Seeding of the mucosal leukocytes in the HALT and trachea of White Leghorn chickens.

Characterising the immune cells of the head-associated lymphoid tissues (HALT) and trachea during maturation in young birds is critical to understanding the immunological responses to avian respiratory diseases and vaccines. Selected mucosal leukocytes of the conjunctiva-associated lymphoid tissue (CALT), Harderian gland (HG), nasal-associated lymphoid tissue (NALT) and trachea from 4-, 6-, 8-, and 10-week-old chickens were enumerated and phenotyped. HG, NALT, and trachea cellularity increased as the birds aged with cell viability varying by tissue. The results showed that the T cell subset numbers, but not B cell numbers, increased in the mucosal tissues of chickens during aging.

Establishment of Swine Primary Nasal, Tracheal, and Bronchial Epithelial Cell Culture Models for the Study of Influenza Virus Infection.

We established primary porcine nasal, tracheal, and bronchial epithelial cells that recapitulate the physical and functional properties of the respiratory tract and have the ability to fully differentiate. Trans-well cultures demonstrated increased transepithelial electrical resistance over time the presence of tight junctions as demonstrated by immunohistochemistry. The nasal, tracheal, and bronchial epithelial cells developed cilia, secreted mucus, and expressed sialic acids on surface glycoproteins, the latter which are required for influenza A virus infection. Swine influenza viruses were shown to replicate efficiently in the primary epithelial cell cultures, supporting the use of these culture models to assess swine influenza and other virus infection. Primary porcine nasal, tracheal, and bronchial epithelial cell culture models enable assessment of emerging and novel influenza viruses for pandemic potential as well as mechanistic studies to understand mechanisms of infection, reassortment, and generation of novel virus. As swine are susceptible to infection with multiple viral and bacterial respiratory pathogens, these primary airway cell models may enable study of the cellular response to infection by pathogens associated with Porcine Respiratory Disease Complex.

Performance and Stability of New Class of Fetal Bovine Sera (FBS) and Its Lyophilized Form in ELISpot and FluoroSpot Assays: Applications for Monitoring the Immune Response in Vaccine, and Cell and Gene Immunotherapy in Clinical Trials.

Interferon-gamma (IFNγ) ELISpot and FluoroSpot are widely used assays to detect functional cell responses in immunotherapy clinical studies. Recognized for their importance in vaccine development studies to quantitate immune responses, these assays have more recently risen to the forefront in cell and gene therapy as well as cancer immunotherapy fields where responses against cancer neoantigens are not easily detectable above assay background. Here, we test a new class of fetal bovine serum (FBS), CultraPure FBS, in ex vivo ELISpot and FluoroSpot assays and cultured FluoroSpot assays following in vitro expansion. Several CultraPure FBS lots that have been specially formulated through the process of lyophilization (lyo-FBS) were compared to liquid CultraPure FBS. We stimulated human PBMCs with antigen-specific peptide pools diluted in media supplemented with liquid CultraPure FBS or lyo-FBS and found equivalent cytokine production with negligible to no assay background with both liquid and lyo-FBS formats. Moreover, the lyo-FBS showed lot-to-lot consistency and 90-day refrigerated (4 °C) stability in both ex vivo direct and in vitro cultured assays. In addition, we present here a method using lyo-FBS for the expansion of low-frequency antigen-specific T cells, mimicking the low frequency seen with cancer neoantigens by utilizing a cultured FluoroSpot assay. Our results demonstrate the presence of Granzyme B, interferon-gamma (IFNγ), and tumor necrosis factor (TNF) production by antigen-specific polyfunctional T cells following a 9-day culture using media supplemented with lyo-FBS.

Pandemic Risk Assessment for Swine Influenza A Virus in Comparative In Vitro and In Vivo Models.

Swine influenza A viruses pose a public health concern as novel and circulating strains occasionally spill over into human hosts, with the potential to cause disease. Crucial to preempting these events is the use of a threat assessment framework for human populations. However, established guidelines do not specify which animal models or in vitro substrates should be used. We completed an assessment of a contemporary swine influenza isolate, A/swine/GA/A27480/2019 (H1N2), using animal models and human cell substrates. Infection studies in vivo revealed high replicative ability and a pathogenic phenotype in the swine host, with replication corresponding to a complementary study performed in swine primary respiratory epithelial cells. However, replication was limited in human primary cell substrates. This contrasted with our findings in the Calu-3 cell line, which demonstrated a replication profile on par with the 2009 pandemic H1N1 virus. These data suggest that the selection of models is important for meaningful risk assessment.