Human immunodeficiency virus type 1 (HIV-1) proviral DNA load in purified CD4+ cells by LightCycler real-time PCR.
BACKGROUND
The human immunodeficiency virus type 1 (HIV-1) proviral DNA persists in infected cells, even after prolonged successful HAART. In the present study, a relative quantification assay of HIV-1 proviral DNA by LightCycler real-time PCR based on SYBR Green I detection was developed in comparison to the number of purified CD4+ cells as estimated by the quantification of the beta-globin gene.


METHODS
The ability of the designed gag primers to quantify HIV-1 Group M and the PCR efficiency were assessed on HIV-1 reference isolate subtypes A, B, C and D. The 8E5 cell line containing a single defective copy of HIV-1 proviral DNA was used as a standard for both the HIV-1 target gene and the beta-globin reference gene. The assay was applied on thirty consecutive patient samples received for RNA viral load determinations and on retrospective samples from fifteen patients undergoing 2 years of structured treatment interruption (STI).


RESULTS
The lower limit of quantification was 50 HIV-1 DNA proviral copies per CD4+ cell sample. The dynamic range was from 50 to 106 HIV-1 DNA copies per CD4+ cell sample with intra- and inter-assay coefficients of variability ranging from 3.1% to 37.1%. The beta-globin reference gene was quantified down to a limit of 1.5 pg of DNA/microl (approximately 5 cells) with intra- and interassay coefficients of variability ranging from 1.8% to 21%. DNA proviral load varies widely among HIV-1 infected patients. Proviral load and plasma viral load rebound were high in STI patients who took longer to achieve an undetectable plasma viral load under therapy. A statistically significant correlation was observed between DNA proviral load and RNA steady state viral load in STI patients (p-value = 0.012).


CONCLUSIONS
We have developed a fast, sensitive and specific relative quantification assay of HIV-1 proviral DNA in purified CD4+ cells. The assay enables the monitoring of HIV-1 proviral load, which may be useful to monitor therapy efficacy especially in patients with undetectable plasma RNA viral load, and allows the exploration of viral reservoirs.
Kabamba-Mukadi
Benoît
B
Laboratoire de référence SIDA, Université Catholique de Louvain, Avenue Hippocrate 54, B1200 Bruxelles, Belgium. Benoit.Kabamba@mblg.ucl.ac.be
Henrivaux
Philippe
P
Ruelle
Jean
J
Delferrière
Nicole
N
Bodéus
Monique
M
Goubau
Patrick
P
D000328:Adult; D023241:Antiretroviral Therapy, Highly Active; D015496:CD4-Positive T-Lymphocytes / Q000821:virology*; D002460:Cell Line; D004279:DNA, Viral / Q000032:analysis* / Q000235:genetics; D005260:Female; D015658:HIV Infections / Q000821:virology*; D015497:HIV-1 / Q000145:classification; D006801:Humans; D008297:Male; D016133:Polymerase Chain Reaction / Q000295:instrumentation* / Q000379:methods; D011533:Proviruses / Q000235:genetics* / Q000302:isolation & purification*; D012367:RNA, Viral / Q000097:blood; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D019562:Viral Load
D004279:DNA, Viral; D012367:RNA, Viral