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Quantitative PCR for the measurement of circulating proviral load in HIV-infected individuals
Clinical and Diagnostic 3(1995)95-104
ELSEVIER
Virology
Quantitative PCR for the measurement of circulating proviral load in HIV-infected individuals Brian Conway *, Doreen Shui-Wah Ko, D. William Cameron Department of Medicine, Ottawa General Hospital, Department ofMicrobiologyand Immunology, University of Ottawa, University of Ottawa AIDS Research Group, Ottawa, Ont. KIH8L6, Canada Received
22 October
1993; revision received 20 April 1994; accepted
26 April 1994
Abstract Background: PCR has been applied extensively as a tool for the detection of HIV. We have previously developed a semi-quantitative microtiter plate assay for the specific detection of HIV PCR products. Objective: To develop and evaluate a novel quantitative assay for the measurement of circulating proviral load in HIV-infected individuals. Study design: We evaluated 70 consecutive, unselected HIV-infected patients, divided into 3 groups, according to their CD4 cell count: greater than 500 cells/u1 (10 subjects); 200-500 cells/pi (31 subjects); less than 200 cells/$ (29 subjects). Peripheral blood mononuclear cell lysates were amplified, and a portion of the product was added to streptavidin-coated wells with a biotinylated capture probe and a horseradish peroxidase-linked reporter probe, complementary to separate regions of the amplified product. Following incubation, readings were taken with an automated plate reader. Products were quantitated by interpolation into a standard curve of serial dilutions of an HIV-containing plasmid, included in each assay. Results: HIV sequences were detected in all 70 clinical samples. Within each patient category, a wide range of proviral loads were observed. However, proviral load/lo6 CD4 cells was associated with disease progression when the patient groups were considered (up to 9.6% infected cells in subjects with CD4 cell counts below 2OO/ul). Conclusions: This quantitative PCR assay will allow the measurement of proviral load in clinical samples. It may be useful in the managment of HIV-infected individuals and the evaluation of the efficacy of antiretroviral therapy. Keywords;
HIV; AIDS; PCR; Proviral load
1. Introduction
The polymerase chain reaction (PCR) has been applied extensively in clinical and research settings as a tool for the detection of HIV (CoutlCe et al., 1991). We have *Corresponding
author.
Fax:
+ 1 (613) 737-8141
092%0197/95/$9.50 0 1995 Elsevier Science B.V. All rights reserved SSDI 0928-0197(94)00026-Q
96
B. Conway et al.IClinical and Diagnostic Virology 3 (1995) 95-104
described a method for the detection of HIV DNA in crude cell lysates using nonradioactive oligonucleotide probes (Conway et al., 1990a). We have further developed a rapid, semi-quantitative microtitre plate assay for the specific detection of HIV PCR products (Conway et al., 1992). In this study, we describe the development of a novel quantitative assay based on this methodology, using a more efficient cell lysis protocol and product detection system. This technique was applied to the evaluation of circulating proviral load in HIV-infected individuals, at various stages of disease.
2. Materials and methods 2.1. Patientpopulation We have studied blood specimens collected from HIV-infected individuals attending the Immunodeficiency Clinic at the Ottawa General Hospital. A total of 70 consecutive, unselected patients were enrolled in this study. CD4 cell counts had been evaluated within one month of study entry by flow cytometry with commercially available monoclonal antibodies. The study subjects were divided into 3 groups according to their circulating CD4 cell count: greater than 500 cells/u1 (10 subjects); 200-500 cells/u1 (3 1 subjects); less than 200 cells/u1 (29 subjects). 2.2. Polymerase chain reaction In preparing specimens for amplification, peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Hypaque centrifugation of non-heparinized anticoagulated blood. An aliquot of 3 x lo6 cells was washed twice in PBS, then lysed in 100 ul of lysis buffer consisting of 10 mM Tris-HCl (pH 7), 2.5 mM MgC12, 0.1% Tween-20 and 0.1% NP-40. Proteinase K (1 mg/ml) was added, and the lysate was incubated at 56°C for 45 min and heated to 94°C for 15 min to inactivate the enzyme. After brief centrifugation, the supernatant was transfered to a fresh tube and stored at 4°C prior to amplification. An aliquot of 10 ul of the cell lysate (equivalent of 0.3 x lo6 cells) was used as a template for amplification. The PCR reaction was carried out in duplicate in a total volume of 50 ul using standard reagents (GeneAmpKit, Perkin-Elmer-Cetus, Norwalk, USA), with 2.0 mM MgC12. Primers designated POL-l/2 (Conway, 1990b), chosen to amplify a 323 base pair segment in the pol region of the HIV genome, were added in a concentration of 0.5 uM each. The amplification protocol consisted of 30 cycles, each with a 1-min denaturation step at 94°C a 1-min annealing step at 45°C and a 1-min extension step at 72°C. Positive amplification controls, included in each assay, consisted of HIV-BHlO cloned into a plasmid vector (5-500 copies). The plasmid DNA was diluted in HIV-seronegative PBMC lysate to approximate the total DNA content of other samples. Negative controls consisted of lysates of cells isolated from the blood of a seronegative volunteer, as well as an aliquot of PCR reaction mixture amplified without sample addition.
