initiation of zidovudine and/or 2',3'-dideoxyinosine therapy. The mean log1o number of proviral HIV-1 copies per 106 CD4+ T cells decreased from 4.3 ± 0.4 at ...
Vol. 31, No. 10
JOURNAL OF CLINICAL MICROBIOLOGY, OCt. 1993, p. 2692-2696 0095-1137/93/102692-05$02.00/0 Copyright C 1993, American Society for Microbiology
Peripheral Blood Mononuclear Cell Human Immunodeficiency Virus Type 1 Proviral DNA Quantification by Polymerase Chain Reaction: Relationship to Immunodeficiency and Drug Effect JOSE G. MONTOYA,t* ROBIN WOOD,t DAVID KATZENSTEIN,t MARK HOLODNY,4 AND THOMAS C. MERIGANt Center for AIDS Research, Stanford University Medical Center, Stanford, California 94305 Received 17 March 1993/Returned for modification 6 May 1993/Accepted 15 July 1993
Human immunodeficiency virus type 1 (HIV-1) proviral DNA from peripheral blood mononuclear cells (PBMCs) was quantitated in 61 HIV-1-seropositive individuals by a nonisotopic polymerase chain reaction assay. Primers from thegag region (SK38, SK39) were used to determine the log1o HIV-1 proviral copy number per 106 CD4+ T lymphocytes (peripheral blood proviral load). A standard curve was generated for each assay by using ACH-2 cell DNA. The peripheral blood proviral load was followed in 15 individuals in a longitudinal study and was measured in 45 individuals in a cross-sectional analysis. Three of four untreated patients who were followed for 14 months had stable PBMC proviral loads and CD4+ T lymphocyte counts; one untreated patient had a sustained increase in PBMC proviral load followed 5 months later by a significant decline in the CD4+ T lymphocyte count. Eleven previously untreated individuals were monitored for 1 year following initiation of zidovudine and/or 2',3'-dideoxyinosine therapy. The mean log1o number of proviral HIV-1 copies per 106 CD4+ T cells decreased from 4.3 ± 0.4 at the baseline to 3.5 ± 0.6 after 2 to 4 months of therapy (P < 0.01). This initial 0.8 loglo fall in the PBMC proviral load after the initiation of therapy was followed by a rise in the PBMC proviral load by the sixth month of therapy. The PBMC proviral load in 45 subjects, both treated (n = 25) and untreated (n = 20), correlated inversely with the CD4+ T lymphocyte count (P < 0.01, R = 0.49). PBMC proviral DNA quantification by a nonisotopic polymerase chain reaction assay correlates with HIV-1 disease progression and could be used to monitor the effect of antiretroviral therapy. a PBMC proviral DNA amplification assay developed and optimized in our laboratory, in 61 HIV-1-seropositive individuals is summarized here, as follows: a cross-sectional study of PBMC proviral load in 45 patients (25 patients on long-term therapy and 20 untreated patients) that correlated the PBMC proviral load with CD4+ T cell counts and the impact of long-term antiretroviral therapy, a longitudinal study of the PBMC proviral load in 4 untreated patients who were followed for 14 to 16 months, and a longitudinal study of the PBMC proviral load in 11 previously untreated individuals starting single or combination nucleoside therapy.
Quantification of human immunodeficiency virus type 1 (HIV-1) in peripheral blood by polymerase chain reaction (PCR) has been reported by using both RNA and DNA amplification (1, 5, 9-11, 13, 15, 17, 18, 21, 22, 24, 25, 27). Measurement of the HIV-1 proviral load may be useful in predicting the progression of disease and the response to currently available and experimental antiretroviral drugs (5, 8, 12, 15, 16). By using quantitative PCR, the amount of HIV-1 proviral DNA in peripheral blood mononuclear cells (PBMCs) has been shown to increase significantly and transiently at the time of symptomatic primary infection (6) and to rise gradually and steadily as the disease progresses
(3, 9, 18, 24, 27).
