表达H5亚型禽流感病毒HA蛋白的重组禽痘病毒的构建及其免疫效力初评
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摘要
近年来由H5N1亚型禽流感病毒（AIV）引起的高致病性禽流感（HPAI）仍然是养禽业和人类健康的重大威胁，中国在防治HPAI的过程中主要采取扑杀和疫苗免疫相结合的策略。当前，在禽流感（AI）疫病防疫中应用的疫苗主要是重组灭活疫苗和病毒载体疫苗。重组灭活疫苗的使用会影响AI的监测和流行病学调查，因此，病毒载体疫苗的研究具有重要意义。
本研究将目前流行于中国南方水禽中的H5N1亚型AIV抗原群的代表株A/Duck/Anhui/1/2006（简称DK/AH/1/2006，属于Clade2.3.4）和A/Duck/Guangdong/S1322/2010(简称DK/GD/S1322/2010，属于Clade2.3.2.c)的HA基因分别克隆到转移载体pSY681-gfp-gpt中，通过感染-转染鸡胚成纤维细胞(CEF)的方法将免疫原HA基因同源重组到鹌鹑化鸡痘弱毒(CVCCAV1003)中，成功构建了2种重组禽痘病毒rFPV-AHHA和rFPV-GD1322HA，western blot鉴定表明它们能够正确表达相应的HA蛋白。
在SPF鸡上对rFPV-AHHA和rFPV-GD1322HA进行免疫效力评价。将3周龄的SPF鸡随机分成12组（8只/组），用106PFU的rFPV-AHHA免疫3组；106PFU的rFPV-GD1322HA免疫5组；106PFU的FPV免疫4组（对照组）。免疫后每周采血，用不同的抗原检测HI抗体。免疫6周后分别用105EID50的HPAIV A/Duck/Hubei/S1513/2010(简称DK/HuB/S1513/2010，属于Clade2.3.2.b)、 A/Duck/Fujian/31/2007(简称DK/FJ/31/2007，属于Clade2.3.4)、A/Chicken/Shandong/A-10/2011（简称CK/SD/A-10/2011，属于Clade7.2）对rFPV-AHHA、rFPV-GD1322HA和FPV免疫组中的第1-3组进行攻毒实验。对于rFPV-GD1322HA免疫组，第4组用于攻毒DK/GD/S1322/2010，第5组用于HI抗体持续期观察。对于FPV免疫组，第4组用于攻毒DK/GD/S1322/2010。最终，通过HI抗体水平测定、病毒分离情况及发病情况和死亡保护情况综合评价rFPV的免疫效果。
重组禽痘病毒rFPV-AHHA免疫后，针对抗原DK/AH/1/2006、DK/HuB/S1513/2010和CK/SD/A-10/2011的HI抗体平均值最高水平分别为4.00log2、3.38log2和4.25log2，病毒攻击后，各组HI抗体水平均有攀升，分别可达4.14log2、7.33log2和8.83log2。免疫鸡对同源病毒DK/FJ/31/2007的攻击能产生100%（7/7）的免疫保护（无死亡，无排毒，无发病）；病毒DK/HuB/S1513/2010攻击后，8只免疫鸡死亡2只，还有1只鸡出现排毒，免疫保护率为75%（6/8）；CK/SD/A-10/2011攻击后免疫保护率为85%（6/7），7只免疫鸡死亡1只，2只鸡出现排毒；而FPV对照组在用不同抗原群的病毒攻击后3天之内全部发病并死亡。结果表明，重组禽痘病毒rFPV-AHHA能完全保护同源病毒的攻击，对异源病毒也能提供部分的免疫保护。
重组禽痘病毒rFPV-GD1322HA免疫后，针对抗原DK/AH/1/2006、DK/HuB/S1513/2010、CK/SD/A-10/2011以及DK/GD/S1322/2010的HI抗体平均值最高水平分别为0.00log2、4.29log2、6.71log2和3.43log2，攻毒后HI抗体水平分别增至1.75log2、6.00log2、8.75log2和8.14log2。免疫鸡对病毒DK/FJ/2007和DK/GD/S1322/2010的攻击能够产生100%的免疫保护；病毒DK/HuB/S1513/2010攻击后，7只免疫鸡死亡1只，1只鸡出现排毒；免疫鸡对CK/SD/A-10/2011的攻击只能提供有限的免疫保护66.7%（4/6），6只免疫鸡死亡2只，1只鸡出现排毒现象。
本研究成功构建了重组禽痘病毒rFPV-AHHA和rFPV-GD1322HA，SPF鸡群中的免疫结果表明，针对不同的抗原，HI抗体水平存在差异；两种重组禽痘病毒都能对同源病毒以及部分异源病毒的致死攻击提供有效的死亡保护，这为进一步系统的免疫效力研究提供了基础数据。
Highly pathogenic avian influenza (HPAI) caused by the H5N1subtype avian influenza virus (AIV)has posed severe threat to the poultry industry and human health. A culling combined with vaccinationstrategy has been adopted in china for the control of HPAI. Currently, inactivated vaccine and livevirus-vectored vaccine have been used in the vaccination. Disturbing to the AIV surveillance andepidemiolog ical investigation might be produced by using of inactivated vaccine.So the study of livevirus-vectored vaccine is of great significance.
