CD8 T Cell Virus Inhibition Assay Protocol

Area of Science:

• Immunology
• Virology
• Clinical Research

Background:

• The human immunodeficiency virus (HIV)-1 viral inhibition assay (VIA) assesses CD8+ T cell-mediated inhibition of HIV replication in CD4+ T cells.
• This assay is crucial for clinical testing of HIV vaccines and immunotherapies, but variations exist in protocols and readouts.
• Existing VIA protocols differ in culture duration, CD4+ T cell purity, HIV strains used, and methods for quantifying viral inhibition.

Purpose of the Study:

• To describe modifications to a 7-day VIA protocol to improve assay reproducibility.
• To introduce independent replicate cultures for HIV-infected CD4+ T cells (HIV-CD4) and HIV-CD4:CD8 T cell cultures.
• To identify methodological and analysis changes for enhanced assay performance.

Main Methods:

• Modified a 7-day VIA protocol, emphasizing independent replicate cultures for HIV-CD4 and HIV-CD4:CD8 T cell cultures.
• Quantified virus inhibition using a ratio of weighted averages of p24+ cells in replicate cultures.
• Calculated 95% confidence intervals for inhibition measurements.

Main Results:

• Introduction of independent replicate cultures significantly improved assay reproducibility.
• Virus inhibition by CD8 T cells in people living with HIV (PLWH) on antiretroviral therapy (ART) remained largely stable over time.
• Identified specific methodological and analysis changes applicable to other VIA protocols.

Conclusions:

• The modified VIA protocol enhances reproducibility for assessing CD8+ T cell-mediated HIV inhibition.
• The stability of CD8 T cell inhibition in PLWH on ART supports the assay's utility for evaluating therapeutic interventions.
• These improvements facilitate more reliable clinical testing of HIV vaccines and immunotherapies.