B. Conway et uL/Clinical and Diagnostic Virology 3 ( 1995) 95-104
97
Following PCR, the reaction mixture was denatured by heating to 94°C for 15 min and placed on ice to prevent renaturation. An aliquot of 10 yl of each sample was then added to a streptavidin-coated microtitre plate well (Catalogue number FP-155, DuPont NEN Research Products, Boston, USA), with one well serving as a substrate blank. Sample wells were overlaid with 100 ul hybridization buffer (6 x SSC, 1% Triton X-100, 0.1% BSA), containing a biotinylated oligonucleotide capture probe (10 uM) and a horseradish-peroxidase-linked oligonucleotide reporter probe (5 PM), both of which were complementary to separate regions of the amplified product (DuPont NEN, Research Products, Boston, USA). Following incubation in the dark at 37°C for 30 min, the plate was washed 6 times with 1% Triton X-100 in PBS and blotted dry. A chromogenic substrate for horseradish peroxidase was added to each well (TM Blue, Trans Genie Science Center for Diagnostic Products, Milford, USA). The plate was incubated in the dark at 37°C for 60 min and color development was stopped with 4 N H2S04. Readings were taken using an automated plate reader (Bio-Rad Laboratories, Chemical Division, Richmond, USA) at 405 nm. A sample was considered positive if the optical density was greater than twice the optical density of the negative controls. An assay was considered valid if the reaction mixture control and seronegative cell lysate control were negative and the plasmid control containing 5 HIV copies was positive. If these conditions were not fulfilled, test sample results were not interpreted, and the PCR reaction was repeated. The results of the plasmid control samples were used to generate a standard curve for the quantitation of clinical samples over a range of 5-500 proviral copies/PCR reaction. To assess the accuracy of quantitation, periodic PCR reactions included a clinical sample previously quantitated at 200-250 proviral copies/lo6 PBMCs. Quantitation was considered accurate if the repeat evaluation remained within this range.
3. Results The plasmid control samples, taken from amplification reactions run on different days, consistently generated a linear relationship, with a standard curve r-value between 0.986-0.996 (Table 1 and Fig. 1). With respect to the interpretation of microtitre plate hybridization results, the range of optical densities of the substrate blank was 0.003-0.005 and the negative controls 0.010-0.013. As shown, samples containing five HIV plasmid copies yielded optical densities of 0.026-0.033. HIV sequences were detected in all 70 clinical samples. Quantitation was possible in all cases within the standard curve (highest value 483 copies/PCR reaction). Samples with optical density values more than twice the mean of the negative controls but less than the five copy standard were judged to contain a single proviral copy, for purposes of analysis. Patients were evaluated in three separate categories according to their circulating CD4 cell count. Proviral load is expressed as copies/lo6 PBMCs and copies/lo6 CD4 cells (Table 2 and Fig. 2). The latter value was calculated based on recent T-lymphocyte population assays performed in a given patient.
98
B. Conway et al.IClinical and Diagnostic
Virology 3 (1995) 95-104
Table 1 Results of amplification of serial dilutions of an HIV-containing plasmid (5-500 copies) following three representative PCR reactions with products detected using the microtitre plate assay HIV Proviral copies
Optical densities Assay 1
Assay 2
Assay 3
0.724 0.285
0.999 0.299
50
0.924 0.299 0.190
10 5
0.050 0.026
0.100 0.042 0.033
0.146 0.064 0.033
500
100
1
0.8 c fz Q) 0.6 m z 0.4 E
0
0.2 0 0
100
200
300
400
500
HIV proviral copies - Series 1 + Series 2 - Series 3 Fig. 1. Curves generated following amplification of 5-500 copies of an HIV-containing plasmid (curve r-values 0.986-0.996).
Within each patient category, a wide range of proviral loads were observed, accounting for the very large standard deviations around the means. When proviral load/lo6 PBMCs was considered, no clear relationship was observed between advancing immune disease and increasing proviral load. In fact, there was a slight decrease in mean and median proviral load when comparing patients with CD4 cell counts below 2OO/ul with those having less advanced immune disease (counts 200-500 ~1). In contrast, we observed a trend towards increasing proviral load/lo6 CD4 cells with progressive immune disease when all three patient groups were considered.