In this study, HA genes from A/Duck/Anhui/1/2006（DK/AH/1/2006) andA/Duck/Guangdong/S1322/2010(DK/GD/S1322/2010) were recombined with FPV（CVCCAV1003) byinfection-transfection method in Chicken embryo fibroblasts (CEF). DK/AH/1/2006andDK/GD/S1322/2010belonged to Clade2.3.4and Clade2.3.2.c respectively. They were both therepresentative strain that now circulating in waterfowl in southern china. After the construction ofrFPV-AHHA and rFPV-GD1322HA, they were indentified by western blot. The result show that HAproteins were correctly expressed by them.
The immune effects of rFPV-AHHA and rFPV-GD1322were assessment in SPF chickens.Three-week-old chickens were randomly divided into twelve groups (Eight chickens of each group).Three groups were vaccinated with106PFU rFPV-AHHA, five groups with106PFU FPV-GD1322HA,and the rest four groups with FPV (Control). Serum for HI antibody detection was collected every weekafter vaccination, and HI antibodies were detected by using different antigens. Six weeks aftervaccination, groups which vaccinated with FPV or rFPV-GD1322HA were challenged with105EID50HPAIV A/Duck/Hubei/S1513/2010(DK/HuB/S1513/2010), A/Duck/Fujian/31/2007(DK/FJ/31/2007),A/Chicken/Shandong/A-10/2011(CK/SD/A-10/2011) and DK/GD/S1322/2010, which belonged toClade2.3.2.b, Clade2.3.4, Clade7.2and Clade2.3.2.c respectively. The fifth group which also vaccinatedwith rFPV-GD1322was treated as a HI duration group. The rFPV-AHHA vaccinated groups werechallenged with these four viruses, except for DK/GD/S1322/2010. The immune effect was evaluatedby HI antibody variation, virus excretion and protection efficiency.
rFPV-AHHA immunized groups, when detected by DK/AH/1/2006, DK/HuB/S1513/2010, andCK/SD/A-10/2011antigens, HI angtibodies were4.00log2,3.38log2and4.25log2respectively. Afterchallenge with viruses, HI antibodies reached4.14log2,7.33log2and8.83log2respectively.100%protection was provided by rFPV-AHHA when challenged with DK/FJ/31/2007. In all seven chickens,no chicken was found dead or virus shedding; After challenged with DK/HuB/S1513/2010, twochichkens were found died and one chicken excreted virus. The protection efficiency was75%(6/8); Inthe other group, when challenged with CK/SD/A-10/2011, there were two chickens dead and onechicken shed virus. So the protection efficiency was85%(6/7); All chickens were died in three days incontrol groups. Those results show that rFPV-AHHA can fully protect chickens from homologous viruschalleng e. But, partially protection was provided by rFPV-AHHA when challenged with heterologousviruses.
rFPV-GD1322HA immunized groups, when detected by DK/AH/1/2006, DK/HuB/S1513/2010,CK/SD/A-10/2011and DK/GD/S1322/2010antigens, HI antibodies were0.00log2,4.29log2,6.71log2and3.43log2respectively. After challenge, HI antibodies reached to1.75log2,6.00log2,8.75log2and8.14log2respectively. rFPV-GD1322HA could protect chickens at leve l of100%, when challenged withDK/FJ/2007and DK/GD/S1322/2010; However, when challenged with DK/HuB/S1513/2010, one deadand one virus shedding were observed in the group; In CK/SD/A-10/2011challenge group, two chickenswere found dead and one chicken excreted virus, the protection efficiency was66.7%(4/6).
