商品疫苗对我国h9.4.2.5分支H9N2亚型禽流感分离株的免疫保护

doi:10.3864/j.issn.0578-1752.2023.15.017

中国农业科学 ›› 2023, Vol. 56 ›› Issue (15): 3040-3050.doi: 10.3864/j.issn.0578-1752.2023.15.017

• 畜牧·兽医 • 上一篇

商品疫苗对我国h9.4.2.5分支H9N2亚型禽流感分离株的免疫保护

麻琦¹(), 和新文¹, 王燕¹, 刘艳晶¹, 潘舒心¹^,³, 侯玉杰¹, 施建忠¹^,³, 邓国华¹^,³, 包红梅¹^,³, 刘景利², 郭兴福², 毛胜刚², 胡井雷², 路通², 杨帆², 田国彬¹^,³, 曾显营¹^,³(), 陈化兰¹^,³()

¹ 中国农业科学院哈尔滨兽医研究所/动物疫病防控全国重点实验室/国家禽类实验动物资源库，哈尔滨 150069
² 哈尔滨维科生物技术有限公司，哈尔滨 150069
³ 农业农村部禽流感等家禽重大疾病防控重点实验室，哈尔滨 150069

收稿日期:2022-07-04 接受日期:2022-09-15 出版日期:2023-08-01 发布日期:2023-08-05
通信作者:
陈化兰，Tel：0451-51997168；E-mail：chenhualan@caas.cn。
曾显营，Tel：0451-51051678；E-mail：zengxianying@caas.cn
联系方式: 麻琦，Tel：0451-51051678；E-mail：13592583529@163.com。
基金资助:
国家重点研发计划(2021YFD1800200); 现代农业产业技术体系(CARS-41)

The Protective Efficacy of Commercial Vaccines Against H9N2 Avian Influenza Virus of Branch h9.4.2.5 Isolated in China

MA Qi¹(), HE XinWen¹, WANG Yan¹, LIU YanJing¹, PAN ShuXin¹^,³, HOU YuJie¹, SHI JianZhong¹^,³, DENG GuoHua¹^,³, BAO HongMei¹^,³, LIU JingLi², GUO XingFu², MAO ShengGang², HU JingLei², LU Tong², YANG Fan², TIAN GuoBin¹^,³, ZENG XianYing¹^,³(), CHEN HuaLan¹^,³()

¹ National Poultry Laboratory Animal Resource Center/State Key Lab for Animal Disease Control and Prevention/Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150069
² Harbin Weike Biotechnology Company Limited, Harbin 150069
³ Key Laboratory for Prevention and Control of Avian Influenza and Other Major Poultry Diseases, Ministry of Agriculture and Rural Affairs, Harbin 150069

