2The Jenner Institute, Oxford University, Oxford, UK ELUCIDATING SPECIFIC RESPONSES AGAINST INFLUENZA IN VACCINATED AND/OR INFECTED CHICKENS USING ELISPOTS ASSAYS Raul Ruiz-Hernandez1, Connor Carson2, Dave Balkissoon1, Karen Staines1, Debra Clayton1, Sarah Gilbert2, Colin Butter1 1 Institute for Animal Health, Compton, Newbury, Berkshire, RG20 7NN, UK 2The Jenner Institute, Oxford University, Oxford, UK Introduction Specific antiviral responses IFN are produced mainly by T lymphocytes expressing the surface antigens CD4 and CD8. A broadly acknowledged method for measuring the immune specific responses is the IFN ELISPOT assay. Although previous studies have employed this techniques in other avian viral infectious diseases , there are no studies that have quantified the specific responses against avian influenza antigens. Materials and Methods ELISA: Recombinant fusion protein chicken IFNγ- Human IgG was employed to detect binding of antibodies. As a negative controls, another fusion protein (chicken DEc205- Human IgG). Coating and detection antibodies were employed between 1-5 µg/ml. Goat anti Mouse antibodies- HRP/Biotine (Southern Biotech) were employed at dilution 1/2000. Colorimetric reaction was measure in NuncMaxiSorp 96 well plates using tetramethylbenzidine (TMB) with ELISA reader at 450 ηm. ELISPOT: Ficollized cells from chicken spleen or peripheral were culture in complete medium (RPMI with 2mM Glutamax-I, supplemented with 10% foetal calf serum, 100 U/ml penicillin, and 100mg/ml streptomycin). IFN-γ ELISpot was carried out as described previously (Ariaans et al. 2008), using a commercially available kit for detection of chicken IFN- γ protein (Chicken IFN-γ ELISA kit, Invitrogen). Briefly, 96-well plates (MAIPS4510, Millipore) were coated overnight at 4oC with Invitrogen capture or EH9 antibody (2µg/ml) diluted in carbonate/bicarbonate buffer, pH 9.6. Plates were washed with PBS/0.1 or 0.05 % Tween 20 between all subsequent steps. Plates were blocked with complete medium. 5×105 splenocytes per well suspended in complete medium were then added, and incubated for 36h at 41oC in 5% CO2, in the presence of non-specific mitogen (10 µg/ml Concanavalin A) or Np+M1 peptide pools (10µg/ml) in triplicate wells. Depending of coating antibody, ELISpot plates were then incubated either with kit-supplied biotinylated detection antibody (1 µg/ml) followed by the supplied streptavidin-horseradish peroxidise conjugate at a 1:2000 dilution or with detection antibody AF10, followed by biotinilated Goat antiMouse IgG2b antibody (AF10 isotype) and avidine-HRP. Plates were developed by incubation with 3-amino-9-ethylcarbazole (AEC). Spots were counted using an ELISpot plate reader. To measure T-cell responses specific to vaccine antigens, 15- to 20-mer peptides overlapping by 10 amino acid residues of were used in IFN-γ ELISpot assays. Peptides were synthesised commercially (Peptide Protein Research, Cambridge, UK) and supplied in lyophilized form. 80 peptides were synthesised spanning the complete Np + M1 insert, and were used in 3 pools for assays. Peptides were added to cultures at a final concentration of 10µg/ml. ANIMALS VACCINATION AND INFECTION: Primary vaccination was carried out by in ovo inoculation of modified Vaccinia virus Ankara (MVA) and non-replicating human Adenovirus 5 (Ad) vectors expressing a fusion of nucleoprotein (Np) and matrix protein (M1) at 17 days embryos of specific-pathogen-free inbred white leghorn chickens (Line 61). MVA and Adenovirus-vectored constructs expressing green fluorescent protein (GFP) were prepared for use as controls. Secondary vaccination (boost) was carried out by intra-muscular injection 4 weeks after primary vaccination. MVA Np+M1 was administered at 1x107 pfu per dose, whereas Ad Np+M1 was given at 1x109 iu per dose. Challenge infection was carried out 4 weeks after boost vaccination, by intra-nasal inoculation of 1×107 pfu LPAI Influenza virus (A/Duck/Singapore/1997/H5N3). Objectives 1) Develop a technology to quantify the specific responses against avian influenza antigens using pair of specific antibodies (our own and commercial antibodies) against chicken IFN. 2) Elucidate specific responses against influenza antigens in vaccination experiments o infection with low pathogenic avian influenza. RESULTS Structure of the reaction Absorvancies results 1 The pair of our EH9/AF10 antibodies were able to detect chicken IFNγ as demonstrated by ELISA: Fig 1.1: Both AF10 and EH9 antibodies recognized a recombinant IFN-γ protein in ELISA experiments Fig 1.2: The specific binding of AF10 (but not EH9) to the recombinant IFN-γ protein could be detected by ELISA We tested a pair of “home –made” antibodies against chicken IFNγ in ELISA experiment before be used in ELISPOTs assay. Fig 1.1. to 1.3 show every step of validation. Fig 1.3: The pair of antibodies EH9/AF10 were able to detect recombinant IFN-γ protein using sandwich ELISA Both pairs of antibodies, EH9/AF10 and Invitrogen Kit ELISA antibodies, detected the same amount of IFNγ producing cells in ELISPOT assays from cultures of chicken splenocytes. 