Development and Evaluation of an Antibody-Dependent Cellular Cytotoxicity (ADCC) Assay for Influenza a Virus

• Main Author: Mehta, Dhwani
• Format: Dissertation
• Language: English
• Published: ProQuest Dissertations & Theses 01-01-2020
• Subjects: Immunology
• ISBN: 9798597090245

Influenza is a global pathogen of major public health impact. Despite the availability of vaccines for seasonal influenza, there are still 3-5 million severe cases of influenza globally each year. Therefore, understanding the mechanisms that the host utilizes to defend and kill the influenza virus is essential. An important host mechanism that has been associated with protection in animal models is Antibody-Dependent Cellular Cytotoxicity (ADCC). ADCC is mediated via innate immune cells that lyse the infected target cells, clearing the infection. There are several in-vitro assays that have been developed to measure influenza-specific ADCC. Most of these assays employ an endpoint that measures a surrogate marker of cell lysis. The major goal of this project was to develop and optimize an ADCC assay for Influenza A virus (IAV) whose endpoint quantification is cellular death due to cytotoxicity, and which can also distinguish responses to the Haemagglutinin (HA) and Neuraminidase (NA) components of 2019-2020 circulating strains. Codon-optimized HA and NA genes were designed, synthesized, and cloned into expression systems allowing stable, inducible expression in a mammalian cell line. An additional reporter construct expressing firefly luciferase and GFP was also stably introduced into the target cells. Robust total and cell surface expression of HA and NA along with luciferase expression was documented upon induction with doxycycline. An in-vitro assay for ADCC was then developed using an immortalized human NK cell line that is mixed with HA or NA-expressing target cells at given effector: target ratios, with lysis measured as the loss of luciferase signal compared to control cells. We next evaluated sera from influenza vaccine recipients in this assay. H1 and H3-specific ADCC responses were readily observed with these sera. N1-specific responses were detectable but appeared weaker in general than either H1 or H3-specific response. Evaluation with additional sera gathered from clinical trials is underway to validate the utility of this assay.