Received:2022-07-04 Accepted:2022-09-15 Published:2023-08-01 Online:2023-08-05

摘要/Abstract

摘要：

【目的】我国批准使用的H9亚型禽流感（avian influenza，AI）商品化灭活疫苗种类繁多，其免疫效果和选用受到养殖者的广泛关注。通过评估不同商品疫苗对我国近期H9N2亚型AIV的免疫保护效果，以期为H9亚型AIV的免疫防控提供科学参考。【方法】依据国家兽药基础数据库疫苗批签发数据，从在售的40种H9亚型AI商品疫苗中选择批签发数量较多的4种疫苗（A—D疫苗），进行免疫攻毒试验。4株h9.4.2.5分支的H9N2亚型分离株CK/XJ/S1204/ 2015、DK/JX/S4512/2017、CK/YN/S1666/2020和CK/NX/S4590/2020为攻毒毒株，分别测定4株病毒的鸡胚半数感染量（EID₅₀）、鸡半数感染量（CID₅₀）和细胞半数感染量（TCID₅₀），以确定动物试验的攻毒剂量和细胞试验的感染剂量。每种疫苗以产品推荐剂量免疫3周龄SPF鸡各40只，同时设同日龄SPF鸡40只接种PBS作为对照组；免疫3周时，采集所有试验鸡血清，测定血凝抑制抗体（HI）和中和抗体（NT）滴度；同时将每种商品疫苗接种40只SPF鸡进行随机分组，每组10只，连同10只对照组鸡，以10 CID₅₀的剂量鼻腔感染H9N2亚型分离株，分别进行商品疫苗对4株病毒的攻毒保护试验。采集攻毒后3、5 d喉头及泄殖腔棉拭子样品，接种10日龄鸡胚检测排毒情况，统计疫苗保护率，比较疫苗对H9N2亚型分离株的免疫保护效果。【结果】 4株H9N2亚型分离株的CID₅₀依次为10^3.5、10^2.5、10^2.5和10^3.5 EID₅₀/0.1 mL。商品疫苗接种后3周，各免疫组SPF鸡血清中针对商品化H9亚型HI试验抗原（CK/SH/10/2001）的HI抗体均在9.4 log₂—11 log₂之间，但针对攻毒株的HI抗体平均效价在4.6 log₂—10.8 log₂之间，不同疫苗免疫组间存在较大差异，最大差异为64倍，各免疫组NT抗体平均效价在6.7 log₂—12.2 log₂之间，最大差异为32倍，对照组鸡的HI抗体和NT抗体均为阴性。以滴鼻方式感染不同H9病毒后，4种疫苗的免疫保护效果存在较大差别，在攻击CK/XJ/S1204/2015毒株的试验中，3种疫苗（B—D疫苗）能为免疫鸡提供80%以上保护；在攻击DK/JX/S4512/ 2017毒株试验中，仅1种疫苗（B疫苗）能为免疫鸡提供80%以上保护；在攻击CK/YN/S1666/2020毒株的试验中，2种疫苗（A和B疫苗）能为免疫鸡提供80%以上免疫保护；而攻击CK/NX/S4590/2020毒株，4种疫苗免疫鸡的保护率均低于80%，而同期各对照组鸡均排毒，排毒率均高于80%。【结论】不同商品疫苗预防近期H9亚型分离株感染的免疫保护效果存在较大差异，疫苗抗原与分离株之间的抗原性差异是免疫保护率降低的主要原因；使用H9N2亚型流行毒株测定免疫后的HI抗体和NT抗体可作为评价商品疫苗免疫保护效果的重要依据。本研究为商品H9疫苗的科学选用提供重要参考。

关键词: H9N2亚型, AIV, 商品疫苗, 免疫效果, 禽流感

Abstract:

【Objective】 There are many commercial inactivated vaccines against the H9 subtype avian influenza approved for use in China, and their protection efficacy and selection have been widely concerned by farmers. In this study, the protection efficacy of main commercial vaccines against H9N2 viruses recently isolated in the field were evaluated, so as to provide the research reference for guiding the prevention and control of H9 subtype AI by immunization in China.【Method】 According to the vaccine batch release of the National Veterinary Drug Basic Information Database in China, 4 commercial vaccines (labeled as A-D) with large batches were selected from 40 kinds of sold H9 commercial vaccines. The four H9N2 subtype AIV, including CK/XJ/S1204/2015, DK/JX/S4512/2017, CK/YN/S1666/2020 and CK/NX/S4590/2020, belonging to h9.4.2.5 branch and isolated at different times and places, were used to evaluate the protection efficacy of selected commercial H9 subtype AI vaccines. The 50% chicken embryo infection (EID₅₀), 50% chicken infection (CID₅₀), and 50% cell infection (TCID₅₀) of the four viruses were tested to determine the challenge dose of the animal experiment and the infection dose of the cell test. 40 3-week-old SPF chickens were injected intramuscularly with 4 kinds of inactivated vaccine, and the groups of 10 similar SPF chickens were inoculated with PBS as control group. 3 weeks post vaccination (p.v.), the serum of all experimental chickens were collected before challenge, the hemagglutination inhibition (HI) and neutralization (NT) antibody titers were detected by HI test and neutralization test; the 40 immunized chickens of each vaccine were randomly divided into four groups, and 10 chickens of each vaccine and 10 control chickens were infected intranasally with the 10 CID₅₀ of each H9N2 virus. Oropharyngeal and cloacal swabs were collected on days 3 and 5 post challenge (p.c.) for virus shedding detection and calculation of the protective rate of each vaccine. 【Result】 The CID₅₀ of the four strains were 10^3.5 EID₅₀/0.1 mL, 10^2.5 EID₅₀/0.1 mL, 10^2.5 EID₅₀/0.1 mL and 10^3.5 EID₅₀/0.1 mL, respectively. At three weeks p.v., the mean HI antibodies titers against the commercial H9 subtype HI test antigen (CK/SH/10/2001) in 4 groups of vaccinated chickens ranged from 9.4 log₂ to 11 log₂, while the mean HI antibodies titers against challenge virus in 4 groups of vaccinated chickens ranged from 4.6 log₂ to 10.8 log₂, and the significant differences of HI antibodies titers were observed among different vaccine groups, for the maximum difference of mean HI antibodies titers were 64-fold among them. The mean NT antibodies titers against challenge virus in 4 groups of vaccinated chickens ranged from 6.7 log₂ to 12.2 log₂, while the maximum difference of mean HI antibodies titers were 32-fold among them, and the HI antibody and NT antibody of the control group were negative. After intranasal infection with different H9 viruses, the immune effects of the four vaccines were quite different. In CK/XJ/S1204/2015 challenged groups, 3 vaccines (B-D) could provide more than 80% protection. In DK/JX/S4512/2017 challenged groups, 1 vaccine (B) could provide more than 80% protection. In CK/YN/S1666/2020 challenged groups, 2 vaccines (A and B) could provide more than 80% protection. In CK/NX/S4590/2020 challenged groups, the protection efficacy of the four vaccines (A-D) were all less than 80%. In the same period, the virus shedding rate of chickens under the control group was more than 8/10. 【Conclusion】 There was a great difference in the immune protection efficacy of the four kinds of commercial vaccines after challenge with recently isolated H9 strains, and the difference of antigenicity between vaccine antigens and isolates was the main reason for the decrease of immune protection efficacy of the commercial vaccines. The titers of HI antibody and NT antibody against H9N2 prevalent strains could be as important data for evaluation of the commercial H9N2 AI vaccines. This study provided the scientific references for selection and usage of the commercial vaccines against H9N2 AI.