4. Discussion PCR is a powerful tool for the detection of HIV. We have described a rapid assay for the detection of HIV PCR products (Conway et al., 1992). The attenuation in
B. Conway et al.lClinical and Diagnostic Virology 3 (1995) 95-104
99
Table 2 Circulating proviral load in peripheral blood mononuclear cells (PBMCs) and CD4 cells in HIV-infected individuals, expressed as a function of the level of CD4 cell depletion CD4 cell count (cells/u1 ) > 500 (n=lO)
200-500
< 200
(n=31)
(n=29)
Proviral copiesll0’ PBMC.s
Mean Median Range
159+283 86.5 3-907
376+383 279 331613
250 & 309 105 3-1306
721+1651 231 3-5335
2507 & 3950 1391 3-17922
10453-18975 5050 9995700
Proviral copies/IO6 CD4 cells
Mean Median Range
,E5
~__~*____-------~-~~~~~~---~-~~~~__~_________________,
2
s ;:
:A ,E4
t’ . .
. .
100
I
.
4
.____*_____________________~__________________________
.
.
.
1
.F
2
.
.__________________________::____________________=____
2
.
:.A..
:A
Q) ,000
s >
E
:
.
(0
u
:
----~-_____________________________________________
0
,o
a
1
. .___*________________________________________________. AA : .
1
I
I
200-500 N=29
N=31
CD4
. I
>500 N=lO
Cell Count
Fig. 2. Individual results of circulating proviral load measurements expressed as a function of the level of CD4 cell depletion, in three discrete categories.
the exponential rate of product accumulation that occurs during late PCR cycles (Innes and Gelfand, 1990) precluded true quantitation over the entire template range studied in our experiments. We reasoned that with a more efficient cell lysis protocol and product detection system, the sensitivity of the assay could be improved. With this in mind, we have modified our DNA extraction procedure to include an initial lysis step with non-ionic detergents in Mg-containing buffer, followed by protein digestion (Higuchi, 1989). This method is widely used in the processing of clinical samples. We have also experimented with alternative non-radioactive probes for product detection. In labelling our reporter probe with horseradish peroxidase,
100
B. Conway et al.IClinical and Diagnostic Virology 3 (1995) 95-104
we have been able to reduce the non-specific binding to the streptavidin plate by 40% as compared with our previously described alkaline phosphatase-linked probes (Conway et al., 1992). With this decreased level of background readings, we were able to increase the hybridization time from 30 to 60 min without affecting the interpretability of our assay results. In this way, with 30 cycles of amplification, we were able to design an assay providing quantitative results with reproducible sensitivity with five proviral copies in the reaction mixture. With this decreased cycle number, the plateau effect was reduced allowing quantitation over a significant range of proviral loads (55500 copies/PCR sample or 17-1667 copies/lo6 PBMCs, given 0.3 x lo6 cells/PCR sample). In our study, all samples could be accurately quantitated within this range. Using the plate reader, objective numerical results were obtained, easily amenable to statistical analysis. This is a distinct advantage compared with other methods relying on comparative intensities of bands on autoradiographs. Although we can accurately distinguish between samples carrying 5, 10 and 50 proviral copies, our methodology is not, strictly-speaking, quantitative. Some have advocated alternative techniques such as the use of competitive templates (Piatak et al., 1992) to achieve a more precise degree of quantitation. It is reasoned that this is necessary as the quantitative nature of PCR is obscured by unpredictable variations in reaction conditions and by inhibitory and/or stimulatory substances in sample preparations (Yang et al., 1993). However, these assays are technically more difficult and involve multiple detection reactions for the same amplified product. It is likely that the degree of quantitation achieved in our protocol is sufficient for the majority of clinical applications, for DNA-based assays. In fact, from the perspective of HIV disease progression, it may be that circulating proviral load behaves as a threshold phenomenon, with loads above lo3 copies/106 CD4 cells associated with clinical deterioration (Conway et al., 1994). Our assay would be entirely appropriate in this context. Further work is underway in our facility to determine the variability of proviral load in clinically stable patients over time as well as the coefficient of variation for multiple repeat amplifications and detections of the same lysed sample. This information will allow us to interpret our future results with more insight and enhance their clinical relevance. For RNA-based methods, an initial reverse transcriptase step is required prior to PCR. As this initial step is essentially non-quantitative, use of competitive templates in the same reaction mixture will be required to approach quantitation (Piatak et al., 1993). Current assays based on this methodology are felt to be accurate within one loglO. Technical improvements may allow for more precise quantitation in the near future. Some groups have suggested that HIV genomes are present in at least 10% of CD4 cells in symptomatic patients (Hsia and Spector, 1991; Michael et al., 1992). However, most groups, using culture (Andrieu et al., 1991; O’Shea et al., 1991; Ho et al., 1992) or PCR (Poznansky et al., 1991) methodologies, have continued to demonstrate infection in 0.01-l% of CD4 cells, depending on disease staging. These latter results are in keeping with those generated in our current study. A recent publication has reported similar results, with O.Ol-10% CD4 cells carrying HIV sequences, depending on the severity of immune disease (Wood et al., 1993). This
