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J Virol. 2016 Jun 29. pii: JVI.01001-16. [Epub ahead of print]
Flow Cytometric and Cytokine ELISpot Approaches to Characterize the Cell-mediated Immune Response in Ferrets Following Influenza Virus Infection.
DiPiazza A1, Richards K1, Batarse F1, Lockard L1, Zeng H2, Garc?a-Sastre A3, Albrecht RA4, Sant AJ5.
Author information
Abstract
Influenza virus infections represent a significant socioeconomic and public health burden worldwide. Although ferrets are considered by many to be ideal for modeling human responses to influenza infection and vaccination, efforts to understand the cellular immune response have been severely hampered by a paucity of standardized procedures and reagents. In this report, we developed flow cytometric and T cell ELISpot approaches to characterize the leukocyte composition and antigen-specific T cell response within key lymphoid tissues following influenza virus infection in ferrets. Through a newly designed and implemented set of serological reagents, we used multi-parameter flow cytometry to directly quantify the frequency of CD4+ and CD8+ T cells, Ig+ B cells, CD11b+ myeloid-derived cells and MHC class II+ APCs both prior to and after intranasal infection with A/California/04/09 (H1N1). We found that the leukocyte composition was altered 10 days post-infection, with notable gains in the frequency of T cells and myeloid cells within the draining lymph node. Furthermore, these studies revealed that the antigen-specificity of influenza-reactive CD4 and CD8 T cells was very broad, with recognition of the viral HA, NA, M1, NS1 and NP proteins and that total reactivity to influenza post-infection represented approximately 0.1% of the circulating PBMCs. Finally, we observed distinct patterns of reactivity between individual animals, suggesting heterogeneity at the MHC locus in ferrets within commercial populations, a finding of considerable interest in efforts to move the ferret model forward for influenza vaccine and challenge studies.
IMPORTANCE:
Ferrets are an ideal animal model to study transmission, disease and vaccine efficacies of respiratory viruses because of their close anatomical and physiological resemblances to humans. However, a lack of reagents has limited our understanding of the cell-mediated immune response following infection and vaccination. In this study, we used cross-reactive and ferret-specific antibodies to study the leukocyte composition and antigen specific CD4 and CD8 T cell responses following influenza A/California/04/09 (H1N1) virus infection. These studies revealed strikingly distinct patterns of reactivity between CD4 and CD8 T cells, which was overlayed with differences in protein-specific responses between individual animals. Our results provide a first, in-depth look at the T cell repertoire in response to influenza infection and suggest there is considerable heterogeneity at the MHC locus, akin to humans and an area of intense research interest.
Copyright ? 2016, American Society for Microbiology. All Rights Reserved.
PMID: 27356897 DOI: 10.1128/JVI.01001-16
[PubMed - as supplied by publisher]
Flow Cytometric and Cytokine ELISpot Approaches to Characterize the Cell-mediated Immune Response in Ferrets Following Influenza Virus Infection.
DiPiazza A1, Richards K1, Batarse F1, Lockard L1, Zeng H2, Garc?a-Sastre A3, Albrecht RA4, Sant AJ5.
Author information
Abstract
Influenza virus infections represent a significant socioeconomic and public health burden worldwide. Although ferrets are considered by many to be ideal for modeling human responses to influenza infection and vaccination, efforts to understand the cellular immune response have been severely hampered by a paucity of standardized procedures and reagents. In this report, we developed flow cytometric and T cell ELISpot approaches to characterize the leukocyte composition and antigen-specific T cell response within key lymphoid tissues following influenza virus infection in ferrets. Through a newly designed and implemented set of serological reagents, we used multi-parameter flow cytometry to directly quantify the frequency of CD4+ and CD8+ T cells, Ig+ B cells, CD11b+ myeloid-derived cells and MHC class II+ APCs both prior to and after intranasal infection with A/California/04/09 (H1N1). We found that the leukocyte composition was altered 10 days post-infection, with notable gains in the frequency of T cells and myeloid cells within the draining lymph node. Furthermore, these studies revealed that the antigen-specificity of influenza-reactive CD4 and CD8 T cells was very broad, with recognition of the viral HA, NA, M1, NS1 and NP proteins and that total reactivity to influenza post-infection represented approximately 0.1% of the circulating PBMCs. Finally, we observed distinct patterns of reactivity between individual animals, suggesting heterogeneity at the MHC locus in ferrets within commercial populations, a finding of considerable interest in efforts to move the ferret model forward for influenza vaccine and challenge studies.
IMPORTANCE:
Ferrets are an ideal animal model to study transmission, disease and vaccine efficacies of respiratory viruses because of their close anatomical and physiological resemblances to humans. However, a lack of reagents has limited our understanding of the cell-mediated immune response following infection and vaccination. In this study, we used cross-reactive and ferret-specific antibodies to study the leukocyte composition and antigen specific CD4 and CD8 T cell responses following influenza A/California/04/09 (H1N1) virus infection. These studies revealed strikingly distinct patterns of reactivity between CD4 and CD8 T cells, which was overlayed with differences in protein-specific responses between individual animals. Our results provide a first, in-depth look at the T cell repertoire in response to influenza infection and suggest there is considerable heterogeneity at the MHC locus, akin to humans and an area of intense research interest.
Copyright ? 2016, American Society for Microbiology. All Rights Reserved.
PMID: 27356897 DOI: 10.1128/JVI.01001-16
[PubMed - as supplied by publisher]