Key words: H9N2 subtype, AIV, commercial vaccine, protective efficacy, avian influenza

麻琦, 和新文, 王燕, 刘艳晶, 潘舒心, 侯玉杰, 施建忠, 邓国华, 包红梅, 刘景利, 郭兴福, 毛胜刚, 胡井雷, 路通, 杨帆, 田国彬, 曾显营, 陈化兰. 商品疫苗对我国h9.4.2.5分支H9N2亚型禽流感分离株的免疫保护[J]. 中国农业科学, 2023, 56(15): 3040-3050.

MA Qi, HE XinWen, WANG Yan, LIU YanJing, PAN ShuXin, HOU YuJie, SHI JianZhong, DENG GuoHua, BAO HongMei, LIU JingLi, GUO XingFu, MAO ShengGang, HU JingLei, LU Tong, YANG Fan, TIAN GuoBin, ZENG XianYing, CHEN HuaLan. The Protective Efficacy of Commercial Vaccines Against H9N2 Avian Influenza Virus of Branch h9.4.2.5 Isolated in China[J]. Scientia Agricultura Sinica, 2023, 56(15): 3040-3050.

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攻毒毒株 Virus	鸡胚半数感染量 EID₅₀	鸡半数感染量 CID₅₀	细胞半数感染量 TCID₅₀
CK/XJ/S1204/2015	10^-8.5/0.1 mL	10^3.5EID₅₀/0.1 mL	10^-6.5/0.1 mL
DK/JX/S4512/2017	10^-8.63/0.1 mL	10^2.5EID₅₀/0.1 mL	10^-7.5/0.1 mL
CK/YN/S1666/2020	10^-8.94/0.1 mL	10^2.5EID₅₀/0.1 mL	10^-6.5/0.1 mL
CK/NX/S4590/2020	10^-8.83/0.1 mL	10^3.5EID₅₀/0.1 mL	10^-7.5/0.1 mL

