The highly pathogenic H5N1 influenza virus causes lethal multi-organ disease in poultry, resulting in significant economic losses and a public health concern in many parts of the world. The greatest threats posed by this virus are its ability to cause mortality in humans, its potential to compromise food supplies, and its possible economic impacts. Viral maintenance in poultry potentiates the risk of human-to-human transmission and the emergence of a pandemic strain through reassortment. An effective, safe poultry vaccine that elicits broadly protective immune responses to evolving flu strains would provide a countermeasure to reduce the likelihood of transmission of this virus from domestic birds to humans and simultaneously would protect commercial poultry operations and subsistence farmers.
DNA vaccines have been shown to elicit robust immune responses in various animal species, from mice to nonhuman primates 
. In human trials, these vaccines elicit cellular and humoral immune responses against various infectious agents, including influenza, SARS, SIV and HIV. In addition to their ability to elicit antibody responses, they also stimulate antigen-specific and sustained T cell responses 
. DNA vaccination has been used experimentally against various infectious agents in a variety of mammals, including cattle (against infectious bovine rhinotracheitis/bovine diarrhea virus, leptospirosis and mycobacteriosis) 
, pigs (against classical swine fever virus and mycoplasmosis) 
, and horses (against West Nile virus and rabies) 
. In addition, DNA vaccines have been tested against avian plasmodium infection in penguins 
and against influenza and infectious bursal disease in chickens 
, duck hepatitis B virus in ducks 
, and avian metapneumovirus and Chlamydia psittaci
in turkeys 
(reviewed in ref. 
). While they have been used in chickens to generate antisera to specific influenza viruses and confer protection against the low pathogenicity H5N2 strain 
, there is only one previous report of a monovalent DNA vaccine effective against H5N1 (and that only against a matched H5N1 isolate) 
; no protection with multivalent DNA vaccines against heterologous strains has been reported.
Development and characterization of a DNA vaccine modality for use in poultry offers a potential countermeasure against HPAI H5N1 avian influenza outbreaks. The virus can infect humans, typically from animal sources, including commercial and wild avian species, livestock, and possibly other non-domesticated animal species 
. While there is marked diversity in the host range of type A influenza viruses, many experts have speculated that a pandemic strain of type A influenza could evolve in avian species or avian influenza viruses could contribute virulent genes to a pandemic strain through reassortment 
. Thus, there is reason to consider vaccination of poultry that would stimulate potent and broad protective immune responses 
. In undertaking such efforts, it is important that there be a differentiation of infected from vaccinated animals 
so that animals can be protected and permit monitoring of new infections using proven and sensitive methodologies.
In this study, we used an automated high capacity needle-free injection device, Agro-Jet® (Medical International Technology, Inc., Denver, CO) to explore the feasibility of DNA vaccination of poultry. After optimization of injection conditions, alternative multivalent DNA vaccine regimens were analyzed and compared for magnitude and breadth of neutralizing antibodies, as well as protective efficacy after challenge in mouse and chicken models of HPAI H5N1 infection. The findings suggest that it is possible to develop a multivalent DNA vaccine for poultry that can protect against multiple HPAI H5N1 strains and that could keep pace with the continued evolution of avian influenza viruses.