H5N1 influenza viruses cause high mortality in avian and mammalian species, including humans. Antigenic drift in H5 sequence poses challenges in the development of vaccine and therapeutic antibody. In this study, a monoclonal antibody 11G12 was produced from inactivated H5N1 immunized mice. Results from IFA, ELISA, HI, and virus neutralization indicated that Mab 11G12 can specifically recognize and neutralize H5 type hemagglutinin from clade 1 and 0 without any cross-reaction to any other clades of H5N1 viruses. Mab 11G12 was used to differentiate and quantify the expression of H5N1 strain A/VietNam/1203/04 from a trivalent vaccine mix in ELISA. Sequencing of escape mutants identified that Mab 11G12 targets a major neutralizing epitope of influenza H5 hemagglutinin. The study indicated that some major neutralizing epitopes in H5s of early strains were mutated due to antigenic drift. 1. Background Highly pathogenic avian influenza H5N1 virus has caused high mortality in birds and humans, raising concerns for the possibility of a future influenza pandemic [1]. In 1997, in Hong Kong, 18 humans were infected and 6 died in the first known case of H5N1 infecting humans [2, 3]. Since the 2004 outbreaks of H5N1 influenza viruses from birds to human in Vietnam and Thailand, newly emerging avian influenza A viruses pose a continued lethal threat, not only to avian species but also to humans [4, 5]. The H5N1 influenza viruses are currently divisible into 10 clades (0 to 9) on the basis of phylogenetic analysis of their hemagglutinin (HA) genes that have evolved in the A/Goose/Guangdong/96-like H5N1 lineage (Clade 0) [6]. Clade 0 includes all the early progenitors which are predominately strains in 1996–2002 from Hong Kong (HK) and China, while clade 1 includes human and bird isolates from Vietnam, Thailand, and Cambodia and bird isolates from Laos and Malaysia [7]. The human isolate A/VietNam/1203/04 (H5N1) from the 2004 outbreak was identified as the most pathogenic isolate in Clade 1.0 [8, 9]. It was widely selected as the vaccine strain for H5N1. The stockpiling of a panel of vaccines with hemagglutinin (HA) antigenic variations, including A/VietNam/1203/2004, A/VietNam/1194/2004, A/Indonesia/05/2005, and A/Anhui/1/2005 vaccine viruses, was recommended by the WHO for vaccine development [7]. However, present vaccine strategies have been hindered by antigenic variation of the influenza strains. Immunity elicited with a single strain from a previous outbreak may not be able to provide sufficient protection against currently circulating H5N1 viruses [10].
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