Volume 5 Supplement 4
Cross reactive cellular immune responses in chickens previously exposed to low pathogenic avian influenza
© Kapczynski et al; licensee BioMed Central Ltd. 2011
Published: 3 June 2011
Avian influenza (AI) infection in poultry can result in high morbidity and mortality, and negatively affect international trade. Because most AI vaccines used for poultry are inactivated, our knowledge of immunity against AI is based largely on humoral immune responses. In fact, little is known about cellular immunity following a primary AI infection in poultry, especially regarding cytotoxic T lymphocytes (CTL’s).
In these studies, major histocompatibility complex (MHC)-defined (B2/B2) chickens were infected with low pathogenic AI (LPAI) H9N2 and clinical signs of disease were monitored over a two weeks period. Splenic lymphocytes from infected and naïve birds were examined for cross reactivity against homologous and heterologous (H7N2) LPAI by ex vivo stimulation. Cellular immunity was determined by cytotoxic lysis of B2/B2 infected lung target cells and proliferation of T cells following exposure to LPAI.
Infection with H9N2 resulted in statistically significant weight loss compared to sham-infected birds. Splenic lymphocytes derived from H9N2-infected birds displayed lysis of both homologous (H9N2) and heterologous (H7N2) infected target cells, whereas lymphocytes obtained from sham-infected birds did not. T cell proliferation was determined to be highest when exposed to the homologous virus.
Taken together these data extend the findings that cellular immunity, including CTL’s, is cross reactive against heterologous isolates of AI and contribute to protection following infection.
Cell-mediated immunity (CMI) is antigen specific immunity mediated by T lymphocytes and has been suggested to be an important factor to the development of protection in chickens vaccinated against viral diseases . The subsets of T lymphocytes: CD4+ helper cells and CD8+ cytotoxic cells constitute the principal cells of the CMI response. For influenza, CD8+ CTL’s play a crucial role in controlling infectious virus from the lungs of mice . A number of recent studies have provided evidence that CMI directed against viral epitopes conserved among influenza A viruses, such as those within the nucleoprotein (NP) and hemagglutinin (HA), may contribute to protection against influenza . In fact, influenza virus NP-specific CTL’s generated through vaccination or introduced by adoptive transfer enhance viral clearance and lead to recovery of the host and protection from death . The objectives of this study were to determine the level of cross reactive CMI against homologous and heterologous isolates of LPAI.
Two-day-of age chickens were received from the USDA-ARS-Avian Disease Oncology Laboratory (East Lansing, Michigan) containing the B2/B2 defined MHC allele (Line 15.6-2). Birds were placed in Horsfall units in BSL3E facilities (USDA-ARS, Athens, Georgia, USA) and provided feed and water ad libitum.
Low pathogenic AI viruses of H9N2 (A/Chicken/NJ/12220/97) and H7N2 (A/Turkey/Virginia/4259/02) subtypes were propagated in the allantoic cavities of 9-11 day of embryonation SPF chicken eggs. Viral titers were determined as previously described .
Two-week-old chickens (6 per group) were inoculated via the intranasal route with 107 EID50 of H9N2 AI virus or sham inoculum in 0.2 ml volume (1/2 each nare) at 2 weeks of age. Chicken were monitored daily for clinical signs of disease and weight loss monitored at 2, 4 and 7 days post inoculation (p.i.) following H9N2 primary infection. Mean weight was compared by ANOVA using the Tukey Test (SigmaStat).
CTL lysis of target cells
Splenic lymphocytes were harvested from B2/B2 infected and control chickens, three each, at 10 days post- infection as previously described . MHC-matched lung cells were obtained as previously described and used as target cells for CTL assay . Lung cells were infected with either H9N2 or H7N2 AI at an MOI of 2 for 16 hours prior to testing. CTL activity was monitored using the CytoTox96 nonradioactive assay (Promega) with the % cells lysed determined by detection of cytosolic lactate dehydrogenase in supernatants from control and infected target cells.
Lymphocyte proliferation as a measure of cellular memory was performed with almarBlue™ (Invitrogen) as previously described, using BPL-inactivated H9N2 (5 µg/ml) or H7N2 (5 µg/ml) . The T cell mitogen, concanavalin A (5 µg/ml), was used as a positive control.
Weight loss following H9N2 infection
Lysis of homologous and heterologous infected B2/B2lung cells
The main objective of the present research was to examine CMI following infection with low pathogenic AI, and to identify cross reactive T cell immunity in chickens. Using birds with a genetically defined MHC haplotype (B2/B2), we found CTL’s isolated from H9N2 infected birds were active against homologous and heterologous isolates of AI based on lysis of MHC-matched target cells and T cell proliferation. In addition, memory T cells demonstrated proliferation in the presence of either isolate tested. These data suggest that antigenic peptides found in both isolates are presented by MHC-class I and II molecules that lead to production of memory CTL’s and stimulated proliferation of T cells, respectively. The proteins containing these peptides remain to be determined, although at least one report has identified a T cell epitope on the HA protein which could activate both chicken CD4+ and CD8+ cells .
For this study birds infected with a LPAI H9N2 isolate were the source of lymphocytes and examining cross reactivity against a heterologous H7N2 isolate. We provide evidence that the H9N2 AI virus can induce cross reactive CMI against the H7N2 AI isolate. Because the majority of vaccines used across the world to protect poultry from AI are inactivated, they must be matched to the field subtype. While these vaccines induce protective antibodies against homologous subtypes based on HA, little to no protective cross reactivity is afforded against heterologous isolates . In contrast, Seo et al. was able to adaptively transfer CD8+ lymphocytes from H9N2-infected chickens and demonstrate protection from H5N1 lethal challenge . Whether the cross reactive CMI demonstrated here could protect against a H7N2 challenge in vivo, remains to be determined.
Taken together these data extend the findings that CMI, including production of CTL’s, is cross reactive against heterologous isolates of AI infection, and contribute to protection following infection.
The authors acknowledge the expert technical assistance of Aniko Zsak and Cam Greene. In addition we recognize Roger Brock and James Doster for assistance with animal care. This research was supported by USDA, ARS CRIS project 6612-32000-053-00D.
This article has been published as part of BMC Proceedings Volume 5 Supplement 4, 2011: Proceedings of the International Symposium on Animal Genomics for Animal Health (AGAH 2010). The full contents of the supplement are available online at http://www.biomedcentral.com/1753-6561/5?issue=S4.
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