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Human immunodeficiency virus type 1 (HIV-1) proviral DNA load in purified CD4+ cells by LightCycler real-time PCR.

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Kabamba-Mukadi, Benoît [UCL] Henrivaux, Philippe Ruelle, Jean [UCL] Delferriere, Nicole [UCL] Bodéus, Monique [UCL] Goubau, Patrick [UCL]

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). CONCLUSION: 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.

Document type Article de périodique (Journal article) – Journal Article
Access type Accès libre
Publication date 2005
Journal information "BMC infectious diseases" - Vol. 5, no. 1, p. 15 (2005)
Peer reviewed yes
e-issn 1471-2334
issn 1471-2334
Publication status Publié
Affiliations UCL - MD/MIGE - Département de microbiologie, d'immunologie et de génétique
UCL - SSS/IREC/MBLG - Pôle de Microbiologie médicale
UCL - (SLuc) Service de microbiologie
MESH Subject Adult ; Antiretroviral Therapy, Highly Active ; CD4-Positive T-Lymphocytes - virology ; Cell Line ; DNA, Viral - analysis - genetics ; Female ; HIV Infections - virology ; HIV-1 - classification ; Humans ; Male ; Polymerase Chain Reaction - instrumentation - methods ; Proviruses - genetics - isolation & purification ; RNA, Viral - blood ; Reproducibility of Results ; Sensitivity and Specificity ; Viral Load
Links
  1. Guidelines for the Use of Antiretroviral Agents in HIV-1-Infected Adults and Adolescents. October 29, 2004, [ http://aidsinfo.nih.gov/guidelines/ ]
  2. Bennett Julie M, Kaye Steve, Berry Neil, Tedder Richard S, A quantitative PCR method for the assay of HIV-1 provirus load in peripheral blood mononuclear cells, 10.1016/s0166-0934(99)00096-8
  3. Chun T.-W., Stuyver L., Mizell S. B., Ehler L. A., Mican J. A. M., Baseler M., Lloyd A. L., Nowak M. A., Fauci A. S., Presence of an inducible HIV-1 latent reservoir during highly active antiretroviral therapy, 10.1073/pnas.94.24.13193
  4. Burgard Marianne, Izopet Jacques, Dumon Béatrice, Tamalet Catherine, Descamps Diane, Ruffault Annick, Vabret Astrid, Bargues Gérard, Mouroux Mireille, Pellegrin Isabelle, Ivanoff Serge, Guisthau Odile, Calvez Vincent, Seigneurin Jean-Marie, Rouzioux Christine, HIV RNA and HIV DNA in Peripheral Blood Mononuclear Cells Are Consistent Markers for Estimating Viral Load in Patients Undergoing Long-Term Potent Treatment, 10.1089/088922200750054666
  5. Milito A., Titanji K., Zazzi M., Surrogate Markers as a Guide to Evaluate Response to Antiretroviral Therapy, 10.2174/0929867033368240
  6. Finzi D., Identification of a Reservoir for HIV-1 in Patients on Highly Active Antiretroviral Therapy, 10.1126/science.278.5341.1295
  7. Garrigue Isabelle, Pellegrin Isabelle, Hoen Bruno, Dumon Béatrice, Harzic Martine, Schrive Marie-Hélène, Séréni Daniel, Fleury Hervé, Cell-associated HIV-1-DNA quantitation after highly active antiretroviral therapy-treated primary infection in patients with persistently undetectable plasma HIV-1 RNA : , 10.1097/00002030-200012220-00006
