Clinical Evaluation of Amplicor HIV-1 Test for Detection of Human Immunodeficiency Virus Type 1 Proviral DNA in Peripheral Blood Mononuclear Cells

Khadir, A; Coutlée, François; Saint-Antoine, Pierre; Olivier, C; Voyer, Hélène; Kessous-Elbaz, Allégria

doi:10.1097/00042560-199507000-00006

Cited by 15 publications

(13 citation statements)

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“…We therefore repeated all time points with this approach, using the three peptide pools that stimulated the highest frequencies of IFN-␥-producing T cells in our initial studies. Only Env 151-175 was recognized at more than one time point (32,38, and 65 mo) and by both CD4 ϩ and CD8 ϩ T cells (Fig. 6, b and c).…”

Section: Risk Behavior and Hiv-1-specific T Cell Responses Over Timementioning

confidence: 89%

“…A medical history, physical examination, complete blood cell count, T cell subset analysis, and HIV-1 testing were performed at the screening visit to confirm eligibility. HIV-1 infection was excluded by the following tests performed at the screening visit and on study day 0: HIV-1/2 ELISA and Western blot (37), HIV-1 plasma RNA RT-PCR (Amplicor HIV-1 Monitor; Roche, Branchburg, NJ) (38), and PBMC DNA PCR (39). HIV-1/2 ELISA and Western blot were repeated every 3 mo.…”

Section: Study Populationmentioning

confidence: 99%

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Most Highly Exposed Seronegative Men Lack HIV-1-Specific, IFN-γ-Secreting T Cells

Hladik¹,

Desbien²,

Lang³

et al. 2003

The Journal of Immunology

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Naturally acquired cellular immunity in individuals who have been exposed to HIV-1 but have remained uninfected may hold clues for the design of an effective HIV vaccine. To determine the presence and nature of such an HIV-1-specific immune response, we evaluated the quantity and fine specificity of HIV-1-reactive IFN-γ-secreting T cells in a group of highly exposed seronegative men having sex with men. All 46 ES reported frequent unprotected anal sex with known HIV-1-infected partners at enrollment, and high risk activities continued in at least one-half of the volunteers for up to >6 years of observation. Despite the high frequency of unprotected anal intercourse and potential HIV-1 exposure, the vast majority of individuals demonstrated no or very low numbers of HIV-1-specific, IFN-γ-secreting T cells. Even when HIV-1 epitopes were presented by peptide-pulsed autologous dendritic cells in 15 of the highest risk volunteers, HIV-1-specific T cells remained infrequent, and the proportion of responders was not significantly different from that in a lower risk seronegative control cohort. Only PBMC from two individuals who have remained uninfected to date exhibited distinctly positive responses. However, these responses rarely persisted over time, single epitope specificities were identified in only one volunteer, and HIV-1-specific memory T cell clones did not expand in vitro. HIV-1-specific, IFN-γ-secreting T cells are thus unlikely to substantially contribute to resistance against infection in most exposed seronegative men having sex with men.

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Section: Risk Behavior and Hiv-1-specific T Cell Responses Over Timementioning

confidence: 89%

Section: Study Populationmentioning

confidence: 99%

Most Highly Exposed Seronegative Men Lack HIV-1-Specific, IFN-γ-Secreting T Cells

Hladik¹,

Desbien²,

Lang³

et al. 2003

The Journal of Immunology

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“…We therefore developed a semiquantitative assay using the Amplicor(TM) HIV-1 qualitative test. This standardized colorimetric PCR assay has been shown to perform well in terms of sensitivity, specificity, and technical flexibility [Whetsell et al, 1992;Khadir et al, 1995;Kovacs et al, 1995;Pane et al, 1995;Sethoe et al, 1995]. The accuracy of our limiting dilution method was checked by using serial dilutions of LAV-8E5 cells, each containing one copy of HIV-1 provirus per cell.…”

Section: Discussionmentioning

confidence: 99%

“…A colorimetric PCR-based assay in kit form, the Amplicor(TM) HIV-1 qualitative test (Roche Diagnostic Systems, Neuilly, France), has been used recently to detect HIV-1 proviral gag sequences in peripheral blood mononuclear cells (PBMC) with good analytical results [Whetsell et al, 1992;Khadir et al, 1995;Kovacs et al, 1995;Pane et al, 1995;Sethoe et al, 1995]. We have used the Amplicor(TM) kit to develop a simple semiquantitative technique for measuring HIV-1 proviral DNA in mononuclear cells, which is based on the limiting dilution method.…”

