Flow Cytometric and Cytokine ELISpot Approaches To Characterize the Cell-Mediated Immune Response in Ferrets following Influenza Virus Infection

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 study, we developed flow cytometric and T cell enzyme-linked immunosorbent spot (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 multiparameter flow cytometry to directly quantify the frequency of CD4؉ and CD8 ؉ T cells, Ig ؉ B cells, CD11b ؉ myeloid-derived cells, and major histocompatibility complex (MHC) class II-positive antigen-presenting cells (APCs) both prior to and after intranasal infection with A/California/ 04/09 (H1N1). We found that the leukocyte composition was altered at 10 days postinfection, 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 virus-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 virus postinfection represented approximately 0.1% of the circulating peripheral blood mononuclear cells (PBMC). 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. IMPORTANCEFerrets are an ideal animal model to study transmission, diseases, 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 were overlaid with differences in protein-specific responses between individual animals. Our results provide a first, indepth look at the T cell repertoire in response to influenza infection and suggest that there is considerable heterogeneity at the MHC locus, which is akin to that in humans and an area of intense research interest.

The immune correlates of protection for an avian influenza H5N1 vaccine in the ferret model using oil-in-water adjuvants

“…The staining and gating strategy is provided as Supplemental Figure 1. CD8 + T-cells were identified by gating on the CD8 + population with the highest mean fluorescence intensity (MFI) as this population had been shown to be the true CD8 + T-cell population15. In gating for the CD4 + T-cells, we had to account for the unexpectedly high background fluorescence of the IFNγ-PE and CD8-APC into the FITC-channel (see Supplemental Figure 1).…”

“…We did not detect any significant CD4 + T-cell numbers in the BALF and the numbers were highly varied in the peripheral blood, particularly for the ferrets in the naïve group with more severe disease. A study published recently15 reported an average of 40% CD4 + and 15% CD8 + T-cells in the BALF of two ferrets infected with the H1N1 virus at day 10 post-infection. Whether the early time-point and the rapidly declining health of our ferrets, or a suboptimal detection antibody, confounded our ability to reliably detect the CD4 + T-cells, is uncertain.…”

Improving immunological insights into the ferret model of human viral infectious disease

“…In addition, an infiltration of CD11b+ and MHC‐II+ cells into lungs between 2 and 5 d.p.i was observed, which could represent lung APCs . CD11b+ cells also increase in secondary lymphoid organs 10 d.p.i, suggesting a coordinated immune response involving both innate and adaptive responses to resolve infection.…”

“…In the absence of cross‐reactive clones, several groups have generated novel monoclonal antibodies specific for ferret cellular markers. For example, novel anti‐ferret CD4‐, CD8‐ and CD5‐specific antibodies were derived by immunising mice with the CD4 ectodomain, whole CD4 protein or ferret thymocytes . Efforts to develop novel antibody‐based reagents to define various ferret immune cell subpopulations are accelerating, particularly through the Centers of Excellence for Influenza Research and Surveillance (CEIRS) network, and are reviewed in detail further below.…”

“…Using existing reagents, innate and adaptive immune responses to influenza infection and immunisation have been studied and provide important insights into ferret antiviral immunity. Following influenza challenge of naïve or vaccinated ferrets, there is a transient increase in CD11b+ expressing PBMCs at 2 d.p.i, which decreased to baseline levels by 3 d.p.i, suggesting a role for monocytes/macrophages/neutrophils or antigen‐presenting cells (APC) during early infection .…”