Jadhao Samadhan J, Lee Chang-Won, Sylte Matt, Suarez David L
Southeast Poultry Research Laboratory, Agricultural Research Service, United States Department of Agriculture, 934 College Station Road, Athens, GA 30605, USA.
Vaccine. 2009 Oct 19;27(44):6247-60. doi: 10.1016/j.vaccine.2009.07.110. Epub 2009 Aug 15.
Highly pathogenic (HP) H5N1 avian influenza has become endemic in several countries in Asia and Africa, and vaccination is being widely used as a control tool. However, there is a need for efficacious vaccines preferably utilizing a DIVA (differentiate infected from vaccinated animals) marker strategy to allow for improved surveillance of influenza in vaccinated poultry. Using a reverse genetics approach, we generated Asian rgH5N9 vaccine strain deriving the hemagglutinin gene from A/chicken/Indonesia/7/2003 (H5N1) with modification of the cleavage site to be low pathogenic (LP) and N9 neuraminidase gene from the North American LP A/turkey/Wisconsin/1968 (H5N9) virus. The recombinant rgH5N9, A/turkey/Wisconsin/1968 (H5N9) A/chicken/Hidalgo/232/1994 (H5N2), and wild type HP A/chicken/Indonesia/7/2003 (H5N1) viruses were used to prepare inactivated oil-emulsified whole virus vaccines. Two weeks after vaccination, chickens were challenged with either Asian HP H5N1 viruses, A/chicken/Indonesia/7/2003 (W.H.O. clade 2.1) or A/chicken/Supranburi Thailand/2/2004 (W.H.O. clade 1.0). The H5 HA1 of the North American vaccine strains exhibited 12% amino acid differences including amino acid changes in the major antigenic sites as compared to the Asian HP H5N1 challenge viruses, serologically exhibited substantial antigenic difference, but still provided 100% protection from mortality. However, challenge virus shedding was significantly higher in chickens immunized with antigenically distinct American lineage vaccines as compared to the antigenically matched Asian rgH5N9 and the wild type Asian H5N1 vaccine. The antibody response to the heterologous subtype neuraminidase proteins were discriminated in vaccinated and infected chickens using a rapid fluorescent 2'-(4-methylumbelliferyl)-alpha-d-N-acetylneuraminic acid sodium salt as substrate for neuraminidase inhibition assay. This study demonstrates the value of using a vaccine containing antigenically matched H5 hemagglutinin for control of HP H5N1 avian influenza in poultry and the potential utility of a heterologous neuraminidase as a DIVA marker.
高致病性（HP）H5N1禽流感已在亚洲和非洲的几个国家成为地方病，疫苗接种正被广泛用作一种控制手段。然而，需要有高效的疫苗，最好采用区分感染动物和接种动物（DIVA）的标记策略，以便更好地监测接种疫苗的家禽中的流感情况。我们采用反向遗传学方法，构建了亚洲rgH5N9疫苗株，其血凝素基因来源于A/鸡/印度尼西亚/7/2003（H5N1），并对裂解位点进行修饰使其具有低致病性（LP），神经氨酸酶基因来源于北美低致病性A/火鸡/威斯康星/1968（H5N9）病毒。用重组rgH5N9、A/火鸡/威斯康星/1968（H5N9）、A/鸡/伊达尔戈/232/1994（H5N2）以及野生型高致病性A/鸡/印度尼西亚/7/2003（H5N1）病毒制备了灭活油乳剂全病毒疫苗。接种疫苗两周后，用亚洲高致病性H5N1病毒、A/鸡/印度尼西亚/7/2003（世界卫生组织2.1分支）或A/鸡/泰国素攀武里/2/2004（世界卫生组织1.0分支）对鸡进行攻毒。与亚洲高致病性H5N1攻毒病毒相比，北美疫苗株的H5 HA1氨基酸差异达12%，包括主要抗原位点的氨基酸变化，血清学上显示出显著的抗原差异，但仍提供了100%的致死保护。然而，与抗原匹配的亚洲rgH5N9和野生型亚洲H5N1疫苗相比，用抗原性不同的美国谱系疫苗免疫的鸡的攻毒病毒排毒量显著更高。以快速荧光2'-（4-甲基伞形酮基）-α-D-N-乙酰神经氨酸钠盐作为神经氨酸酶抑制试验的底物，区分接种疫苗和感染鸡对异源亚型神经氨酸酶蛋白的抗体反应。本研究证明了使用含有抗原匹配的H5血凝素的疫苗来控制家禽高致病性H5N1禽流感的价值，以及异源神经氨酸酶作为DIVA标记物的潜在用途。