Section: Introductionmentioning

confidence: 99%

Quantification of HIV-1 proviral DNA by a standardized colorimetric PCR-based assay

et al. 1998

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A simple method was developed for measuring human immunodeficiency virus type 1 (HIV-1) proviral DNA in mononuclear cells based on the commercially available Amplicor(TM) HIV-1 polymerase chain reaction (PCR) assay and the limiting dilution method. The lowest limit of detection was four proviral genomes per 10(6) cells. The accuracy was demonstrated by using serial dilutions of LAV-8E5 cells, and the interassay variability was 0.2 log. The technique was used to measure HIV-1 proviral DNA in the peripheral blood mononuclear cells (PBMC) of 18 antiretroviral drug-naive HIV-1-positive individuals before and 4 weeks after initiating double nucleoside therapy. The DNA proviral titers at baseline (median = 3.45, range = 2.11-4.7 log copies/10(6) cells) were 2.08 log greater than the infectivity titers, but there was a correlation between these two parameters (r = 0.63, P = 0.009). The mean decrease in the proviral DNA titer after 4 weeks of therapy was 0.31 log, whereas the decrease in the infectivity titer was 0.81 log and the decrease in the plasma RNA concentration was 1.29 log. The technique was also used to measure HIV-1 proviral DNA in the PBMC of 11 patients who had undetectable plasma HIV-1 RNA after being placed on combination antiretroviral therapy. Although proviral DNA remained detectable in all patients after 36 weeks of treatment, a gradual decline with an estimated half-life of 21-58 weeks was observed. The reliability of this simple and convenient colorimetric PCR-based technique indicates its suitability for assessing the effect of current antiretroviral regimens on the latent reservoirs of provirus.

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“…A medical history, physical examination, complete blood cell count, T-cell subset analysis, and HIV-1 testing were done at the screening visit to confirm eligibility. HIV-1 infection was excluded by the following tests done at the screening visit and study day 0: HIV-1/2 enzyme-linked immunosorbent assay (ELISA) and Western blot (50), HIV-1 plasma RNA reverse transcription-PCR (Amplicor HIV-1 Monitor; Roche Molecular Systems) (19), and peripheral blood mononuclear cell (PBMC) DNA PCR (41). HIV-1/2 ELISA and Western blot were repeated every 3 months, and HIV-1 PBMC DNA PCR was performed again 4 weeks and 8 weeks after enrollment and then repeated yearly.…”

Section: Methodsmentioning

confidence: 99%

Combined Effect of CCR5-Δ32 Heterozygosity and the CCR5 Promoter Polymorphism −2459 A/G on CCR5 Expression and Resistance to Human Immunodeficiency Virus Type 1 Transmission

Hladik

Liu²,

Speelmon

et al. 2005

J Virol

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Exposed seronegative individuals (ES) with persistent high-risk sexual behavior may be less susceptible to human immunodeficiency virus type 1 (HIV-1) infection because they carry the chemokine receptor (CR) gene alleles CCR5 open reading frame (ORF) ⌬32, CCR5 promoter ؊2459G, or CCR2 ORF 64I (CCR2-64I), all of which have been found to diminish HIV-1 infectivity and/or disease progression. To investigate this, we determined the haplotypes for these three genetic loci in 93 ES and 247 low-risk control individuals. To test if protective haplotypes exert their effect by modulating CR expression, we measured the protein expression of CCR5 and CXCR4 on circulating CD4؉ T cells and CD14 ؉ monocytes in 71 ES and 92 controls. To avoid investigator bias, the analysis was performed without knowledge of each subject's risk and genotype. The CCR5 ؊2459G allele was significantly enriched in ES Caucasian men, who constituted the majority (84%) of the ES cohort, compared to the control Caucasian men (P ‫؍‬ 0.02). This increase was mostly attributable to a higher frequency of the ؊2459 A/G versus the ؊2459 A/A genotype in individuals heterozygous for the ⌬32 allele (P ‫؍‬ 0.012). No protective influence of the CCR2-64I allele was observed. The haplotypes CCR5 ORF ⌬32/CCR5 ؊2459A (in complete linkage disequilibrium) and CCR5 ORF wt/CCR5 ؊2459G had a cumulative negative effect on the expression of CCR5, since we measured significantly reduced CCR5 densities on both T-helper cells and monocytes only when both haplotypes were present. Densities of CCR5 on lymphocytes and monocytes were correlated (r ‫؍‬ 0.59; P < 0.0001), indicating concordance of CCR5 expression patterns across different cell types. We conclude that the CCR5 ORF ⌬32/wt-CCR5 ؊2459 A/G genotype combination offers an advantage in resisting sexual HIV-1 transmission and that this effect is mediated by a relative paucity of CCR5 on potential target cells of HIV-1.

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Clinical Evaluation of Amplicor HIV-1 Test for Detection of Human Immunodeficiency Virus Type 1 Proviral DNA in Peripheral Blood Mononuclear Cells

Cited by 15 publications

References 0 publications

Most Highly Exposed Seronegative Men Lack HIV-1-Specific, IFN-γ-Secreting T Cells

Most Highly Exposed Seronegative Men Lack HIV-1-Specific, IFN-γ-Secreting T Cells

Quantification of HIV-1 proviral DNA by a standardized colorimetric PCR-based assay

Combined Effect of CCR5-Δ32 Heterozygosity and the CCR5 Promoter Polymorphism −2459 A/G on CCR5 Expression and Resistance to Human Immunodeficiency Virus Type 1 Transmission

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