Abstract
Avian influenza virus (AIV) emerged and has continued to re-emerge, continuously posing great threats to animal and human health. The detection of hemagglutination inhibition (HI) or virus neutralization antibodies (NA) is essential for assessing immune protection against AIV. However, the HI/NA-independent immune protection is constantly observed in vaccines’ development against H7N9 subtype AIV and other subtypes in chickens and mammals, necessitating the analysis of the cellular immune response. Here, we established a multi-parameter flow cytometry to examine the innate and adaptive cellular immune responses in chickens after intranasal infection with low pathogenicity H7N9 AIV. This assay allowed us to comprehensively define chicken macrophages, dendritic cells, and their MHC-II expression, NK cells, γδ T cells, B cells, and distinct T cell subsets in steady state and during infection. We found that NK cells and KUL01+ cells significantly increased after H7N9 infection, especially in the lung, and the KUL01+ cells upregulated MHC-II and CD11c expression. Additionally, the percentages and numbers of γδ T cells and CD8 T cells significantly increased and exhibited an activated phenotype with significant upregulation of CD25 expression in the lung but not in the spleen and blood. Furthermore, B cells showed increased in the lung but decreased in the blood and spleen in terms of the percentages or/and numbers, suggesting these cells may be recruited from the periphery after H7N9 infection. Our study firstly disclosed that H7N9 infection induced local and systemic cellular immune responses in chickens, the natural host of AIV, and that the flow cytometric assay developed in this study is useful for analyzing the cellular immune responses to AIVs and other avian infectious diseases and defining the correlates of immune protection.
Highlights
Avian influenza A (H7N9) virus has posed a dual challenge to public health and the poultry industry since its emergence in 2013 in China [1]
We developed a multi-parameter flow cytometry by using a combination of antibodies to chicken markers Bu-1, monocyte/macrophage (KUL01), CD45, major histocompatibility complex class II (MHC-II), CD11c, CD3, CD8α, CD8β, TCRγδ, CD4, and CD25 to comprehensively numerate innate and adaptive immune cell populations in chicken at steady state and after H7N9 infection, leading to an in-depth analysis of the cellular composition and dynamics within lymphoid and non-lymphoid tissues
By outputting forward scatter (FSC) versus side scatter (SSC), lymphocytes were located in a dot-plot (Figure 1F) and Bu-1+ B cells were defined (Figure 1I)
Summary
Avian influenza A (H7N9) virus has posed a dual challenge to public health and the poultry industry since its emergence in 2013 in China [1]. Many vaccine candidates against AIV H7N9 subtype, including inactivated vaccine [4] and HA-carrying recombinant vector vaccine [5,6] have been developed and experimentally shown protection against H7N9 in mice and humans [7]. These vaccines induced a low level of hemagglutination inhibition (HI), and viral neutralizing antibodies (NA) titers against the. H7N9 subtype that was lower than the standard of vaccine evaluation (>24 ) for other influenza A subtypes (e.g., H1N1 and H5N1) [8] and seasonal influenza viruses They were not well correlated with the immune protection conferred by these vaccines [5,6]. In chickens, our previous studies showed that recombinant Newcastle disease virus (NDV) vaccine carrying H7N9 HA gene (rNDV-H7N9 HA) provided complete protection but did not induce a high level of HI and NA [9,10]
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