Assessment of Route of Administration and Dose Escalation for an Adenovirus-Based Influenza A Virus (H5N1) Vaccine in Chickens

Highly pathogenic avian influenza (HPAI) virus causes one of the most economically devastating poultry diseases. An HPAI vaccine to prevent the disease in commercial and backyard birds must be effective, safe, and inexpensive. Recently, we demonstrated the efficacy of an adenovirus-based H5N1 HPAI vaccine (Ad5.HA) in chickens. To further evaluate the potential of the Ad5.HA vaccine and its cost-effectiveness, studies to determine the minimal effective dose and optimal route of administration in chickens were performed. A dose as low as 10⁷ viral particles (vp) of adenovirus-based H5N1 vaccine per chicken was sufficient to generate a robust humoral immune response, which correlated with the previously reported level of protection. Several routes of administration, including intratracheal, conjunctival, subcutaneous, and in ovo routes, were evaluated for optimal vaccine administration. However, only the subcutaneous route of immunization induced a satisfactory level of influenza virus-specific antibodies. Importantly, these studies established that the vaccine-induced immunity was cross-reactive against an H5N1 strain from a different clade, emphasizing the potential of cross-protection. Our results suggest that the Ad5.HA HPAI vaccine is safe and effective, with the potential of cross-clade protection. The ease of manufacturing and cost-effectiveness make Ad5.HA an excellent avian influenza vaccine candidate with the ability to protect poultry from HPAI virus infection. Considering the limitations of the influenza vaccine technology currently used for poultry applications, any effort aimed at overcoming those limitations is highly significant.Influenza A virus is a segmented, negative-strand RNA virus that belongs to the family Orthomyxoviridae, which is divided into subtypes based on serological reactions of the two surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA). Thus far, 16 different HA subtypes (H1 to H16) and 9 different NA subtypes (N1 to N9) (11, 34) have been identified. Each of the subtypes has been isolated from waterfowl species, the natural hosts of all known influenza A viruses. These birds are the reservoir for the spread of influenza virus worldwide in wild birds and poultry (19, 34). Avian influenza (AI) virus strains are further classified into low and highly pathogenic avian influenza (LPAI and HPAI, respectively) viruses based on their pathogenicity.Continued outbreaks of HPAI viruses of the H5 and H7 subtypes in poultry in Asia, Europe, Africa, and Canada represent a serious risk for animal and public health worldwide. Avian influenza is one of the greatest concerns for public health that has emerged from an animal reservoir in recent times. Since the late 1990s, the number of outbreaks of avian influenza in poultry has dramatically increased. For example, in 2008-2009, 2,770 outbreaks occurred in Vietnam, 1,143 in Thailand, 1,084 in Egypt, and 219 in Turkey; outbreaks have also occurred in many other countries worldwide (http://www.oie.int/eng/en_index.htm).In 2008-2009, 20 million poultry died or were depopulated because of HPAI outbreaks. This had devastating consequences for the international poultry industry and raised concerns about the potential for transmission to humans and the possible pandemic spread of lethal disease. Culling represents the first line of defense against avian influenza virus; however, continuing outbreaks over the last 6 years revealed that implementation of culling at the farm level was insufficient to halt the spread of disease.Vaccines, in conjunction with other measures of prevention and management, may represent an alternative to preemptive culling to achieve a reduction in the rate of transmission by reducing the susceptibility of healthy flocks at risk. Although vaccination has been recommended by the World Organization for Animal Health (OIE) and the Food and Agriculture Organization (FAO) to control AI, few effective AI vaccines are available (http://www.oie.int/eng/avian_influenza/OIE_FAO_Recom_05.pdf). Conventional inactivated vaccines containing the same viral subtype as field virus (with differing degrees of antigenic similarity) (4, 5, 22), inactivated vaccines generated through reverse genetic techniques (18, 33), and recombinant vaccines (3, 15, 21, 23) have been tested. However, production constraints associated with conventional inactivated influenza virus vaccines that are manufactured in eggs could severely hinder control of an emerging AI virus with pandemic potential (7).We investigated recombinant replication-defective adenoviruses as possible influenza vaccine vehicles for poultry. Recombinant adenovirus-based vaccines are highly effective inducers of both humoral immunity and cellular immunity in mammals and have shown promise as vaccine vehicle candidates against numerous infectious pathogens (2, 12, 14, 24, 32). Previously, we generated Ad5.HA, an E1/E3-deleted human adenovirus serotype 5 (Ad5)-based vector that expresses the codon-optimized hemagglutinin (HA) gene from the influenza A/Vietnam/1203/04 (H5N1) virus (13). The Ad5.HA vaccine induced humoral and cellular immune responses against HA and protected against influenza virus challenges in both mice and chickens. We have now extended our studies to determine the efficacy of Ad5.HA immunization in chickens when administered at different dosages via different routes.