免疫保护效力研究

Protective efficacy of DNA recombination plasmid against H5 clade 2.3.4.4 avian influenza virus

为研制H5亚型禽流感病毒(AIV)流行株的DNA疫苗,本研究将目前流行的H5亚型2.3.4.4分支AIV代表株A/Duck/Guizhou/S4184/2017(H5N6)[简称DK/GZ/S4184/2017(H5N6)]的HA基因经密码子优化后克隆至载体pCAGGs中构建重组质粒pCA-S4184,经PCR和测序鉴定正确后转染293T细胞,利用间接免疫荧光试验和western blot鉴定。结果显示,该HA基因可以在293T细胞中瞬时表达。将15μg/只的重组质粒pCA-S4184经肌肉注射免疫3周龄SPF鸡,3周后以相同的剂量加强免疫,1周后再通过滴鼻途径分别以10^(5)EID_(50)/0.1 mL/只的同源高致病性AIV(HPAIV)DK/GZ/S4184/2017(H5N6)株和同分支异源HPAIV DK/GD/S1110/2019(H5N6)、DK/GX/1/2018(H5N6)、DK/HuN/SE0110/2018(H5N6)株攻毒,首免及攻毒后每周采血,检测每组鸡的HI抗体水平。在攻毒后14 d的观察期内每天记录各组鸡的发病和死亡情况,攻毒后第3 d、5 d和7 d采集所有鸡的喉头和泄殖腔拭子进行病毒的分离及其滴度的测定。结果显示,加强免疫后各组鸡的平均HI抗体滴度为1:8~1:11,攻毒后HI抗体效价均显著增加;pCA-S4184可以对SPF鸡提供抵抗致死性同源病毒和同分支异源病毒攻击的能力,保护率达到100%,且能显著降低SPF鸡的排毒量甚至不排毒。随后以15μg/只的pCA-S4184两次免疫3周龄SPF鸡,免疫后每隔2周采血检测HI抗体的动态消长规律并在首免后第12周和24周,分别随机选取8只免疫鸡和8只对照鸡通过滴鼻途径以10^(5)EID_(50)/0.1 mL/只感染同源HPAIV DK/GZ/S4184/2017(H5N6)株,并在攻毒后第3 d、第5 d和第7 d采集所有鸡的喉头和泄殖腔拭子,分离病毒并测其滴度,以评估该重组质粒在免疫持续期间所诱导的免疫保护效果。结果显示,重组质粒仍可以使免疫鸡完全抵抗同源病毒的攻击。本研究为重组质粒pCA-S4184作为防控H5亚型2.3.4.4分支禽流感的候选DNA疫苗提供了实验依据。

The candidate DNA vaccine strain needs to be reconstructed according to the epidemiological investigation of H5 high pathogenic avian influenza virus (HPAIV).In this study,the codon optimized HA gene of the latest isolated from DK/GZ/S4184/2017(H5N6),a representative strain of clade 2.3.4.4 HPAIVs,was subcloned into pCAGGS vector to construct the recombinant plasmid pCA-S4184 as the DNA vaccine candidate against AIV.The correct plasmid p CA-S4184 was transfected into293T cells after PCR and sequencing identification.The results showed that the HA gene could be expressed instantaneously in293T cells by IFA and western blot detection.In addition,three-week-old SPF chickens were inoculated intramuscularly with the recombinant plasmid pCA-S4184 at a dose of 15μg/bird,and boosted with the same dose after three weeks to evaluate the protective efficacy of this recombinant plasmid.One week post-booster,the immunized chickens were respectively challenged intranasally with a dose of 10^(5)EID_(50)/0.1mL/bird of homologous HPAIV DK/GZ/S4184/2017(H5N6) and heterologous HPAIV DK/GD/S1110/2019(H5N6),DK/GX/1/2018(H5N6) and DK/HuN/SE0110/2018(H5N6).Blood samples were collected weekly after the first immunization and challenge,and the HI antibody titer of each group was detected.The incidence and death of experimental chickens in each group were recorded every day during the observation period of 14 days after challenge,and oropharyngeal and cloacal swabs were collected on day 3,5,and 7 post-challenge for virus isolation and titer determination.Our results showed that the average HI antibody titer of each group was 1:8-1:11 after booster immunization,which increased significantly after challenge.Recombinant plasmid p CA-S4184 was able to provide for SPF chickens with 100%protection against fatal HPAIV homologous and heterologous viruses,and was also able to markedly decrease the amount of SPF chicken shedding virus or even no shedding virus.Then three-week-old SPF chickens were immunized twice with the same dose of pCA-S4184 as the above experiment,and the wing venous blood was collected every two weeks after immunization to detect the dynamic fluctuation of the HI antibody titer.At the 12th and 24th weeks after the first immunization,8 immunized chickens and 8 control chickens were randomly selected and intranasally challenged with the homologous virus DK/GZ/S4184/2017(H5N6) at the dose of 10^(5)EID_(50)/0.1mL/bird,and the oropharyngeal and cloacal swabs were collected on day 3,5,and 7 post-challenge for virus isolation and titer detection to evaluate the immunoprotective effect induced by the recombinant plasmid during the duration.The result showed that the recombinant plasmid pCA-S4184 can still provide complete protection for immunized chickens against homologous virus.Our research provided the experimental evidence for the recombinant plasmid pCA-S4184 as a candidate DNA vaccine strain to prevent and control H5subtype clade 2.3.4.4 AIV.