HA基因密码子及表达载体优化的H5亚型禽流感DNA疫苗免疫保护效果比较

为评估HA基因密码子优化及不同表达载体对H5亚型禽流感DNA疫苗免疫效果的影响，pCIHA5、pCAGGHA5 、pCIoptiHA5和 pCAGGoptiHA5分别以100 和10 μg剂量一次免疫3周龄SPF鸡，4周后分别用100 LD50的高致病力禽流感病毒(HPAIV) A/Goose/GuangDong/1/96(H5N1)[GD/1/96(H5N1)]鼻腔途径进行攻击，观察发病与死亡情况，分别于攻毒后第3、5和7无采集喉头及泄殖腔拭子进行病毒分离、滴定检测排毒情况，同时检测免疫后、攻毒前及攻毒后血清HI抗体、AGP抗体的动态变化，对4种疫苗单次免疫的免疫保护效果进行比较评估。结果表明，100 μg剂量组中， pCAGGoptiHA5和pCAGGHA5 均可对免疫鸡形成100％完全保护（不发病、不致死和不排毒），pCIoptHA5(75%) 和pCIHA5(50%)仅可形成部分免疫保护；10 μg剂量免疫组中， pCAGGoptiHA5和pCAGGHA5 可对免疫鸡形成100％的保护（不发病和不致死），pCIoptiHA5可形成对免疫鸡的部分免疫保护(75%)，而pCIHA5则基本不能形成免疫保护。结果表明，HA基因密码子的优化以及鸡β-actin启动子表达载体pCAGGS可显著提高H5亚型禽流感DNA疫苗诱导的保护性抗体免疫反应水平，增强免疫保护效果同时证明，通过密码子和载体的优化，可使H5亚型禽流感DNA疫苗有效免疫保护剂量降低至10 μg，其推广应用已具备充分的经济可行性。pCAGGoptiHA5作为免疫效果良好、成本低廉的优化DNA疫苗，有望成为预防H5亚型高致病力禽流感的高效、安全新型基因工程疫苗。

Comparison of Protection Effects of H5 Subtype Avian Influenza DNA Vaccine Optimized with HA Gene and Expressive Vector

To evaluate the effect of H5 subtype avian influenza DNA vaccine optimized with HA gene codon and expressive vectors, groups of 3-week-old SPF chickens were intramuscular inoculated singly with 100 and 10μg of pCIHA5, pCAGGHA5, pCIoptiHA5 and pCAGGoptiHA5 in 200 μL volume, respectively. A group of chickens were injected with 200 μL PBS as controls. Sera were collected every week after vaccination for detecting the HI and AGP antibodies. After four weeks the all chickens were challenged with 100 LD50 of highly pathogenic A/Goose/GuangDong/1/96(H5N1)[Gs/GD/1/96(H5N1)], Oropharyngeal and cloacal swab specimens were collected from all the chickens 3, 5 and 7 days after inoculation for titration of virus in eggs, respectively, and they were observed daily for disease signs and deaths for 2 weeks. Results showed that in the 100 μg groups, pCAGGoptiHA5 and pCAGGHA5 vaccinated chickens were completely protected from virus challenge (no disease signs, no virus shedding and no deaths), while only partial protection were obtained in pCIoptiHA5 (75%) and pCIHA5(50%). And in the 10 μg groups, pCAGGoptiHA5 and pCAGGHA5 vaccinated chickens were protected from virus challenge (no disease signs and no deaths), and pCIoptiHA5(75%) vaccinated chickens were partially protected from virus challenge , while pCIHA5(50%) vaccinated chickens were all died after challenge. Results indicated that codon optimization of HA gene and β-actin promoter expression vector PCAGGS could observably enhance the protection effciency of H5 subtype avian influenza DNA vaccine. And the construction pCAGGoptiHA5 could protect chickens from lethal H5N1 virus challenge even at the low dose of 10 μg, implies the potential commercialization of avian influenza DNA vaccine in the future.