4 Detection of specific responses from chickens against influenza antigens vaccine and infection experiments through ELISPOTs assays. 2 We evaluated ex vivo the cellular specific immune responses against influenza antigens (pools peptides that covered NP and M proteins) of chicken vaccinate by IFN gamma ELISPOT, before and after infection with low pathogenic avian influenza virus (LPAI).The time points for evaluation were 10 days post vaccination boost ( 1st arrow) and 2 week after infection of vaccinated birds (3rd arrow).1 Week after infection, we compared the specific responses in peripheral blood cells and splenocytes from same birds (2nd arrow). We compared the spots detected form the same samples using the 2 two different pairs of antibodies EH9/AF10 antibodies Invitrogen antibodies Fig 4.1 Virally vectored vaccination against influenza proteins induced specific IFN gamma producing cells on splenocytes of vaccinated chickens. Figure show the net values (minus background) in SFU values of ELISPOTs assays of cells of samples 10 days after boost (5 birds per each group). Both homologous (MVA/MVA or Ad/Ad) and heterologous (MVA/Ad or Ad/MVA) vaccination regimen induced specific responses on birds. Fig 2.1: ELISPOT of chicken splenocytes using EH9/AF10 (left) or Invitrogen (right). Below title of each pair of antibodies is represented the scheme of the ELISPOT reaction. Adjacent photographs show Lower part show the resulting wells No stimulated (only media) and wells stimulated with Concanavalin A (stimulated). Fig 2.2: Number of spots detected (SFU, spot forming unit) in ELISPOTs from the same samples using the pair of antibodies EH9/AF10 and Invitrogen antibodies. Results form a same sample of chicken splenocytes (n=9 per each pair) using different volume of substrate (60-80-100 µl). The volume of substrate didn’t affect the results. Fig 4.2 Comparison of IFN gamma positive cells from spleen and peripheral blood cells from the same birds. This representative samples come from a heterologous vaccinated birds (MVA-Ad) 1 week after infection with LPAI virus. As observed, most of IFN gamma reactive cells are mostly present in splenocytes. And almost any specific IFN reactive cells were present. 3 Calibration ELISPOT for the best reagent and cell culture conditions In order to maximize the detection of specific chicken IFNγ producing cells with our the previous antibodies, we tested different samples, conditions and reagents. Fig 3.1: Titration and testing of several conditions and reagents of ELISPOTs using the pair of antibodies EH9/AF10 and Invitrogen ELISA. We tested substrate reagent tetramethylbenzidine (TMB) and 3-amino-9-ethylcarbazole (AEC)., figure upper left. Different tissue samples as splenocytes or peripheral blood mononuclear cells (PBLs) as method of isolation using extraction of cells with Ficoll of slow centrifugation method (slow spin), figure upper right. Adittionaly, optimum number of cells is each well check (bottom figure). Number underneath figures indicate SFU (spot forming units) Fig 4.3 Specific IFN gamma producing cells in infected birds appeared to be higher on splenocytes of vaccinated birds in comparison with PBS vaccine control birds (left figure, net spots units after sustracting background values of wells without peptides). However, non-specific boosting of IFN-γ ELIspot responses by exposure to LPAI challenge virus was observed in Np+M1 vaccinated birds, as shown by high spot counts in unstimulated control ELIspot wells in these birds (with media only, right figure), significantly higher than in control PBS vaccinated chickens. Conclusion Both EH9/AF10 or Invitrogen commercial antibodies could be employed for IFN gamma ELISPOT assays. Specific IFN responses are detected mostly in splenocytes in comparison with blood mononuclear cells in immunized chickens. Viral-vectored vaccination expressing Np+M1 constructs are immunogenic in chickens, promoting a Type-1 cellular immune response characterized by antigen-specific production of IFN-γ as demonstrated here by ex vivo ELISPOTs. Subsequent exposure to live virus boosted non-specific IFN-γ ELISpot responses in Ad-MVA and MVA-MVA Np+M1 vaccinated birds, but not in controls, confirming vaccine immunogenicity. References: ELISPOT and intracellular cytokine staining: novel assays for quantifying T cell responses in the chicken. Ariaans, M. P. Dev Comp Immunol. 2008;32(11):1398-404. Epub 2008 Jun 11. Acknowledgements: This work was supported by Jenner Institute and BBSRC. Raul Ruiz-Hernandez and Connor Carson have a Jenner grant both contributed equally to this work.