Background
During the past 40 years, the number and frequency of highly pathogenic avian influenza (HPAI) outbreaks in poultry worldwide has steadily increased [
1,
2]. In North America, a HPAI clade 2.3.4.4c H5N
x HPAI epizootic began in November 2014 when Eurasian HPAI H5N8 and H5N2 was detected in wild and backyard birds [
3‐
5]. The following year, infections with the H5N2 HPAI virus devastated poultry production in the Midwest of USA, primarily affecting egg-laying chickens and turkeys, with approximately 47 million poultry dead or euthanized [
6]. A stamping-out policy was employed to control the epizootic, without vaccination, which was declared over in June 2015. In January of 2022, isolation of H5N1 clade 2.3.4.4b HPAI occurred in an American Widgeon in South Carolina (
https://www.aphis.usda.gov/aphis/newsroom/stakeholder-info/sa_by_date/sa-2022/hpai-sc). This event began on the east coast, primarily in wild birds, and subsequently infected commercial and backyard poultry. Over 171 commercial and 108 backyard flocks were confirmed positive for H5Nx HPAI, affecting more than 37.5 million birds at that time. (
https://www.aphis.usda.gov/aphis/ourfocus/animalhealth/animal-disease-information/avian/avian-influenza/hpai-2022).
During the H5Nx outbreak in 2015, multiple studies developed and tested commercial and experimental H5 vaccines against avian influenza virus (AIV) to demonstrate protective efficacy against the clade 2.3.4.4 H5Nx HPAI virus [
7‐
11]. When used properly and in conjunction with other control measures, vaccines can be an effective method to control clinical disease and reduce virus transmission, and has led to successful HPAI eradication [
12,
13]. More recently, effective application of vaccines for either H5 or H7 HPAI have significantly reduced some HPAI viruses in China, particularly H7N9 [
14,
15]. One key aspect of this vaccine approach is the continued updating of seed strain utilized in the vaccine to match field virus HA as it continually evolves.
Previous HPAI epizootics have occurred in vaccinated chicken flocks [
13]. This may be the result of improper administration, lack of timely seroconversion before HPAI exposure, antigenic mismatch or use of poor quality vaccine. Qualities of a proper poultry vaccine include efficacy, safety, onset of immunity, ease of application, decrease virus replication and transmission potential, cost, maternal antibody consideration, and the ability to distinguish infected versus vaccinated animals (DIVA strategy) [
12,
13].
Our previous vaccine studies in chickens demonstrated that older USDA H5 vaccine bank strains (e.g. Tk/WI/68(H5N9)) provided limited protection against clade 2.3.4.4 H5N2 HPAI, signifying the need for new vaccines [
7]. In addition, we have demonstrated variable protection with a replication-deficient alphavirus vaccine (RP-H5) and a recombinant turkey herpes virus vaccine (rHVT-AI) following clade 2.3.4.4 H5 challenge in laying hens and turkey [
8,
9]. No vaccine studies have been performed with broiler chickens. Although major differences in response from layer chickens are not anticipated, the short production life of commercial broilers of 6–7 weeks requires a different strategy for vaccination to provide earlier protection. Specifically, the vaccination at day of age, if effective, allows for more efficient vaccination at the hatchery before birds are placed in the field and potentially offers earlier protection.
The objective of the current study was to investigate protection of broiler-type birds using recombinant AI vaccines delivered in ovo or at day-of-age followed by challenge at 3 or 6 weeks of age with a 2015 North American HPAI virus. The study demonstrates that a single dose of rHVT-AI or RP-H5 provided above 90% protection following lethal challenge. In addition, a dose response was observed with the RP-H5 vaccine when delivered at one-day of age. A combination of the two vaccines delivered at day of age also protected chickens from a H5N2 HPAIV challenge. The results provide a framework for consideration of HPAI vaccination of broilers in the U.S.
Discussion
Vaccination of poultry is a cost-effective approach to disease prevention; however, its application for AI is not yet globally warranted for a myriad of reasons [
19,
20]. Besides cost, trade issues and lack of protective immunity to prevent infection and transmission are key considerations against AI vaccination. In addition, the overwhelming majority of vaccines, including inactivated and recombinant, provide systemic immunity, but not mucosal immunity, and thus do not prevent infection [
12]. The proper use of vaccines, as a component of an AI control strategy, must be added to traditional control methods (e.g., stamping out, animal movement control, increased biosecurity, and increased surveillance). AI vaccines should be antigenically matched to the field virus, and ideally would be amenable to mass-administration techniques to large numbers of poultry to make them more cost effective [
21].
