Previously, we generated a recombinant adenovirus encoding the HA protein derived from the A/VN/1203/04 (H5N1) HPAI virus strain (13
). Evaluation of the efficacy of the adenovirus-based influenza vaccine in challenge studies confirmed that a single subcutaneous inoculation in chickens provided complete protection against a lethal challenge with the influenza A/VN/1203/04 (H5N1) virus in combination with a significant reduction of virus isolates in cloacal and oral swabs. Furthermore, high HI antibody responses to influenza virus were generated in chickens and were correlated with protection, with chickens with low HI titers showing incomplete protection when HI titers were lower than 4 (log2
). Our goal for the present study was to expand upon these findings and investigate different routes of administration and the minimal dosage required by correlating the induced HI titer in order to manufacture a cost-effective poultry vaccine. Considering the use of chickens as broilers, breeders, or layers, the site of injection of the adenovirus-based vaccine is an important question to address. Although a human adenovirus-based vaccine is replication incompetent, the direct injection of such a vaccine into the “meat” would not be favorable and would require further safety investigations. In breeders and layers, it would be more acceptable, since vaccine traces are not expected to be detected in eggs and offspring. Toro et al. described an interesting vaccination technology that proposed the use of robotic injectors for in ovo
immunization of an adenovirus-based vaccine encoding the H5N9-derived hemagglutinin (28
). Therefore, we also tested the in ovo
immunization route for comparison. Among the different routes of administration studied here (intratracheal, conjunctival, subcutaneous, and in ovo
), only the subcutaneous route for Ad5.HA was able to induce an HI titer correlating with protection. Despite the previously reported successful in ovo
immunization using an adenovirus-based vaccine, we failed to consistently detect an HI titer in the hatched chickens after egg injection of high doses of Ad5.HA. One possible explanation for this unfavorable outcome could be the technique used to administer in ovo
vaccines. The success of in ovo
vaccination depends not only on the manner in which the vaccine is applied but also on the timing of the injection in relation to the stage of embryonic development and the exact site of injection (route) in the developing egg. An in ovo
injection can access five different areas of the egg during late stage incubation. These areas include the air cell, the allantoic sac, the amniotic fluid, the body of the embryo, and the yolk sac. Each area represents a distinct route of vaccine administration to the embryo, and these in turn represent distinct types of vaccine and antigen presentation to the avian immune system. Additionally, these in ovo
compartments change quickly during the window of injection timing. Thus, we do not exclude the possibility that a different route and/or timing could lead to a successful in ovo
application of adenovirus-based vaccines.
Using the subcutaneous route of administration, subsequent dose escalation studies were performed with Ad5.HA; in these studies, 107
vp were sufficient to induce a high HI titer similar to those induced by higher doses (1010
, and 108
vp) of Ad5.HA. Remarkably, the antibodies elicited were cross-reactive with a heterotypic H5N1 influenza virus strain from a different clade. These data indicate potential resistance to other circulating H5N1 influenza virus strains in the immunized chicken, as observed with other poultry vaccines (4
). We hypothesize that Ad5.HA at the dosage of 107
vp will provide protection against an influenza A/VN/1203/04 (H5N1) virus infection because HI titers correlate with the previously reported level of protection in chickens (13
). However, challenge studies that measure viral titers in cloacal and oral swabs in chickens are warranted to determine the impact of subcutaneous vaccination with 107
vp of Ad5.HA on protection and viral shedding (sterilizing immunity).
Current licensed avian influenza (AI) virus vaccines used in poultry around the world include the inactivated oil adjuvanted whole-virus vaccine and a recombinant fowlpox virus-vectored vaccine with an H5 AI virus antigen insert. Historically, AI virus strains selected for manufacturing inactivated vaccine have been based on low-pathogenicity viruses obtained from field outbreaks that have homologous HA proteins. HPAI virus strains have rarely been used to manufacture inactivated vaccines because they require specialized, high-biocontainment manufacturing facilities. More importantly, HPAI virus strains are difficult to grow in eggs due to their toxicity, which results in poor yields. The fowlpox-vectored H5 vaccine is safe and effective in chickens and has the advantage of early application on the first day of life (3
). However, this vaccine cannot be used in older animals, as their immunity to fowlpox virus prevents the development of effective immunity (27
In addition to the vaccines mentioned above, other novel recombinant vaccine technologies for use in poultry, such as the reverse-genetics-produced live attenuated influenza A virus vaccine (18
) and a Newcastle disease virus (NDV)-based live attenuated vaccine (15
), have recently been developed and show promising results. Reverse-genetics-produced vaccines, which sometimes take longer to generate, have the advantage of being able to overcome egg toxicity. Moreover, biocontainment for the production process could be lowered and would result in easier and less costly manufacturing. Using an NDV virus as a vector encoding the H5N1 HA protein, Steel et al. developed a bivalent vaccine strategy that induced protective immune responses against both H5N1 and NDV infections (26
). In addition to resulting in protection in H5N1 challenges and reduction of viral shedding, they both are effective when delivered in ovo
, which would reduce the cost of administration dramatically.
Among the strategies for vaccinating poultry against influenza, the use of adenovirus-based influenza vaccines may complement current technologies and offer some advantages. First, adenovirus is known to produce strong humoral and cell-mediated immune responses that are not confounded by natural preexisting immunity to the viral vector. However, the efficacy of sequential immunizations with Ad5-based vaccines in poultry could potentially be reduced by the induction of Ad5-specific neutralizing antibodies after the first immunization. Studies performed both with mice and with humans have shown that the level of neutralizing antibodies, route of administration, and serotype all played important roles in the effective delivery of an adenovirus-based vaccine (10
). This suggests that vector-specific immunity may be overcome by enhancing the vaccine dosage or by using alternative human and animal adenovirus serotypes. However, given the short lifespan of poultry (particularly for the broiler), we believe this might constitute a relatively minor limitation in this vaccine target population.
In comparison to the use of live attenuated influenza vaccines, adenoviral technology based on a recombinant incompetent human pathogen does not replicate in avian species and therefore poses no risk of reactivation in the host.
The second advantage is that the culture of recombinant adenovirus is both rapid and efficient. The ability to scale up to large quantities makes this technology one of the most promising DNA-based platforms for vaccination. Adenovirus-based vaccines, either in solution or lyophilized, can be stored at room temperature or at 4°C for up to 2 years without substantial loss of viral titer (8
). The ability to store the vaccine at room temperature in a lyophilized form, the low-dose application (107
vp/chicken), and the high production yield (with one cell producing 1,000 to 10,000 vp) all lower the burden of cost for vaccination at all stages of vaccine production and administration.
From an initial cost evaluation analysis that we performed for large-scale manufacturing of an adenovirus-based vaccine, we estimated the cost of 1 to 5 cents per dose (data not shown), which is within the acceptable cost range for poultry vaccines (6
In conclusion, our findings support the development of replication-defective adenovirus-based vaccines for the prevention of HPAI. An adenovirus-based vaccine could be additional ammunition in the current vaccine armamentarium to control the disease and to reduce the economic damage.