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An infected chicken kidney cell co-culture ELISpot for enhanced detection of T cell responses to avian influenza and vaccination.

Ruiz-Hernandez R, Peroval M, Boyd A, Balkissoon D, Staines K, Smith A, Butter C - J. Immunol. Methods (2014)

Bottom Line: We have developed a method incorporating infected chicken kidney cells (CKC) in culture with splenocytes in an IFNγ ELISpot assay to enumerate ex vivo responses against influenza virus antigens.Splenocytes from birds challenged with influenza showed specific responses to the influenza virus, with responding cells being mainly CD8 positive.The utility of the assay was also demonstrated in the detection of an antigen specific enhancement of IFNγ producing cells from birds vaccinated with recombinant Fowlpox vectored influenza nucleoprotein and matrix protein.

View Article: PubMed Central - PubMed

Affiliation: The Pirbright Institute, Compton Laboratory, Compton, Newbury, Berkshire RG20 7NN, UK.

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IFNγ co-culture ELISpot using CKC infected with recombinant MVA and splenocytes from birds challenged with LPAI. ELISpot responses in splenocytes from infected birds from unvaccinated (PBS group, n = 3) or birds vaccinated with a recombinant vaccine Fowlpox (F9 group, n = 4), or FowlpoxNpM1 (F9-NpM1 group, n = 4). Splenocytes were cultured with either irradiated MVAGFP infected CKC (black dot) or irradiated MVANpM1 (white circle) infected CKC. Lines indicate samples from the same spleen under the two previous conditions. Results were expressed as spot forming units (SFU) per million cells. Significance of the data is represented as follows: * = p < 0.05.
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f0025: IFNγ co-culture ELISpot using CKC infected with recombinant MVA and splenocytes from birds challenged with LPAI. ELISpot responses in splenocytes from infected birds from unvaccinated (PBS group, n = 3) or birds vaccinated with a recombinant vaccine Fowlpox (F9 group, n = 4), or FowlpoxNpM1 (F9-NpM1 group, n = 4). Splenocytes were cultured with either irradiated MVAGFP infected CKC (black dot) or irradiated MVANpM1 (white circle) infected CKC. Lines indicate samples from the same spleen under the two previous conditions. Results were expressed as spot forming units (SFU) per million cells. Significance of the data is represented as follows: * = p < 0.05.

Mentions: Three of the four F9-NpM1 vaccinated birds challenged with influenza showed IFNγ responses that distinguished them from F9 vaccinated and challenged birds (Fig. 5) (40.0 ± 12.5 vs. 3.0 ± 1.9, p < 0.05). The majority of responses in the F9-NpM1 vaccinated birds were greater with CKC infected with MVA-NpM1 fusion transgene. Some responses were also observed with F9-NpM1 vaccinated birds when APCs were infected with MVA-GFP (although this result was not significant). Our results with vaccinated birds thus suggest that this technique can be used to evaluate vaccine induced responses, and additionally that recombinant viruses expressing appropriate transgenes can be used to replace influenza infection of CKC. This permits the analysis of more defined antigen specific responses while reducing the requirement to handle live influenza virus in the laboratory.


An infected chicken kidney cell co-culture ELISpot for enhanced detection of T cell responses to avian influenza and vaccination.

Ruiz-Hernandez R, Peroval M, Boyd A, Balkissoon D, Staines K, Smith A, Butter C - J. Immunol. Methods (2014)

IFNγ co-culture ELISpot using CKC infected with recombinant MVA and splenocytes from birds challenged with LPAI. ELISpot responses in splenocytes from infected birds from unvaccinated (PBS group, n = 3) or birds vaccinated with a recombinant vaccine Fowlpox (F9 group, n = 4), or FowlpoxNpM1 (F9-NpM1 group, n = 4). Splenocytes were cultured with either irradiated MVAGFP infected CKC (black dot) or irradiated MVANpM1 (white circle) infected CKC. Lines indicate samples from the same spleen under the two previous conditions. Results were expressed as spot forming units (SFU) per million cells. Significance of the data is represented as follows: * = p < 0.05.
© Copyright Policy - CC BY
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4334094&req=5

f0025: IFNγ co-culture ELISpot using CKC infected with recombinant MVA and splenocytes from birds challenged with LPAI. ELISpot responses in splenocytes from infected birds from unvaccinated (PBS group, n = 3) or birds vaccinated with a recombinant vaccine Fowlpox (F9 group, n = 4), or FowlpoxNpM1 (F9-NpM1 group, n = 4). Splenocytes were cultured with either irradiated MVAGFP infected CKC (black dot) or irradiated MVANpM1 (white circle) infected CKC. Lines indicate samples from the same spleen under the two previous conditions. Results were expressed as spot forming units (SFU) per million cells. Significance of the data is represented as follows: * = p < 0.05.
Mentions: Three of the four F9-NpM1 vaccinated birds challenged with influenza showed IFNγ responses that distinguished them from F9 vaccinated and challenged birds (Fig. 5) (40.0 ± 12.5 vs. 3.0 ± 1.9, p < 0.05). The majority of responses in the F9-NpM1 vaccinated birds were greater with CKC infected with MVA-NpM1 fusion transgene. Some responses were also observed with F9-NpM1 vaccinated birds when APCs were infected with MVA-GFP (although this result was not significant). Our results with vaccinated birds thus suggest that this technique can be used to evaluate vaccine induced responses, and additionally that recombinant viruses expressing appropriate transgenes can be used to replace influenza infection of CKC. This permits the analysis of more defined antigen specific responses while reducing the requirement to handle live influenza virus in the laboratory.

Bottom Line: We have developed a method incorporating infected chicken kidney cells (CKC) in culture with splenocytes in an IFNγ ELISpot assay to enumerate ex vivo responses against influenza virus antigens.Splenocytes from birds challenged with influenza showed specific responses to the influenza virus, with responding cells being mainly CD8 positive.The utility of the assay was also demonstrated in the detection of an antigen specific enhancement of IFNγ producing cells from birds vaccinated with recombinant Fowlpox vectored influenza nucleoprotein and matrix protein.

View Article: PubMed Central - PubMed

Affiliation: The Pirbright Institute, Compton Laboratory, Compton, Newbury, Berkshire RG20 7NN, UK.

Show MeSH
Related in: MedlinePlus