In this study, two recombinant viruses rFPV-AHHA and rFPV-GD1322HA were constructedsuccessfully. Their protection efficiency in SPF chickens show that the HI antibody could be differentwhen detected with different antigens. But rFPV-AHHA and rFPV-GD1322HA could protect chickensfrom death when challenged with homologous virus as well as some heterologous viruses. All thoseresults will provide basic data for the further study of immune effect.
本研究将目前流行于中国南方水禽中的H5N1亚型AIV抗原群的代表株A/Duck/Anhui/1/2006（简称DK/AH/1/2006，属于Clade2.3.4）和A/Duck/Guangdong/S1322/2010(简称DK/GD/S1322/2010，属于Clade2.3.2.c)的HA基因分别克隆到转移载体pSY681-gfp-gpt中，通过感染-转染鸡胚成纤维细胞(CEF)的方法将免疫原HA基因同源重组到鹌鹑化鸡痘弱毒(CVCCAV1003)中，成功构建了2种重组禽痘病毒rFPV-AHHA和rFPV-GD1322HA，western blot鉴定表明它们能够正确表达相应的HA蛋白。
在SPF鸡上对rFPV-AHHA和rFPV-GD1322HA进行免疫效力评价。将3周龄的SPF鸡随机分成12组（8只/组），用106PFU的rFPV-AHHA免疫3组；106PFU的rFPV-GD1322HA免疫5组；106PFU的FPV免疫4组（对照组）。免疫后每周采血，用不同的抗原检测HI抗体。免疫6周后分别用105EID50的HPAIV A/Duck/Hubei/S1513/2010(简称DK/HuB/S1513/2010，属于Clade2.3.2.b)、 A/Duck/Fujian/31/2007(简称DK/FJ/31/2007，属于Clade2.3.4)、A/Chicken/Shandong/A-10/2011（简称CK/SD/A-10/2011，属于Clade7.2）对rFPV-AHHA、rFPV-GD1322HA和FPV免疫组中的第1-3组进行攻毒实验。对于rFPV-GD1322HA免疫组，第4组用于攻毒DK/GD/S1322/2010，第5组用于HI抗体持续期观察。对于FPV免疫组，第4组用于攻毒DK/GD/S1322/2010。最终，通过HI抗体水平测定、病毒分离情况及发病情况和死亡保护情况综合评价rFPV的免疫效果。
重组禽痘病毒rFPV-AHHA免疫后，针对抗原DK/AH/1/2006、DK/HuB/S1513/2010和CK/SD/A-10/2011的HI抗体平均值最高水平分别为4.00log2、3.38log2和4.25log2，病毒攻击后，各组HI抗体水平均有攀升，分别可达4.14log2、7.33log2和8.83log2。免疫鸡对同源病毒DK/FJ/31/2007的攻击能产生100%（7/7）的免疫保护（无死亡，无排毒，无发病）；病毒DK/HuB/S1513/2010攻击后，8只免疫鸡死亡2只，还有1只鸡出现排毒，免疫保护率为75%（6/8）；CK/SD/A-10/2011攻击后免疫保护率为85%（6/7），7只免疫鸡死亡1只，2只鸡出现排毒；而FPV对照组在用不同抗原群的病毒攻击后3天之内全部发病并死亡。结果表明，重组禽痘病毒rFPV-AHHA能完全保护同源病毒的攻击，对异源病毒也能提供部分的免疫保护。
重组禽痘病毒rFPV-GD1322HA免疫后，针对抗原DK/AH/1/2006、DK/HuB/S1513/2010、CK/SD/A-10/2011以及DK/GD/S1322/2010的HI抗体平均值最高水平分别为0.00log2、4.29log2、6.71log2和3.43log2，攻毒后HI抗体水平分别增至1.75log2、6.00log2、8.75log2和8.14log2。免疫鸡对病毒DK/FJ/2007和DK/GD/S1322/2010的攻击能够产生100%的免疫保护；病毒DK/HuB/S1513/2010攻击后，7只免疫鸡死亡1只，1只鸡出现排毒；免疫鸡对CK/SD/A-10/2011的攻击只能提供有限的免疫保护66.7%（4/6），6只免疫鸡死亡2只，1只鸡出现排毒现象。
本研究成功构建了重组禽痘病毒rFPV-AHHA和rFPV-GD1322HA，SPF鸡群中的免疫结果表明，针对不同的抗原，HI抗体水平存在差异；两种重组禽痘病毒都能对同源病毒以及部分异源病毒的致死攻击提供有效的死亡保护，这为进一步系统的免疫效力研究提供了基础数据。
Highly pathogenic avian influenza (HPAI) caused by the H5N1subtype avian influenza virus (AIV)has posed severe threat to the poultry industry and human health. A culling combined with vaccinationstrategy has been adopted in china for the control of HPAI. Currently, inactivated vaccine and livevirus-vectored vaccine have been used in the vaccination. Disturbing to the AIV surveillance andepidemiolog ical investigation might be produced by using of inactivated vaccine.So the study of livevirus-vectored vaccine is of great significance.
