may be important for induction of IBV-specific immunity and may contribute to persistence of the vaccine in the host. The S1 sequence variant of the spike protein of IBV that persisted in chickens after vaccinations and that resembled the non-attenuated parent sequence of the IBV ArkDPI vaccine strain was inserted into a replication-incompetent adenovirus vector serotype 5 (Ad5). Ad5 is a common cold virus, and some of its early genes have been removed to make it replication-incompetent. The S1 sequence variant inserted into the Ad5 vector was named component 2 (C2). A single ocular administration of Ad5 vector-expressing C2 increased IBV-specific IgA and IgG antibodies to IBV in plasma, IBV-neutralizing antibody levels, and circulating IBV specific B cells. Lymphocytes from
Ad5-C2-immunized chickens, when stimulated in vitro with LPS (LPS activates B cells and stimulates B cell division) secreted IBV-specific antibodies. The plasma IgG response to the whole IBV virus after Ad5-C2 immunization could be divided into three groups: high, medium, and low responders. More detailed analyses of these antibody responses were performed using the above-outlined 17 amino acid long, overlapping peptide array covering the S1 sequence. The high responder group contained highly cross-reactive IgG antibodies. The medium IgG group recognized six B-cell epitopes. As noted earlier, a B-cell epitope is the part of the S1 protein that is recognized by antibodies after vaccination with IBV, and by using overlapping peptides covering the S1 sequence we precisely determined the locations of these sites. The IgG antibodies in the medium IBV response group had a weak response to three of the six recognized Bcell epitopes. The low responder group recognized only two peptides of the S1 sequence. Going from low to high IgG responder groups, the IgA antibodies in these groups recognized two to four peptides in the host attachment domain of the S1 sequence. An additional cluster of IgAantibody-recognized peptides was located down from the host attachment domain towards the carboxy-terminus of the protein. The number of
peptides recognized in this cluster by IgA increased with decreasing IgG responses to the whole virus. This observation is consistent with a less-focused humoral IgA response, possibly due to limited antigen exposure by the somewhat low titer of Ad5-C2 available.
Conclusion Persistence of the Arkansas serotype of IBV remains a problem in the poultry industry, because the control of IBV is a complex problem that involves many variables. These variables include the variant subpopulation of the live-attenuated ArkDPI IBV vaccine; the continuous changing nature of this RNA virus in the field, enabling immune escape; the presence of immunosuppressive viruses that hamper induction of protective immunity; and our limited understanding of the role of cell-mediated versus humoral immunity on the control of this virus, as well as the role of systemic versus mucosal immunity in protection against this virus. Despite the complexity of this problem, progress is being made and hopefully will lead to the design of a highly cross-protective IBV vaccine in the future.
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