Expression of HIV Envelope on HEK293T Cells for Subsequent Antibody Dependent Cellular Cytotoxicity Testing
Presenter Type
UNO Undergraduate Student
Major/Field of Study
Biology
Other
Biology
Advisor Information
Dr. Paul W Denton
Location
MBSC Ballroom Poster # 1007 - U
Presentation Type
Poster
Start Date
24-3-2023 1:00 PM
End Date
24-3-2023 2:15 PM
Abstract
Expression of HIV Envelope on HEK293T Cells for Subsequent Antibody Dependent Cellular Cytotoxicity Testing
Nathan Booher1, Jaden Nienhueser1, Paul W. Denton PhD1
1Department of Biology, University of Nebraska at Omaha
Introduction
Human Immunodeficiency virus (HIV) affects nearly 40 million individuals worldwide. While antiretroviral therapy is effective against the virus, this therapy is a lifelong commitment. Thus, there is a strong global effort to develop interventions that are able to, at minimum, functionally cure this infection. Functional cure is when the body’s immune system is trained to control the HIV infection in the absence of ongoing antiretroviral therapy. Thus, multiple strategies are being investigated (e.g., immunotherapies in NCT03837756) to boost anti-HIV immunity. Our question is how such interventions may impact natural killer (NK) cell functions – as NK cells may be a critical component in controlling HIV in the absence of antiretroviral therapy. This project focuses on developing an ex vivo strategy for measuring the impact of relevant immunotherapies on human NK cell killing capacities with a particular focus on NK cell-mediated antibody dependent cellular cytotoxicity (ADCC).
Methods
To accomplish our goals, we need target cells for NK cells to kill. The target cells need to express HIV envelope that can be bound by an ADCC-inducing antibody (e.g., 3BNC117). We are utilizing the plasmid Q842.D16 to transfect HEK293T cells causing the cells to express HIV-1 envelope (gp160). We are using a non-ADCC antibody that does not compete with the ADCC antibody for a binding site (e.g., PG9) to identify HIV-Env positive target cells to measure their death in a killing assay. To make PG9 useful in this context, we labeled the antibody with a fluorophore that can be detected in the flow cytometer used to measure cell killing in our assay. The HIV Reagent Program graciously provided Q842.D16, PG9, and 3BNC117.
Results
Using a Nanodrop 2000, unlabeled PG9 was determined to have a protein concentration of 3.1E-4 mM and a degree of labeling (DOL) of 8.36 units. The DOL of PG9 was in the acceptable range (4-9 units). Initial target cell labeling experiments were encouraging. However, subsequent data suggest that PG9 is non-specifically binding to on the surface of 293T cells. Next steps are to find an appropriate blocking strategy for this step in the procedure such that we have reproducible target cell recognition in the killing assay.
Discussion
Despite the discouraging setback in target cells recognition, we are optimistic that this technical challenge will be overcome. For example, we are investigating an alternative strategy of identifying HIV-Env positive cells by having the envelope expression vector also express green fluorescent protein to bypass the need for a second antibody altogether. Details about this concept will also be presented in the poster. The work in this poster builds on the initial steps of the process described in the partner poster presented by Jaden Nienhueser.
Scheduling
9:15-10:30 a.m., 10:45 a.m.-Noon, 1-2:15 p.m., 2:30 -3:45 p.m.
Expression of HIV Envelope on HEK293T Cells for Subsequent Antibody Dependent Cellular Cytotoxicity Testing
MBSC Ballroom Poster # 1007 - U
Expression of HIV Envelope on HEK293T Cells for Subsequent Antibody Dependent Cellular Cytotoxicity Testing
Nathan Booher1, Jaden Nienhueser1, Paul W. Denton PhD1
1Department of Biology, University of Nebraska at Omaha
Introduction
Human Immunodeficiency virus (HIV) affects nearly 40 million individuals worldwide. While antiretroviral therapy is effective against the virus, this therapy is a lifelong commitment. Thus, there is a strong global effort to develop interventions that are able to, at minimum, functionally cure this infection. Functional cure is when the body’s immune system is trained to control the HIV infection in the absence of ongoing antiretroviral therapy. Thus, multiple strategies are being investigated (e.g., immunotherapies in NCT03837756) to boost anti-HIV immunity. Our question is how such interventions may impact natural killer (NK) cell functions – as NK cells may be a critical component in controlling HIV in the absence of antiretroviral therapy. This project focuses on developing an ex vivo strategy for measuring the impact of relevant immunotherapies on human NK cell killing capacities with a particular focus on NK cell-mediated antibody dependent cellular cytotoxicity (ADCC).
Methods
To accomplish our goals, we need target cells for NK cells to kill. The target cells need to express HIV envelope that can be bound by an ADCC-inducing antibody (e.g., 3BNC117). We are utilizing the plasmid Q842.D16 to transfect HEK293T cells causing the cells to express HIV-1 envelope (gp160). We are using a non-ADCC antibody that does not compete with the ADCC antibody for a binding site (e.g., PG9) to identify HIV-Env positive target cells to measure their death in a killing assay. To make PG9 useful in this context, we labeled the antibody with a fluorophore that can be detected in the flow cytometer used to measure cell killing in our assay. The HIV Reagent Program graciously provided Q842.D16, PG9, and 3BNC117.
Results
Using a Nanodrop 2000, unlabeled PG9 was determined to have a protein concentration of 3.1E-4 mM and a degree of labeling (DOL) of 8.36 units. The DOL of PG9 was in the acceptable range (4-9 units). Initial target cell labeling experiments were encouraging. However, subsequent data suggest that PG9 is non-specifically binding to on the surface of 293T cells. Next steps are to find an appropriate blocking strategy for this step in the procedure such that we have reproducible target cell recognition in the killing assay.
Discussion
Despite the discouraging setback in target cells recognition, we are optimistic that this technical challenge will be overcome. For example, we are investigating an alternative strategy of identifying HIV-Env positive cells by having the envelope expression vector also express green fluorescent protein to bypass the need for a second antibody altogether. Details about this concept will also be presented in the poster. The work in this poster builds on the initial steps of the process described in the partner poster presented by Jaden Nienhueser.