Improvements to the hemagglutination inhibition test for serological assessment of recombinant fowlpox-H5-avian-influenza vaccination in chickens and its use along with an agar gel immunodiffusion test for differentiating infected from noninfected vaccinated animals.

In general, avian influenza (AI) vaccines protect chickens from morbidity and mortality and reduce, but do not completely prevent, replication of wild AI viruses in the respiratory and intestinal tracts of vaccinated chickens. Therefore, surveillance programs based on serological testing must be developed to differentiate vaccinated flocks infected with wild strains of AI virus from noninfected vaccinated flocks in order to evaluate the success of vaccination in a control program and allow continuation of national and international commerce of poultry and poultry products. In this study, chickens were immunized with a commercial recombinant fowlpox virus vaccine containing an H5 hemagglutinin gene from A/turkey/Ireland/83 (H5N8) avian influenza (AI) virus (rFP-H5) and evaluated for correlation of immunological response by hemagglutination inhibition (HI) or agar gel immunodiffusion (AGID) tests and determination of protection following challenge with a high pathogenicity AI (HPAI) virus. In two different trials, chickens immunized with the rFP-H5 vaccine did not develop AGID antibodies because the vaccine lacks AI nucleoprotein and matrix genes, but 0%-100% had HI antibodies, depending on the AI virus strain used in the HI test, the HI antigen inactivation procedure, and whether the birds had been preimmunized against fowlpox virus. The most consistent and highest HI titers were observed when using A/turkey/Ireland/83 (H5N8) HPAI virus strain as the beta-propiolactone (BPL)-inactivated HI test antigen, which matched the hemagglutinin gene insert in the rFP-H5 vaccine. In addition, higher HI titers were observed if ether or a combination of ether and BPL-inactivated virus was used in place of the BPL-inactivated virus. The rFP-H5 vaccinated chickens survived HPAI challenge and antibodies were detected by both AGID and HI tests. In conclusion, we demonstrated that the rFP-H5 vaccine allowed easy serological differentiation of infected from noninfected birds in vaccinated populations of chickens when using standard AGID and HI tests.

Efficacy of two H5N9-inactivated vaccines against challenge with a recent H5N1 highly pathogenic avian influenza isolate from a chicken in Thailand.

The objective of this study was to compare the efficacy of two avian influenza (AI) H5-inactivated vaccines containing either an American (A/turkey/Wisconsin/68 H5N9; H5N9-WI) or a Eurasian isolate (A/chicken/Italy/22A/98 H5N9; H5N9-It). Three-week-old specific pathogen-free chickens were vaccinated once and challenged 3 wk later with a H5N1 highly pathogenic AI (HPAI) virus isolated from a chicken in Thailand in 2004. All unvaccinated challenged birds died within 2 days, whereas 90% and 100% of chickens vaccinated with H5N9-WI and H5N9-It, respectively, were protected against morbidity and mortality. Both vaccines prevented cloacal shedding and significantly reduced oral shedding of the challenge HPAI virus. Additional chickens (vaccinated or unvaccinated) were placed in contact with the directly challenged birds 18 hr after challenge. All unvaccinated chickens in contact with unvaccinated challenged birds died within 3 days after contact, whereas unvaccinated chickens in contact with vaccinated challenged birds either showed a significantly delayed mortality or did not become infected. All vaccinated contacts were protected against clinical signs, and most chickens did not shed detectable amount of HPAI virus. Altogether, these data indicate that both vaccines protected very well against morbidity and mortality and reduced or prevented shedding induced by direct or contact exposure to Asian H5N1 HPAI virus.

Efficacy of a fowlpox-vectored avian influenza H5 vaccine against Asian H5N1 highly pathogenic avian influenza virus challenge.

A recombinant fowlpox-avian influenza (AI) H5 vaccine (rFP-AIV-HS) expressing the hemagglutinin of the A/turkey/Ireland/1378/83 H5N8 AI isolate has been used in Central America since 1998 to control H5N2 low pathogenicity AI. Previously, this vaccine was shown to induce full protection against a panel of H5 highly pathogenic (HP) AI isolates, including HPAI H5N1. Here, we evaluate the efficacy of rFP-AIV-H5 against escalating doses of HPAI H5N1 A/chicken/ SouthKorea/ES/03 isolate and against the HPAI H5N1 A/chicken/Vietnam/0008/2004 isolate. In both studies, 1-day-old specific pathogen-free (SPF) chickens were vaccinated by subcutaneous route with rFP-AIV-H5 and challenged 3 wk later by the oronasal route. In the first study, full protection was observed up to a challenge dose of 6.5 log10 embryo infectious dose (EID50), and the 50% chicken infectious dose was estimated to be 3.1 and 8.5 log10 EID50 in the control and the rFP-AIV-H5-vaccinated group, respectively. A 2-4 log10 and > 4 log10 reduction of oral and cloacal shedding was observed in rFP-AIV-H5 vaccinated birds, respectively. The rFP-AIV-H5 vaccine induced hemagglutination inhibition antibodies (5.2 log2) detectable with homologous H5N8 antigen. In the second study, rFP-AIV-HS-vaccinated chicks were fully protected against morbidity and mortality after challenge with the 2004 Vietnam isolate, whereas unvaccinated chickens died within 2 days of challenge. Shedding in cloacal swabs was detected in all unvaccinated controls but in none of the rFP-AIV-H5-vaccinated chickens. Together, these results confirm the excellent level of protection induced by rFP-AIV-H5 in SPF chickens against two recent Asian HPAI H5N1 isolates.

Development and use of fowlpox vectored vaccines for avian influenza.

The avian influenza (AI) vaccine designated TROVAC-AIV H5 (TROVAC-H5) contains a live recombinant fowlpox rec. (FP) recombinant (recFP), expressing the hemagglutinin (HA) gene of an AI H5 subtype isolate. This recombinant vaccine was granted a license in the United States for emergency use in 1998 and full registration in Mexico, Guatemala, and El Salvador where over 2 billion doses have been administered. One injection of TROVAC-H5 protects chickens against AI-induced mortality and morbidity for at least 20 weeks, and significantly decreases shedding after challenge with a wide panel of H5-subtype AI strains, regardless of neuraminidase subtype. Recently, excellent protection was demonstrated against 2003 and 2004 Asian highly pathogenic H5N1 isolates. Whereas TROVAC-H5 AI H5 efficacy was not inhibited by anti-AI or anti-fowlpox maternal antibodies (passive immunity), protection to AI was significantly decreased in chickens previously vaccinated or infected with FP (active immunity). Advantages of the TROVAC-H5 vaccine over inactivated AI vaccines are: (a) single administration at 1 day of age and early onset (1 week) of protection, (b) easy monitoring of AI infection in vaccinated flocks with agar gel precipitation (AGP) and enzyme-linked immunosorbent assay (ELISA) used as tests to differentiate infected from vaccinated animals (DIVA tests), and (c) no residue problem due to adjuvant. These features make TROVAC-H5 an ideal AI vaccine for routine administration of day-of-age chicks in hatcheries. RecFP expressing HA from three lineages of H7 subtype (Eurasian, American, and Australian) were also tested for efficacy against a highly pathogenic avian influenza (HPAI) Eurasian HPAI H7N1. Only the recFP expressing the Eurasian H7 gene provided sufficient protection indicating that the breadth of protection induced by recFP is apparently restricted for H7 isolates. The fowlpox vector technology can also be used for the production of an emergency vaccine: once the HA sequence of an emerging AI virus is known, recFP can be rapidly generated. TROVAC-H5 has recently been shown to be immunogenic in cats and could therefore also be considered for use in mammals.