title: Improved quantification of HIV-1-infected CD4+ T cells using an optimised method of intracellular HIV-1 gag p24 antigen detection.
abstract: 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.
authors.affiliation: Oxford NIHR Biomedical Research Centre, University of Oxford, Oxford, UK.
authors.forename: Hongbing
authors.initials: H
authors.lastname: Yang
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Yorke
authors.forename: Gemma
authors.initials: G
authors.lastname: Hancock
authors.forename: Genevieve
authors.initials: G
authors.lastname: Clutton
authors.forename: Nellia
authors.initials: N
authors.lastname: Sande
authors.forename: Brian
authors.initials: B
authors.lastname: Angus
authors.forename: Redmond
authors.initials: R
authors.lastname: Smyth
authors.forename: Johnson
authors.initials: J
authors.lastname: Mak
authors.forename: Lucy
authors.initials: L
authors.lastname: Dorrell
mesh_terms: D015415:Biomarkers / Q000378:metabolism; D018791:CD4 Lymphocyte Count / Q000379:methods*; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology / Q000821:virology*; D002469:Cell Separation / Q000379:methods*; D002478:Cells, Cultured; D003902:Detergents; D004797:Enzyme-Linked Immunosorbent Assay; D005404:Fixatives; D005434:Flow Cytometry; D005557:Formaldehyde; D016655:HIV Core Protein p24 / Q000378:metabolism*; D015658:HIV Infections / Q000175:diagnosis* / Q000276:immunology / Q000821:virology; D015497:HIV-1 / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D000432:Methanol; D017830:Octoxynol; D011092:Polyethylene Glycols; D011108:Polymers; D011237:Predictive Value of Tests; D012680:Sensitivity and Specificity; D013997:Time Factors; D016707:Tissue Fixation
chemical_list: D015415:Biomarkers; D003902:Detergents; D005404:Fixatives; D016655:HIV Core Protein p24; D011108:Polymers; D005557:Formaldehyde; D011092:Polyethylene Glycols; D017830:Octoxynol; C010615:Nonidet P-40; C003043:paraform; D000432:Methanol