  8. Ngo-Giang-Huong Nicole, Deveau Christiane, Da Silva Isabelle, Pellegrin Isabelle, Venet Alain, Harzic Martine, Sinet Martine, Delfraissy Jean-François, Meyer Laurence, Goujard Cécile, Rouzioux Christine, Proviral HIV-1 DNA in subjects followed since primary HIV-1 infection who suppress plasma viral load after one year of highly active antiretroviral therapy : , 10.1097/00002030-200104130-00001
  9. Pellegrin Isabelle, Caumont Anne, Garrigue Isabelle, Merel Patrick, Schrive Marie‐Hélène, Fleury Hervé, Dupon Michel, Pellegrin Jean‐Luc, Ragnaud Jean‐Marie, Predictive Value of Provirus Load and DNA Human Immunodeficiency Virus Genotype for Successful Abacavir‐Based Simplified Therapy, 10.1086/345860
  10. Saitoh Akihiko, Hsia Karen, Fenton Terence, Powell Christine A., Christopherson Cindy, Fletcher Courtney V., Starr Stuart E., Spector Stephen A., Persistence of Human Immunodeficiency Virus (HIV) Type 1 DNA in Peripheral Blood Despite Prolonged Suppression of Plasma HIV‐1 RNA in Children, 10.1086/340614
  11. Wong J. K., Recovery of Replication-Competent HIV Despite Prolonged Suppression of Plasma Viremia, 10.1126/science.278.5341.1291
  12. Aleman Soo, Visco-Comandini Ubaldo, Lore Karin, Sonnerborg Anders, Long-Term Effects of Antiretroviral Combination Therapy on HIV Type 1 DNA Levels, 10.1089/088922299310142
  13. Hockett Richard D, Saag Michael S, Kilby J.Michael, Sfakianos Greg, Wakefield Theresa B, Bucy R.Pat, Stability in the HIV vDNA pool in peripheral CD4+ T cells of untreated patients by single tube quantitative PCR, 10.1016/s0166-0934(00)00139-7
  14. Kostrikis L. G., Touloumi G., Karanicolas R., Pantazis N., Anastassopoulou C., Karafoulidou A., Goedert J. J., Hatzakis A., Quantitation of Human Immunodeficiency Virus Type 1 DNA Forms with the Second Template Switch in Peripheral Blood Cells Predicts Disease Progression Independently of Plasma RNA Load, 10.1128/jvi.76.20.10099-10108.2002
  15. Christopherson C, Kidane Y, Conway B, Krowka J, Sheppard H, Kwok S: PCR-based assay to quantify human immunodeficiency virus type 1 DNA in peripheral blood mononuclear cells. J Clin Microbiol. 2000, 38: 630-634.
  16. Comandini Ubaldo Visco, Sönnerborg Anders, Vahlne Anders, Yun Zhibing, Quantification of HIV-1 proviral DNA from peripheral blood mononuclear cells using a high throughput four-competitor competitive PCR, 10.1016/s0166-0934(97)00153-5
  17. Gratzl Stephanie, Moroni Christoph, Hirsch Hans H., Quantification of HIV-1 viral RNA and proviral DNA by isotopic competitive PCR, 10.1016/s0166-0934(97)00064-5
  18. Guenthner PC, Hart CE: Quantitative, competitive PCR assay for HIV-1 using a microplate-based detection system. BioTechniques. 1998, 24: 810-816.
