Amplification of the HIV Envelope Expressing Plasma Q842.D16 for the Purpose of Evaluating Human Natural Killer Cell-mediated Killing of HIV Positive Cells
Presenter Type
UNO Undergraduate Student
Major/Field of Study
Biology
Other
Molecular & Biomedical Biology/Medical Humanities
Advisor Information
Paul W. Denton PhD.
Location
MBSC Ballroom Poster # 804 - U
Presentation Type
Poster
Start Date
24-3-2023 10:30 AM
End Date
24-3-2023 11:45 AM
Abstract
Amplification of the HIV Envelope Expressing Plasma Q842.D16 for the Purpose of Evaluating Human Natural Killer Cell-mediated Killing of HIV Positive Cells
Jaden Nienhueser1, Nathan Booher1, Paul W. Denton PhD1
1Department of Biology, University of Nebraska at Omaha
Background: 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 a way to express HIV envelope on the surface of target cells. For this, we needed to logarithmically amplify the small plasmid stock provided by the NIH HIV Reagent Program. To do this, the HIV envelope gene containing (and ampicillin resistance cassette containing) plasmid Q842.D16 was transformed into competent Stbl2 Escherichia coli. Next, we grew transformed bacteria on LB agar plates supplemented with ampicillin. Following incubations, a colony of transformed bacteria was isolated from each plate. The colonies were amplified in LB broth supplemented with ampicillin. The most rapidly growing culture, based upon absorbance at 600 nm (Nanodrop 2000), was carried to the subsequent step of plasmid collection using a midi-prep kit (Zymo # D4200). Restriction enzyme fragmentation and subsequent gel electrophoresis was conducted to confirm the identity of the plasmid.
Results: Our data confirmed that we amplified and purified the Q842.D16 plasmid. Our plasmid yield was ~660 ng/µL in ~3 mL, a quantity sufficient for long term use in the laboratory.
Discussion: The overarching purpose of the processes completed for this abstract was to acquire an abundance of the Q842.D16 plasmid for transfecting potential target cells in our laboratory’s natural killer cell functional assay. We accomplished this goal. The next direction of the research is the focus of the partner poster being presented by Nathan Booher.
Scheduling
9:15-10:30 a.m., 10:45 a.m.-Noon
Amplification of the HIV Envelope Expressing Plasma Q842.D16 for the Purpose of Evaluating Human Natural Killer Cell-mediated Killing of HIV Positive Cells
MBSC Ballroom Poster # 804 - U
Amplification of the HIV Envelope Expressing Plasma Q842.D16 for the Purpose of Evaluating Human Natural Killer Cell-mediated Killing of HIV Positive Cells
Jaden Nienhueser1, Nathan Booher1, Paul W. Denton PhD1
1Department of Biology, University of Nebraska at Omaha
Background: 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 a way to express HIV envelope on the surface of target cells. For this, we needed to logarithmically amplify the small plasmid stock provided by the NIH HIV Reagent Program. To do this, the HIV envelope gene containing (and ampicillin resistance cassette containing) plasmid Q842.D16 was transformed into competent Stbl2 Escherichia coli. Next, we grew transformed bacteria on LB agar plates supplemented with ampicillin. Following incubations, a colony of transformed bacteria was isolated from each plate. The colonies were amplified in LB broth supplemented with ampicillin. The most rapidly growing culture, based upon absorbance at 600 nm (Nanodrop 2000), was carried to the subsequent step of plasmid collection using a midi-prep kit (Zymo # D4200). Restriction enzyme fragmentation and subsequent gel electrophoresis was conducted to confirm the identity of the plasmid.
Results: Our data confirmed that we amplified and purified the Q842.D16 plasmid. Our plasmid yield was ~660 ng/µL in ~3 mL, a quantity sufficient for long term use in the laboratory.
Discussion: The overarching purpose of the processes completed for this abstract was to acquire an abundance of the Q842.D16 plasmid for transfecting potential target cells in our laboratory’s natural killer cell functional assay. We accomplished this goal. The next direction of the research is the focus of the partner poster being presented by Nathan Booher.