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Network of expertise on animal influenza

OIE - World Organization for Animal HealthFAO - Food and Agriculture Organization of the United Nations

OFFLU Avian Influenza Vaccine Efficacy project in Egypt

Highly pathogenic avian influenza (HPAI) subtype H5N1 is currently endemic in Egypt, with the first outbreaks reported in February 2006. In efforts to control the disease, vaccination of commercial flocks was introduced at the end of March 2006, and mass vaccination of household poultry was conducted from May 2007 until 2009. As in other countries applying vaccination against HPAI, these vaccination efforts met with variable success, and to increase understanding of how to improve the use of vaccination as part of an overall control strategy, the Food and Agriculture Organization of the United Nations (FAO) – in close collaboration with national agencies – implemented a three-year (2008 to 2011) World Organisation for Animal Health (OIE)/FAO network of expertise on animal influenza (OFFLU) technical project entitled Vaccine Efficacy for the Control of Avian Influenza (AI) in Egypt (OSRO/EGY/801/USA). The project aimed to promote the appropriate use of efficacious poultry vaccines as part of a comprehensive strategy to combat HPAI through understanding the characteristics and epidemiology of circulating A/H5N1 viruses; determining the efficacy of available AI poultry vaccines; and supporting the development of sustainable national systems to monitor viral evolution and ensure vaccine efficacy.

National partners included laboratories with national diagnostic responsibilities (Central Laboratory for Quality of Poultry Production – CLQP) and those responsible for veterinary vaccine quality (Central Laboratory for Evaluation of Veterinary Biologics – CLEVB). International partners were recognized leaders in influenza research: the Southeast Poultry Research Laboratory (SEPRL) of the United States Department of Agriculture (USDA) (which is the OIE Collaborating Centre in Research on Emerging Avian Diseases) was responsible for laboratory capacity building activities for CLQP, conducting of and training in laboratory challenge trials, and evaluation of procedures for registration and licensing of poultry vaccines for AI; and the Erasmus Medical Centre undertook virus relationship analysis using antigenic cartography.

The project also benefited from concurrent work conducted by the Istituto Zooprofilattico Sperimentale delle Venezie (IZSVe, Italy) and the Friedrich-Loeffler-Institut (FLI, Germany), which are OIE/FAO reference centres for avian/animal influenza and Newcastle disease. Technical collaboration between IZSVe and the public and private sectors in Egypt (particularly universities and vaccine manufacturers), and the OIE twinning project between FLI and CLQP, entitled Promotion of Rapid Molecular Diagnosis and Characterization of Avian influenza and Newcastle Disease Viruses, contributed data and shared information that enabled the OFFLU project to develop an even broader perspective on the Egyptian H5N1 situation. The project was funded through the United States Agency for International Development (USAID), which had funded a similar project implemented by FAO in Indonesia. Lessons learned from these projects and other FAO national programmes in affected countries contribute to the understanding and control of HPAI.

Characteristics and epidemiology of circulating A/H5N1 viruses

To improve understanding of the evolution of the H5N1 HPAI virus in Egypt and assess the impact of AI vaccines used in poultry, biologic and genetic characterization and analysis of H5N1 HPAI viruses were conducted at both national and international reference laboratories. Between 2006 and 2008, a total of 1 592 cases of H5 AI were detected using real-time reverse transcription polymerase chain reaction (RT-PCR) (confirmation of N1 was conducted on a subset of samples), and 586 cases were confirmed during 2009/2010. A total of 540 virus isolates were obtained, predominantly from household poultry: 157 from 2006 to 2008; 160 from 2009; and 223 from 2010. Genetic analysis was conducted on isolates from 2008 to 2010; SEPRL conducted sequencing of 27 isolates and supported the laboratory staff at CLQP in completing 170 H5 genes and 71 N1 genes. The data from the majority of the genes sequenced by CLQP and SEPRL have been submitted to public databases such as Genbank. Phylogenetic analysis of the Egyptian viruses indicated two major groupings – “classical” and “variant”. The classical viruses prevailed in household poultry, most of which is unvaccinated; these viruses belong to clade 2.2.1, according to the updated World Health Organization (WHO) unified nomenclature for H5N1 and demonstrated few mutations compared with the initial introduced virus from 2006. The variant viruses were predominantly detected in the commercial sector, where vaccination is routinely applied, and appeared in late 2007 (Arafa et al., submitted). These variants belong to a newly designated clade and demonstrated a higher mutation rate (Cattoli et al., 2011) than classical viruses. It is to be noted that these variant viruses have not been associated with human infections, apart from one human case in 2009 (109 human cases were reported by WHO between 2008 and 2 November 2011). While these findings have improved the epidemiological understanding of the HPAI situation in Egypt (Arafa et al., 2010; submitted), they also highlight the need to include representative sampling from all poultry production sectors to ensure a more complete understanding of the situation, especially in efforts to monitor vaccine efficacy. Antigenic cartography (Smith et al., 2004) quantifies the antigenic differences between the haemagglutinin (HA) proteins of tested viruses based on haemagglutination inhibition (HI) assay data. The data are displayed in a map format to enable visualization of the antigenic distances between the viruses. This method was applied to aid the selection of candidate challenge and vaccine strains for further in vivo testing. (Antigenic cartography data should not be regarded as the only criterion for predicting vaccine efficacy).  Reference strains for reagent production were selected from the Egyptian strains, based on the year of isolation and biological characterization, and shared among the partners. Technical staffs from CLPQ were trained in assay techniques, data management and analysis of the cartography results, and participated in reagent production with SEPRL, using a harmonized protocol.