  19. Izopet Jacques, Tamalet Catherine, Pasquier Christophe, Sandres Karine, Marchou Bruno, Massip Patrice, Puel Jacqueline, Quantification of HIV-1 proviral DNA by a standardized colorimetric PCR-based assay, 10.1002/(sici)1096-9071(199801)54:1<54::aid-jmv8>3.0.co;2-o
  20. Jurriaans Suzanne, Dekker John T., de Ronde Anthony, HIV-1 viral DNA load in peripheral blood mononuclear cells from seroconverters and long-term infected individuals : , 10.1097/00002030-199207000-00004
  21. Stieger M., Démollière C., Ahlborn-Laake L., Mous J., Competitive polymerase chain reaction assay for quantitation of HIV-1 DNA and RNA, 10.1016/0166-0934(91)90095-h
  22. Zhao Y., Yu M., Miller J. W., Chen M., Bremer E. G., Kabat W., Yogev R., Quantification of Human Immunodeficiency Virus Type 1 Proviral DNA by Using TaqMan Technology, 10.1128/jcm.40.2.675-678.2002
  23. ERIKSSON LARS E., LEITNER THOMAS, WAHREN BRITTA, BOSTROM ANN-CHARLOTTE, FALK KERSTIN I., A multiplex real-time PCR for quantification of HIV-1 DNA and the human albumin gene in CD4+ cells, 10.1034/j.1600-0463.2003.1110605.x
  24. Gibellini D, Quantitative detection of human immunodeficiency virus type 1 (HIV-1) proviral DNA in peripheral blood mononuclear cells by SYBR green real-time PCR technique, 10.1016/s1386-6532(03)00169-0
  25. Girard PM, Katlama Ch, Pialoux G: VIH. Paris: Doin. 2001
  26. The Entrez Nucleotides database. [ http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=Nucleotide ]
  27. CHEINGSONG-POPOV RACHANEE, OSMANOV SALADIN, PAU CHOU-PONG, SCHOCHETMAN GERALD, BARIN FRANCIS, HOLMES HARVEY, FRANCIS GARRY, RUPPACH HORST, DIETRICH URSULA, LISTER SIMON, WEBER JONATHAN, Serotyping of HIV Type 1 Infections: Definition, Relationship to Viral Genetic Subtypes, and Assay Evaluation, 10.1089/aid.1998.14.311
  28. Folks TM, Powell D, Lightfoote M, Koenig S, Fauci AS, Benn S, Rabson A, Daugherty D, Gendelman HE, Hoggan MD: Biological and biochemical characterization of a cloned Leu-3-cell surviving infection with the acquired immune deficiency syndrome retrovirus. J Exp Med. 1986, 280-90. 10.1084/jem.164.1.280.
  29. Alizon Marc, Wain-Hobson Simon, Montagnier Luc, Sonigo Pierre, Genetic variability of the AIDS virus: Nucleotide sequence analysis of two isolates from African patients, 10.1016/0092-8674(86)90860-3
  30. Clavel F, Guetard D, Brun-Vezinet F, Chamaret S, Rey M., Santos-Ferreira M., Laurent A., Dauguet C, Katlama C, Rouzioux C, al. e., Isolation of a new human retrovirus from West African patients with AIDS, 10.1126/science.2425430
  31. Guyader Mireille, Emerman Michael, Sonigo Pierre, Clavel François, Montagnier Luc, Alizon Marc, Genome organization and transactivation of the human immunodeficiency virus type 2, 10.1038/326662a0
  32. Rey Marie-Anne, Krust Bernard, Laurent Anne G., Guétard Denise, Montagnier Luc, Hovanessian Ara G., Characterization of an HIV-2-related virus with a smaller sized extracellular envelope glycoprotein, 10.1016/0042-6822(89)90242-0
  33. Schmit J.-C., Cogniaux J., Hermans P., Van Vaeck C., Sprecher S., Van Remoortel B., Witvrouw M., Balzarini J., Desmyter J., De Clercq E., Vandamme A.-M., Multiple Drug Resistance to Nucleoside Analogues and Nonnucleoside Reverse Transcriptase Inhibitors in an Efficiently Replicating Human Immunodeficiency Virus Type 1 Patient Strain, 10.1093/infdis/174.5.962
  34. Advanced Biological Laboratories S.A. [ http://www.ablnetworks.org/viroscorer/virosc_1.html ]
Bibliographic reference Kabamba-Mukadi, Benoît ; Henrivaux, Philippe ; Ruelle, Jean ; Delferriere, Nicole ; Bodéus, Monique ; et. al. Human immunodeficiency virus type 1 (HIV-1) proviral DNA load in purified CD4+ cells by LightCycler real-time PCR.. In: BMC infectious diseases, Vol. 5, no. 1, p. 15 (2005)
Permanent URL http://hdl.handle.net/2078.1/10139