Cookies on this website

We use cookies to ensure that we give you the best experience on our website. If you click 'Accept all cookies' we'll assume that you are happy to receive all cookies and you won't see this message again. If you click 'Reject all non-essential cookies' only necessary cookies providing core functionality such as security, network management, and accessibility will be enabled. Click 'Find out more' for information on how to change your cookie settings.

The capacity of CD8+ T cells to inhibit HIV-1 replication in vitro strongly correlates with virus control in vivo. Post-hoc evaluations of HIV-1 vaccine candidates suggest that this immunological parameter is a promising benchmark of vaccine efficacy. Large-scale analysis of CD8+ T cell antiviral activity requires a rapid, robust and economical assay for accurate quantification of HIV-1 infection in primary CD4+ T cells. Detection of intracellular HIV-1 p24 antigen (p24 Ag) by flow cytometry is one such method but it is thought to be less sensitive and quantitative than p24 Ag ELISA. We report that fixation and permeabilisation of HIV-infected cells using paraformaldehyde/50% methanol/Nonidet P-40 instead of a conventional paraformaldehyde/saponin-based protocol improved their detection across multiplicities of infection (MOI) ranging from 10(-2) to 8×10(-5), and by nearly two-fold (p<0.001) at the optimal MOI tested (10(-2)). The frequency of infected cells was strongly correlated with p24 Ag release during culture, thus validating its use as a measure of productive infection. We were also able to quantify infection with a panel of HIV-1 isolates representing the major clades. The protocol described here is rapid and cost-effective compared with ELISA and thus could be a useful component of immune monitoring of HIV-1 vaccines and interventions to reduce viral reservoirs.

Original publication

DOI

10.1016/j.jim.2013.03.001

Type

Journal article

Journal

Journal of immunological methods

Publication Date

05/2013

Volume

391

Pages

174 - 178

Addresses

Oxford NIHR Biomedical Research Centre, University of Oxford, Oxford, UK.

Keywords

CD4-Positive T-Lymphocytes, Cells, Cultured, Humans, HIV-1, HIV Infections, Polyethylene Glycols, Octoxynol, Methanol, Formaldehyde, Polymers, HIV Core Protein p24, Fixatives, Detergents, CD4 Lymphocyte Count, Enzyme-Linked Immunosorbent Assay, Flow Cytometry, Cell Separation, Tissue Fixation, Sensitivity and Specificity, Predictive Value of Tests, Time Factors, Biomarkers