Challenge tests

Trials to determine vaccine efficacy through challenge testing against both classical and variant strains were conducted at both SEPRL and CLQP, and benefited from additional information from trials conducted separately at FLI (Grund et al., 2011) and IZSVe (Terregino et al., 2010). In laboratory trials, birds were inoculated using either commercially available inactivated vaccines or experimental inactivated vaccines generated from classical or variant Egyptian strains. The level of protection and the extent of virus shedding after challenge were then evaluated. All trials under the OFFLU project – and those carried out by FLI, IZSVe, CLQP and the Veterinary and Agrochemical Research Centre (VAR – Belgium) (Rauw et al.,2011), where a classical sub-lineage Egyptian virus was used for challenge – demonstrated 100 percent clinical protection under laboratory conditions, regardless of the vaccine strain used. For trials using challenge viruses from the variant sub-lineage, protection afforded from vaccination was highly variable (ranging from nearly 80 percent to complete vaccine failure of 0 percent protection in a few cases), as was the level of virus shedding post-challenge. However, the data also suggest that vaccines with sufficient antigen content to produce high titres in the bird could be protective even against viruses with large antigenic and genetic differences (e.g.,variants). This highlights the importance of the antigenic content of commercial vaccines in stimulating the appropriate immune response.

Laboratory capacity building

Laboratory capacity building activities and technology transfer under this project aimed to support the rapid and accurate diagnosis of H5N1 HPAI in government veterinary laboratories and to ensure the sustainability of ongoing surveillance and monitoring activities. Laboratory staff at CLQP and CLEVB received training on-site and abroad (at SEPRL) on genetic sequencing for H5N1 HPAI and phylogenetic analysis; antigenic characterization and production of standardized reagents; safety, potency, purity testing, and challenge testing of H5 AI vaccines; and laboratory biosafety and biosecurity. Recommendations were provided, related to the CLQP and CLEVB facilities: protocols for pathogen characterization, vaccine potency and efficacy determination; and virus surveillance.

Recommendations and perspectives

Project partners contributed their expertise through joint data analysis and technical review of strategies and policies, developed recommendations and indicated necessary actions. These outcomes were delivered at two meetings (January 2009 in Cairo, Egypt, and June 2011 in Rome, Italy), which engaged other national stakeholders. Project activities helped strengthen national laboratory capacity, improve biosafety procedures in national partner laboratories, and support ongoing virus detection and characterization through the provision of laboratory equipment, reagents and other consumables. Support to the development of a new laboratory information management system made it possible to monitor and respond to disease outbreaks and analyse epidemiologic data in conjunction with laboratory data. The outcome of biologic, genetic and antigenic analyses of Egyptian viruses contributed to understanding of these viruses and identification of potential candidate vaccine strains for use in poultry. CLQP should continue the surveillance and characterization of influenza viruses, to develop a complete understanding of the epidemiology of H5N1 HPAI in Egypt and ensure sound disease control planning and informed decision-making. The data generated by such surveillance and characterization are used to validate diagnostic tests and enable early detection of emerging viruses with specific mutations that have major phenotypic implications. Collection and integration of data on vaccine efficacy and effectiveness at both the laboratory and the field levels (e.g., post-vaccination monitoring, vaccination data analysis) are still needed in Egypt. Given the country’s current socio-economic situation, external funding may be required in the short term to continue this important work. There is also need for increased cooperation between government and industry, to improve the virus data and epidemiologic information regarding commercial poultry. The engagement of other stakeholders – such as private laboratories, Egyptian universities and other institutions conducting laboratory analysis in the country, including New Medical Research Unit 3 (NAMRU3) – should be encouraged to allow the sharing of data and information that can contribute to disease control efforts. The integration of epidemiological information with virus characterization data could also contribute to better early risk assessments, leading to appropriate response actions in both the animal and the human sectors and to a more effective national AI control programme. The national animal health sector should play an active and leading role in improving these linkages, in line with the four-way linking framework proposed by FAO and WHO (A four-way linking workshop was conducted in Egypt from 26 to 28 September 2011). Although this study shows that most AI poultry vaccines currently used in Egypt appear to confer acceptable levels of protection when applied appropriately, the proper handling and application of vaccines in the field are critical if vaccination is to be an effective part of a comprehensive control programme. Based on field data, the actual coverage of vaccination campaigns appears to be low, suggesting that application of AI vaccination in Egypt needs to be improved. Vaccination of day-old chicks (DOCs) with an effective vaccine (if and when available) may significantly decrease the virus load and subsequent circulation along the poultry value chain. The application of DOC vaccines at both traditional and modern hatcheries represents an efficient intervention that could have a significant impact on the control of H5N1 HPAI. FAO encourages the ongoing efforts of pharmaceutical companies to develop vaccines for use in DOCs. FAO continues to emphasize that biosecurity remains one of the most important and critical tools in the fight against H5N1 HPAI; as a supportive measure to reduce virus load in the poultry population and the environment, vaccination should be applied as part of a comprehensive control programme. When a nation opts to apply AI vaccination, a clear plan for ongoing surveillance and postvaccination monitoring must be in place (FAO, 2011). This project contributed to the development of enhanced capacity to undertake laboratory-based activities in support of field investigations and surveillance in Egypt, but inputs for implementing the recently revised HPAI surveillance programme based on the value chain and covering all sectors of poultry production are still required.