In ovo vaccination is an efficacious and convenient method of vaccinating embryonating embryos in the hatchery with over 80% of US broiler industry utilizing this approach to control Marek’s disease virus [
22]. Advantages of
in ovo vaccine include decreased labor, time, and costs and facilitates uniform administration of vaccine dose into hundreds of eggs per minute. A consideration for this type of vaccination program is the shorter life span of broiler type birds. Earlier studies have successfully demonstrated efficacy following
in ovo vaccination with AI challenge [
23,
24]. In addition, numerous experimental studies have demonstrated the efficacy of the rHVT-AI vaccine in poultry, with protection from HPAI ranging from 60 to 100% [
7,
25‐
30]. The rHVT-AI vaccine appears to induce lower antibody titers compared to traditional inactivated vaccines [
25]. However, in these studies the antigen used for challenge was not matched to the rHVT-AI insert used for vaccination, or the HI test, and therefore this may have contributed to lower titers observed in these studies and others [
7,
8,
25,
27]. We have previously demonstrated these types of recombinant vaccines induce cross-reactive cellular immunity to the HA, with broader protection from various H5 HPAI lineages [
25]. The combined humoral and cellular immunity in that study resulted in significantly reduced viral shedding after challenge.
The aim of this study was to investigate the protective efficacy of three recombinant H5 vaccines for application in commercial broilers against North American clade 2.3.4.4c H5N2 HPAI virus challenge. Overall, the results demonstrate that
in ovo vaccination with a single dose of the rHVT-AI or two doses of RP-H5 adequately protected the birds and decreased the virus shedding. The results also demonstrate that the RP-H5 did not induce immunity when delivered only
in ovo, based on serology results and lack of protection, and may indicate the vaccine is not suited for
in ovo application. The RP-H5 was not designed for use in ovo application, but we included it as an option because to see if it could facilitate a mass vaccination approach if this route of administration was effective. It is worth noting that antigenic matching did not appear to offer increased protection as both the RG-H5 and RP-H5 given alone did not demonstrate better protection. Finally, combining both vaccines (rHVT-AI and RP-H5) and applying them at one day of age also provided good protection. It should be noted that a higher challenge dose of virus was used based on a higher bird infectious dose of North American 2.3.4.4c H5 viruses determined by Bertran, et al. [
5]. This higher dose may have contributed to higher levels of virus shedding observed in vaccinated groups after challenge.
The recombinant alphavirus vaccine, RP-H5, has also been demonstrated to provide protection against HPAI challenge, although better protection has been demonstrated when this vaccine is used in a prime-boost schedule [
8,
9,
31,
32]. The results here demonstrated the inability of the vaccine to provide protection when delivered
in ovo. These results occurred most likely from a failure of antigen expression and/or induced immunity in immunologically immature animals, as no detectable antibodies were observed pre-challenge. One possibility was the vaccine was delivered to the amniotic fluid rather parenterally into the bird. Delivering the vaccine inoculum to the amniotic fluid is technically an oral vaccine and this route of delivery could have contributed to the failure of protection. Providing a second dose of RP-H5 vaccine subcutaneously at 3 weeks of age decreased viral shedding, increased antibody levels, and protected against HPAI challenge at 6 weeks of age. When the vaccines were applied (rHVT-AI and RP-H5) in combination at one day of age, protection at 6 weeks of age with decreased virus shedding was observed. However, it is difficult to assess the contribution of the RP-H5 in this schedule as
in ovo application with the rHVT-AI alone provided protection at 6 weeks.
Reverse genetics derived vaccines for avian influenza have been used globally for many years in countries that allow for AI vaccination, including China, Egypt, Hong Kong, Indonesia Vietnam, and Mexico [
33]. One advantage to these vaccines is the ability to directly match the HA in the vaccine to the outbreak strain. In our previous studies, the RG-H5 vaccine provided complete protection against HPAI challenge in chickens and turkeys when vaccinated at 3 weeks of age [
7,
8]. In these studies, vaccination at one-day of age only provided some protection (62%) against challenge. It is likely that age of the bird at vaccination, not being immunologically mature, contributed to the lack of protection observed in these studies compared to the previous studies because the birds in all three studies received an equal dose and adjuvant for vaccination as well as the challenge isolate and dose. Previous vaccination studies of day old chicks support the idea that their immune response is greatly reduced and the immune response will be reduced [
34,
35]. The RP-H5 vaccine did demonstrate a dose-based difference in immune response with birds receiving the 10
8 vaccine dose had better protection than the 10
7 dose, but it still wasn’t completely protective. Despite the obvious advantages of vaccinating birds in the hatchery, the use of killed or non-replicating vaccines has severe limitations when working with day old chicks.
In conclusion, this study provides support for the use of recombinant vaccines for protection of broiler chickens should a similar outbreak occur, and emergency vaccination is considered. During the recent U.S. outbreaks of clade 2.3.4.4 HPAI, vaccination was considered, however many variables such as trade issues, lack of vaccine efficacy studies, and commodity group interest, resulted in non-deployment in the field. While not tested in these studies, use of a DIVA strategy to distinguish vaccinated from infected animals can be utilized with recombinant vaccine technology [
36‐
38]. The ability to utilize active surveillance in vaccinated flocks is a critical consideration of a vaccination program. Although nearly 47 million birds died or were destroyed in recent outbreaks, the triggers to employ AI vaccination in the U.S are not defined. Here, vaccination with the rHVT-H5
in ovo provided the best protection. While two doses of RP-H5 provided protection, the goal of a broiler vaccination program would be to achieve protection in a single application. These data provided support for consideration of recombinant vaccine protection of broiler birds from the current H5Nx HPAI outbreak.
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