攻毒毒株 Virus	疫苗编号 Vaccine number	攻毒后3 d和5 d各组鸡排毒结果：排毒数/总数（Log₁₀EID₅₀/mL） Virus shedding detection on days 3 and 5 post challenge(p.c.) No. of chickens shedding virus/Total chickens (Log₁₀EID₅₀/mL)				保护率 Protection ratio
		3 d		5 d
		喉头 Oropharynx	泄殖腔 Cloaca	喉头 Oropharynx	泄殖腔 Cloaca
CK/XJ/S1204/2015	A	5/10（2.9）	3/10（3.3）	9/10（3.3）	3/10（2.1）	10%
	B	0/10	0/10	2/10（2.9）	0/10	80%
	C	2/10（2.9）	0/10	0/10	0/10	80%
	D	0/10	0/10	1/10（3.0）	0/10	90%
	PBS	10/10（4.3）	0/10	10/10（3.1）	5/10（3.0）	/
DK/JX/S4512/2017	A	5/10（3.7）	0/10	10/10（4.9）	7/10（3.2）	0
	B	0/10	0/10	0/10	0/10	100%
	C	4/10（4.6）	3/10（2.3）	9/10（4.2）	7/10（3.1）	10%
	D	3/10（4.9）	1/10（1.3）	10/10（4.6）	10/10（3.2）	0
	PBS	10/10（5.9）	9/10（3.1）	10/10（5.0）	10/10（3.2）	/
CK/YN/S1666/2020	A	0/10	0/10	0/10	0/10	100%
	B	0/10	0/10	0/10	0/10	100%
	C	10/10（5.5）	6/10（2.9）	7/10（4.9）	7/10（2.4）	0
	D	3/10（4.5）	1/10（3.3）	6/10（5.5）	2/10（2.8）	40%
	PBS	10/10（6.1）	8/10（2.9）	10/10（6.5）	9/10（3.1）	/
CK/NX/S4590/2020	A	10/10（4.8）	4/10（2.4）	10/10（5.5）	9/10（3.1）	0
	B	4/10（4.8）	2/10（2.4）	9/10（5.3）	4/10（3.2）	10%
	C	10/10（4.9）	3/10（2.4）	10/10（5.4）	8/10（2.9）	0
	D	8/10（4.7）	2/10（2.4）	10/10（5.5）	8/10（3.0）	0
	PBS	10/10（6.3）	6/10（2.5）	10/10（6.6）	6/10（3.8）	/

疫苗编号 Vaccine number	攻毒毒株 Challenge virus	HI抗体效价 HI antibody titers n=6（log₂）			保护率 Protection ratio
疫苗编号 Vaccine number	攻毒毒株 Challenge virus	CK/SH/10/2001	攻毒毒株 Challenge virus	攻毒毒株 Challenge virus	保护率 Protection ratio
A	CK/XJ/S1204/2015	9.3	9.3	8.9	10%
	DK/JX/S4512/2017	10.2	5.3	6.7	0
	CK/YN/S1666/2020	10.0	8.0	10.9	100%
	CK/NX/S4590/2020	10.2	6.3	8.3	0
B	CK/XJ/S1204/2015	10.2	10.2	11.1	80%
	DK/JX/S4512/2017	9.8	9.8	10.0	100%
	CK/YN/S1666/2020	10.3	10.8	12.2	100%
	CK/NX/S4590/2020	9.3	8.6	8.4	10%
C	CK/XJ/S1204/2015	9.5	9.5	10.0	80%
	DK/JX/S4512/2017	10.5	8.0	8.8	10%
	CK/YN/S1666/2020	9.5	4.6	7.6	0
	CK/NX/S4590/2020	9.3	5.1	7.0	0
D	CK/XJ/S1204/2015	10.8	10.8	10.9	90%
	DK/JX/S4512/2017	11.0	7.0	8.4	0
	CK/YN/S1666/2020	9.8	7.5	8.4	40%
	CK/NX/S4590/2020	10.2	6.8	8.6	0

病毒 Virus	血清交叉抗体滴度 Cross-reactive HI antibody titers of different antisera (log₂)
病毒 Virus	CK/SH/10/2001	CK/XJ/S1204/2015	DK/JX/S4512/2017	CK/YN/S1666/2020	CK/NX/S4590/2020
CK/SH/10/2001	1024	256	256	64	64
CK/XJ/S1204/2015	256	256	32	16	32
DK/JX/S4512/2017	32	64	2048	128	64
CK/YN/S1666/2020	64	128	512	512	512
CK/NX/S4590/2020	64	128	256	128	512

商品疫苗对我国h9.4.2.5分支H9N2亚型禽流感分离株的免疫保护

The Protective Efficacy of Commercial Vaccines Against H9N2 Avian Influenza Virus of Branch h9.4.2.5 Isolated in China

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