Abdelwhab, E.M., Grund, C., Aly, M.M., Beer, M., Harder, T.C. & Hafez, H.M. 2011. Influence of maternal immunity on vaccine efficacy and susceptibility of day old chicks against Egyptian highly pathogenic avian influenza H5N1. Veterinary microbiology, 2011, Epub ahead of print.

Abdelwhab, E.M., Grund, C., Aly, M.M., Beer, M., Harder, T.C. & Hafez, H.M. 2011. Multiple dose vaccination with heterologous H5N2 vaccine: immune response and protection against variant clade 2.2.1 HPAI H5N1 in broiler breeder chicken. Vaccine, 29: 6219– 6225.

Arafa, A., Suarez, D.L., Hassan, M.K. & Aly, M.M. 2010. Phylogenetic analysis of HA and NA genes of HPAI-H5N1 Egyptian strains isolated from 2006 to 2008 indicates heterogeneity with multiple distinct sublineages. Avian Diseases, 54: 345–349.

Arafa, A., Selim, A., Kholosy, S.G., Hassan, M.K., Aly, M.M., Abdel-Wanees, S., Suarez, D. & Swayne D. submitted. Evolution of highly pathogenic avian influenza H5N1 viruses in Egypt indicating progressive adaptation.

Cattoli, G., Fusaro, A., Monne, I., Coven, F., Joannis, T., El-Hamid, H.S.A., Hussein, A.A., Cornelius, C., Amarin, N.M., Mancin, M., Holmes, E.C. & Capua, I. 2011. Evidence for differing evolutionary dynamics of A/H5N1 viruses among countries applying or not applying avian influenza vaccination in poultry. Vaccine, Epub ahead of print.

FAO. 2011. Assessment of the impact of avian influenza vaccination in household and commercial poultry sectors in Egypt. Consultancy by M. Peyre. Rome. 53 pp.

Grund, C., el Abdelwhab, E.M., Arafa, A.S., Ziller, M., Hassan, M.K., Aly, M.M., Hafez, H.M., Harder, T.C. & Beer, M. 2011. 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. Vaccine, 29(33): 5567–5573.

Kilany, W.H., Abdelwhab, E.M., Arafa, A.-S., Selim, A., Safwat, M., Nawar, A.A., Erfan, A.M., Hassan, M.K., Aly, M.M. & Hafez, H.M. 2011. Protective efficacy of H5 inactivated vaccines in meat turkey poults after challenge with Egyptian variant highly pathogenic avian influenza H5N1 virus. Veterinary Microbiology, 150: 28–34.

Kilany, W.H., Palya, V., Aly, M.M., Hassan, M.K., El Fetou, A.A. & Gardin, Y. in press. Evaluation of the efficacy of recombinant HVT-H5 vaccine in commercial broiler chickens carrying maternal derived antibody (MDA) under field conditions in Egypt. Prepared for the 8th International Symposium on Avian Influenza, London,
1 to 4 April 2012.

Rauw, F., Palya, V., Van Borm, S., Welby, S., Tatar-Kis, T., Gardin, Y., Dorsey, K.M., Aly, M.M., Hassan, M.K., Soliman, M.A., Lambrecht, B. & van den Berg, T. 2011. Further evidence of antigenic drift and protective efficacy afforded by a recombinant HVT-H5 vaccine against challenge with two antigenically divergent Egyptian clade 2.2.1 HPAI H5N1 strains. Vaccine, 29(14): 2590 –2600.

Smith, D.J., Lapedes, A.S., de Jong, J.C., Bestebroer, T.T., Rimmelzwaan, G.F., Osterhaus, A.D.M.E. & Fouchier, R.A.M. 2004. Mapping the antigenic and genetic evolution of influenza virus. Science, 305: 371–376.

Terregino, C., Toffan, A., Cilloni, F., Monne, I., Bertoli, E., Castellanos, L., Amarin, N., Mancin, M. & Capua, I. 2010. Evaluation of the protection induced by avian influenza vaccines containing a 1994 Mexican H5N2 LPAI seed strain against a 2008 Egyptian H5N1 HPAI virus belonging to clade 2.2.1 by means of serological and in
vivo tests. Avian Pathol., 39(3): 215–222.

Contributors: Gwenaelle Dauphin (FAO), Mia Kim (FAO), Yilma Jobre (FAO), Juan Lubroth (FAO)

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