In this study, HA genes from A/Duck/Anhui/1/2006（DK/AH/1/2006) andA/Duck/Guangdong/S1322/2010(DK/GD/S1322/2010) were recombined with FPV（CVCCAV1003) byinfection-transfection method in Chicken embryo fibroblasts (CEF). DK/AH/1/2006andDK/GD/S1322/2010belonged to Clade2.3.4and Clade2.3.2.c respectively. They were both therepresentative strain that now circulating in waterfowl in southern china. After the construction ofrFPV-AHHA and rFPV-GD1322HA, they were indentified by western blot. The result show that HAproteins were correctly expressed by them.
The immune effects of rFPV-AHHA and rFPV-GD1322were assessment in SPF chickens.Three-week-old chickens were randomly divided into twelve groups (Eight chickens of each group).Three groups were vaccinated with106PFU rFPV-AHHA, five groups with106PFU FPV-GD1322HA,and the rest four groups with FPV (Control). Serum for HI antibody detection was collected every weekafter vaccination, and HI antibodies were detected by using different antigens. Six weeks aftervaccination, groups which vaccinated with FPV or rFPV-GD1322HA were challenged with105EID50HPAIV A/Duck/Hubei/S1513/2010(DK/HuB/S1513/2010), A/Duck/Fujian/31/2007(DK/FJ/31/2007),A/Chicken/Shandong/A-10/2011(CK/SD/A-10/2011) and DK/GD/S1322/2010, which belonged toClade2.3.2.b, Clade2.3.4, Clade7.2and Clade2.3.2.c respectively. The fifth group which also vaccinatedwith rFPV-GD1322was treated as a HI duration group. The rFPV-AHHA vaccinated groups werechallenged with these four viruses, except for DK/GD/S1322/2010. The immune effect was evaluatedby HI antibody variation, virus excretion and protection efficiency.
rFPV-AHHA immunized groups, when detected by DK/AH/1/2006, DK/HuB/S1513/2010, andCK/SD/A-10/2011antigens, HI angtibodies were4.00log2,3.38log2and4.25log2respectively. Afterchallenge with viruses, HI antibodies reached4.14log2,7.33log2and8.83log2respectively.100%protection was provided by rFPV-AHHA when challenged with DK/FJ/31/2007. In all seven chickens,no chicken was found dead or virus shedding; After challenged with DK/HuB/S1513/2010, twochichkens were found died and one chicken excreted virus. The protection efficiency was75%(6/8); Inthe other group, when challenged with CK/SD/A-10/2011, there were two chickens dead and onechicken shed virus. So the protection efficiency was85%(6/7); All chickens were died in three days incontrol groups. Those results show that rFPV-AHHA can fully protect chickens from homologous viruschalleng e. But, partially protection was provided by rFPV-AHHA when challenged with heterologousviruses.
rFPV-GD1322HA immunized groups, when detected by DK/AH/1/2006, DK/HuB/S1513/2010,CK/SD/A-10/2011and DK/GD/S1322/2010antigens, HI antibodies were0.00log2,4.29log2,6.71log2and3.43log2respectively. After challenge, HI antibodies reached to1.75log2,6.00log2,8.75log2and8.14log2respectively. rFPV-GD1322HA could protect chickens at leve l of100%, when challenged withDK/FJ/2007and DK/GD/S1322/2010; However, when challenged with DK/HuB/S1513/2010, one deadand one virus shedding were observed in the group; In CK/SD/A-10/2011challenge group, two chickenswere found dead and one chicken excreted virus, the protection efficiency was66.7%(4/6).
In this study, two recombinant viruses rFPV-AHHA and rFPV-GD1322HA were constructedsuccessfully. Their protection efficiency in SPF chickens show that the HI antibody could be differentwhen detected with different antigens. But rFPV-AHHA and rFPV-GD1322HA could protect chickensfrom death when challenged with homologous virus as well as some heterologous viruses. All thoseresults will provide basic data for the further study of immune effect.
引文
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