Induction of mucosal immunity in the avian Harderian gland with a replication-deficient Ad5 vector expressing avian influenza H5 hemagglutinin

Abstract

The chicken Harderian gland (HG) plays an important role in adaptive immune responses upon ocular exposure to avian pathogens such as avian influenza (AI). To determine the role of HGs in generating immunity, chickens were immunized ocularly with an adenovirus (Ad5) vector expressing the AI hemagglutinin H5 gene. The Ad5-H5 vector induced H5 transgene expression and induced H5- and Ad5-specific IgA and IgG spot-forming cells (SFCs) in the HGs. The IgA and IgG SFC peaked on day 9 forAd5 and day 11 for the H5 protein. In addition, Ad5- and H5-specific antibodies were induced in serum. IgA in chicken tears was predominantly dimeric, while in serum monomeric IgA was most abundant. Analysis of HG mRNA confirmed expression of the polymeric immunoglobulin receptor (plgR). These data demonstrated the importance of HGs to generate mucosal and systemic immunity to AI following ocular Ad5-H5 administration to chickens.

Fig. 1

Expression of avian influenza hemagglutinin in chicken Harderian glands 9 days after ocular Ad5-H5 exposure. White leghorns were exposed to 2.5 × 10⁸ infectious units of adenovirus expressing the AI hemagglutinin gene serotype 5. The tissues were fixed and 5 μm sections were obtained. The slides were stained with an anti-H5 affinity-purified rabbit-anti H5 antibodies. The Ad5-H5 exposed Harderian glands but not the controls expressed H5.

Fig. 2

Hemagglutination-inhibition (HI) titer in plasma of chickens’ ocular immunized with Ad5-H5. Plasma samples were collected from control chickens (n = 11) and chickens immunized two (n = 15) or three (n = 9) times with 2.5 × 10⁸ I.U. of Ad5-H5. HI titers of <1.0 Log₂ were arbitrarily assigned a titer of 0. No HI titer was detected in control chickens while the HI titer increased with each Ad5-H5 administration tested 14 days after ocular administration.

Fig. 3

Induction of Ad5-specific antibodies after ocular administration of Ad5-H5. Ad5-specific antibodies were detected by ELISA as described. The highest dilution with an OD₄₀₅ of .100 or more above background was defined as the endpoint-titer. No IgG or IgA antibody levels were detected in tears and serum from control chickens.

Fig. 4

IgA secreting cells in the Harderian glands after ocular immunization. Lymphocytes isolated from the Harderian glands 9 days after immunization were isolated and loaded on antigen-coated ELISPOT plates and were incubated overnight in a CO₂ incubator. Antibody secreting cells were detected as described in Section 2. Illustrated is a representative section of a single well containing Harderian gland lymphocytes derived from control or ocular challenged chickens.

Fig. 5

H5- and Ad5-specific IgG antibody secreting cells in the Harderian glands after ocular immunization with Ad5-H5. Lymphocytes were isolated from the Harderian glands (HDGL) at various days after Ad5-H5 administration and were analyzed for antibody secreting cells for H5 and Ad5. Indicated are the mean numbers of IgG spot-forming cells per 10⁶ lymphocytes and one standard error. Both Ad5-specific and H5-specific IgG antibodies are produced in the Harderian glands after ocular Ad5-H5 administration.

Fig. 6

H5- and Ad5-specific IgA antibody secreting cells in the Harderian glands after ocular immunization with Ad5-H5. Lymphocytes were isolated from the Harderian glands at various days after Ad5-H5 administration and were analyzed for IgA antibody secreting cells specific for H5 and Ad5. Indicated are the mean numbers of IgA spot-forming cells per 10⁶ lymphocytes and one standard error. Both Ad5-specific and H5-specific IgA antibodies are produced in the Harderian glands and peak on day 9 and 11 after Ad5-H5 administration.

Fig. 7

Polymeric-Ig receptor expression in the Harderian glands of chickens. Total RNA was isolated from the Harderian glands of three chickens using Tri-reagent (Molecular Research, Inc.) according manufactures protocols. The RNA was reverse transcribed (+) or not (−). The resulting PCR products and a 100 bp DNA ladder were separated on a 1.5% agarose gel and stained with ethidium bromide. A 400 bp amplicon was observed in reverse transcribed (+) samples confirming that pIgR mRNA is produced in the Harderian glands. No product was observed when the reverse transcription was eliminated (−).

Fig. 8

Immunoprecipitation of chicken IgA in tears and serum. Tears and serum were immunoprecipitated with biotinylated mouse-anti-chicken IgA monoclonal antibody and streptavidin-conjugated sepharose beads. The immunoprecipitants were analyzed by SDS–polyacrylamide gel electrophoresis (SDS–PAGE) on a 4–12% pre-cast gradient gel. The proteins were visualized using the Silver SNAP^® silverstain kit according to manufacturer protocols. Abbreviations used: monomeric IgA = mIgA; polymeric IgA = pIgA; tetrameric IgA = tIgA.