MATERIALS AND METHODS from 61 ambulatory and asymptomatic patients Specimens participating in AIDS Clinical Trials Group clinical trials at the Center for AIDS Research at Stanford University Medical Center in 1988 were available. All specimens were obtained from the patients with informed consent, and the study was approved by the institutional review board of the Stanford University Medical Center. Citrated blood was obtained by peripheral venipuncture, and PBMCs were separated by Ficoll-Hypaque density gradient centrifugation. Cells were stored at - 180°C. DNAs extracted from the PBMCs of seronegative individuals were used as negative controls in each assay. Cells were digested overnight at 55°C in proteinase K buffer, and proteinase K was inactivated by heating the samples at 95°C. DNA was extracted with phenol-chloroform (23), dissolved in 55 ,ll of TE buffer (10 mM Tris-HCl [pH 8.0], 0.5 mM EDTA), and quantitated by measuring the A260. DNA equivalent to approximately 200,000 PBMCs (1.2 ,g) per sample was
PBMC proviral DNA amplification has also been used to assess the viral load response to therapy with immunomodulators or nucleoside compounds (4, 8). Both Clark et al. (4) and Aoki et al. (2) reported significant decreases in the amount of PBMC proviral DNA in drug-naive patients commencing zidovudine with recombinant interleukin-2 and 2',3'-dideoxyinosine (ddI) therapy, respectively. However, Donovan et al. (7) found no significant change in the amount of PBMC proviral DNA in six patients after 5 to 14 months of zidovudine therapy. Study of the peripheral blood proviral load (log1o HIV-1 copy number per 106 CD4+ T lymphocytes), determined by *
Corresponding author.
t Present address: Division of Infectious Diseases (S-156), Stan-
ford University Medical Center, Stanford, CA 94305. t Present address: Infectious Diseases Section (III-ID), Veterans Affairs Medical Center, Palo Alto, CA 94304.
2692
VOL. 31, 1993
amplified in a PCR mixture containing 15 mM Tris-HCl, 75 mM KCl, 2.5 mM MgCl2, 0.4 ,ug of human placental DNA, 0.01% gelatin, 250 ,M (each) deoxynucleoside triphosphates, 50 pmol each of the gag primer SK39 and biotinylated SK38 (20), and 2.5 U of Taq DNA polymerase (AmpliTaq DNA polymerase; Perkin-Elmer Cetus, Emerville, Calif.) in a total volume of 100 pl at pH 8.4. Amplification was carried out for 40 cycles in a GeneAmp PCR System 9600 (Perkin-Elmer Cetus). Each cycle consisted of three steps of 30 s each at 94, 60, and 72°C, with a 10-min final extension step at 72°C. PCR products were quantitated by using a colorimetric enzyme-linked immunosorbent assay format (14). Briefly, DNA was denatured at 95°C and rapidly cooled on ice, and 5 ,ul of PCR product from each sample was added to avidin-coated 96-well microplates. Hybridization solution containing horseradish peroxidase-labeled SK19 probe was added, and the plates were incubated at 42°C for 1 hour. By using a Biomek 1000 Automated Workstation (Beckman Instruments, Palo Alto, Calif.), each well was washed 20 times, o-phenylenediamine (horseradish peroxidase substrate) was added, and after adding 1 N H2SO4 to stop the reaction, the optical density at 490 nm was measured. A half loglo dilution series of ACH-2 DNA containing one HIV-1 copy per genome (AIDS National Reagent Program, National Institutes of Health, Bethesda, Md.) was coamplified in duplicate with each PCR assay in order to establish a relationship between HIV-1 copy number and the optical density at 490 nm. The log1o of the optical density versus the log1o of the HIV-1 copy number generated a standard curve for each assay (14). This standard curve was linear between 10 and 1,000 copies of input DNA, with a mean R value for all standard curves of 0.98 and a standard error of 0.006. The HIV-1 copy number was calculated for each experimental sample by interpolation of the linear portion of the standard curve. HIV-1 copy number was normalized to 106 CD4+ T lymphocytes by using the percentage of CD4+ T lymphocytes in peripheral blood of the same experimental sample. The percentage of CD4+ T lymphocytes in peripheral blood was measured independently by the Stanford University Flow Cytometry Laboratory by flow cytometry of total blood. The viral load was expressed as log1o HIV copy number per 106 CD4+ T lymphocytes by the following formula: log1o HIV copy number/106 CD4 cells = PBMC log1o HIV copy number + log1o (100/percent CD4 cells). An analysis of HIV-1 proviral DNA amplification in lymphocyte subsets demonstrated that 93% of the HIV-1 proviral DNA was in the CD3+-CD4+ cell population. The contribution to the HIV-1 proviral DNA signal was minimal by the CD45+CD20+ and the CD45+-CD14+ cell subsets (26). Statistical analysis. Linear regression analysis between the half log1o dilution series of ACH-2 cell DNA and the optical density at 490 nm was performed by using the LINEAR program from the statistical package for Lotus 1-2-3 (Data Management Branch of the Division of Computer Research and Technology, National Institutes of Health). Student's t test for paired and unpaired data was used to compare the differences in the log1o HIV-1 copy number per 106 CD4+ T lymphocytes within individuals over time and between groups of patients. Significance was defined as a P value of
