Highly pathogenic avian influenza virus H5N1 from Egypt escapes vaccine-induced immunity but confers clinical protection against a heterologous clade 2.2.1 Egyptian isolate
Highlights
► Egyptian HPAIV H5N1 lineage 2.2.1var escape immunity induced by heterologous H5N2 vaccines. ► Clinical protection was achieved only against classic Egyptian strains (H5N1 2.2.1pro). ► In spite of clinical protection considerable shedding of 2.2.1pro virus was observed. ► A homologous Egyptian clade 2.2.1var vaccine induced sterile immunity. ► Silent virus spread and consecutive vaccine-driven evolution of HPAIV H5N1 pose evident risks.
Introduction
Highly pathogenic avian influenza viruses (HPAIV) pose a severe threat to poultry industry worldwide. Due to a certain zoonotic potential of these viruses human populations may be at risk [1]. An HPAIV lineage of subtype H5N1 which originated in Southeast Asia in 1996/1997 has spread across Eurasia since 2003 and entered Africa in 2005 [2]. Despite intense attempts to eradicate the virus endemic status is now reported from Indonesia and Egypt where continuous viral circulation is likely associated with silently infected domestic ducks [3]. Endemic infection of poultry increases risks of sporadic human infections, the majority of which had a fatal outcome [4].
In Egypt, poultry possesses considerable importance as a source of animal protein for human consumption. Before the incursion of HPAIV H5N1 to Egypt in 2006, a huge poultry population of about 1 billion heads was reared for home consumption and trade to other Middle East and African countries [5]. About 75% of the poultry was raised in commercial farms. Almost all poultry enterprises are located in the Nile valley, the majority in the Nile delta, paralleling the human population density. Keeping of mixed backyard poultry flocks amounts to about 25% of the countries’ poultry population and is intimately intertwined with human rural and urban life. Due to cultural preferences in consuming fresh meat the majority of poultry is traded via life bird markets; slaughterhouses cover only about 30% of the total trading loads. Marketing is associated with frequent and, outside the Nile delta, long-distance transport of poultry.
After the incursion of HPAIV H5N1 into Egypt in 2006, possibly by wild migratory ducks [6], attempts to limit the spread and eradicate H5N1 were concentrated in a test-and-cull control strategy. Commercial flocks require a certificate stating freedom from HPAIV after testing for viral RNA before general trading restrictions are lifted. In parallel, and to limit mass depopulation of poultry, blanket vaccination campaigns in commercial and then in backyard holdings (free of charge) were launched using several standard H5 vaccines from different vendors and sources. Despite these concerted efforts, the virus persisted in poultry and evolved into phylogenetically distinguishable, co-circulating lineages [2], [7], [8]. As a surrogate marker of virus circulation, human infections continue to be registered [9].
In poultry flocks with a high level of population immunity after vaccination against subtype H5 influenza virus infection, transmission and spread of HPAI H5 virus is reduced below a reproduction rate (R0) of 1 which eventually leads to eradication of the infection [10]. At the same time, and especially in the case of incomplete or waning population immunity, caused, e.g. by maternally derived antibodies [11] or antigenically distantly related vaccine strains, a strong selection pressure is imposed to which influenza A viruses respond swiftly with the generation and expansion of antigenic drift and neutralization escape mutants. Such variants are capable of circumnavigating vaccine-induced immunity [12], [13]. This dynamic process, the results of which can be depicted by antigenic cartography [14], is the basis for updating human seasonal influenza virus vaccine in a bi-annual fashion [15]. Generation of escape mutants in poultry vaccinated against subtype H5 influenza A viruses has been first observed in the follow-up phase of HPAIV H5N2 outbreaks in Mexico in the 1990s [16]. Also, among the HPAIV H5N1 viruses of Asian origin, clades 2.3.4 (“Fujian”) and 7 (“Shanxi”) are believed to represent such vaccine-escape strains [17]. In Egypt, since 2007, recurrent outbreaks of HPAIV H5N1 in vaccinated flocks were reported with increasing frequency and the efficacy of the vaccine potency currently in use in Egypt was challenged [18]. Sequence analysis of the circulating viruses revealed the emergence of variant strains with considerable genetic (subclade 2.2.1) and antigenic distances to the originally introduced strain of classic clade 2.2 [7].
Here we examined the potency of different inactivated commercial and experimental whole H5 virus vaccines to protect against representative challenge viruses derived from two antigenically widely distinct HPAIV H5N1 “variant” (2.2.1 var) and “classic” (2.2.1 pro) lineages which are currently co-circulating in Egypt.
Section snippets
Origin of viruses
Egyptian HPAIV H5N1 isolated in embryonated chicken eggs at the National laboratory for veterinary quality control on poultry production (NLQP), Animal Health and Research Institute at Giza, Egypt, were obtained during routine surveillance of poultry holdings in Egypt. Other viruses were taken from the virus repository at the Friedrich-Loeffler-Institut (FLI). All viruses used in this study for antigen or vaccine production or for challenge were grown in 9–11 day-old embryonated eggs from
Co-circulation of several phylogenetically distinct lineages of clade 2.2.1 in Egypt
The phylogenetic tree of hemagglutinin gene sequences of HPAIV H5N1 from Egypt has continued to branch and expand in 2009. Currently, viruses from at least four discernable lineages within clade 2.2.1 (Fig. 1, colored branches) are co-circulating while other sublineages seem to be extinct (black branches). In particular, one group of viruses (red branches) has evolved to considerable phylogentic distance and has been regarded as “variant” lineage 2.2.1 (2.2.1var) [27]. This group is
Discussion
We have shown that phylogenetically discernable lineages of HPAI H5N1 viruses co-circulating in poultry in Egypt have antigenically distinguishable features influencing protection efficacy of commercial AIV H5 vaccines. The phylogenetic distance is reflected antigenically particularly for viruses of the clade 2.2.1 referred to as “variant” viruses in relation to the more conservedly reacting “classic” HPAIV H5N1 isolates of clade 2.2.1pro. Challenge experiments proved that variant strains
Acknowledgements
We are grateful to technical staff at the NLQP, Cairo, and the OIE and NRL AI, Isle of Riems, for continuing excellent work. Parts of this study have been funded by an O.I.E. Twinning grant to NLQP and OIE/NRL AI and by funds from Intervet (Ltd.) to H.M. Hafez.
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Both authors contributed equally to this work.