B. Conway et al./Clinical and Diagnostic Virology 3 (1995) 95-104
101
group has also shown that the viral load carried by CD4 cells can be accurately estimated from the load in the total mononuclear cells and the percentage of circulating CD4 cells, as we have done. Other recent studies have shown that, in general terms, 0.01-l% CD4 cells in the circulation are infected (as assessed by quantitative PCR), with this number occasionally reaching 10% in advanced AIDS (Bieniasz et al., 1993; Kozal et al., 1993). Reports using in situ PCR suggest that as many as 13”/0of circulating PBMCs may be infected with HIV in some patients with advanced AIDS (Bagasra et al., 1992). These findings await independent confirmation, using more conventional assays. Viral load is clearly associated with the severity of HIV-related immune disease (Ho et al., 1989), and disease progression in a given individual is associated with an increasing viral load ( Andrieu et al., 199 1). The CD4 cell count is widely accepted as a surrogate marker for immune disease (Hamilton et al., 1992). In a recent publication, four patients were followed longitudinally over 668 years, with serial determination of viral load in the circulation by quantitative culture and PCR (Connor et al., 1993). A clear relationship between changing proviral load and deterioration in clinical disease status was observed. In these initially healthy individuals, only 0.01-o. 1% CD4 cells were infected. Only in one individual were 5~10% CD4 cells infected, in association with rapid progression of immune disease. It has been suggested that, especially when circulating viral titres (by quantitative culture) are low, the relationship between proviral DNA load and infectious virus titre may be variable, and that one may not necessarily be able to deduce one value having measured the other (Bieniasz et al., 1993). However, in studies such as the one mentioned above, both appear to correlate with disease progression. A difference between the two measures, if present, likely reflects the presence of non-viable virions in the circulation, detected by PCR but not by culture. The significance of such a phenomenon remains to be elucidated. In our own work, we have clearly demonstrated an association between CD4 cell counts and proviral load/O6 CD4 cells. We observed no such association between these counts and proviral load/lo6 PBMCs. It is likely that in these patient groups, an increased proviral load/IO6 CD4 cells is balanced by a decreased proportion of CD4 cells in the circulating mononuclear cell pool. As with other studies, we have shown a great inter-patient variability between individuals with comparable or dissimilar CD4 cell counts (Jurrians et al., 1992). This precludes the use of this measurement as an individual marker of the severity of immune disease. However, serial measurements of viral and proviral load have been proposed as virologic markers that may be useful as endpoints in therapeutic trials of antiviral agents (Coombs et al., 1991). To date, studies have failed to demonstrate a reduction in proviral load in patients on zidovudine therapy (Ho et al., 1989; Donovan et al., 1991). In our cross-sectional study, the effect of intercurrent antiretroviral therapy was not taken into account. Thus, we cannot comment on the impact it may have had on our results. Potentially, zidovudine use in patients with CD4 cell counts below 5OO/ul (as is practiced in our centre) could have decreased our ability to differentiate proviral load levels between individuals with very mild immune disease and all others. Alternatively, it could have had no effect on our results, if proviral load is not modified by currently available therapy.
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B. Conway et al./Clinical and Diagnostic Virology 3 (1995) 95-104
Reduction in viral load in treated patients has been shown by some groups (Collier et al., 1990; Semple et all, 1991), but not others (Coombs et al., 1991). Impressive decreases in circulating viral load following initiation of antiviral therapy have recently been demonstrated using novel RNA PCR assays with competitive templates (Piatak et al., 1993). It is hoped that with the development of rapid, simple, sensitive tests such as the quantitative PCR assay we have described, the precise measurement of viral and proviral load in clinical samples will be possible. This may be useful in the management of HIV-infected individuals and the evaluation of the efficacy of antiretroviral therapy. At the present time, quantitative proviral load PCR assays are not universally recommended in the assessment of the clinical response to many nucleoside analogues (Hammer et al., 1993). This will certainly need to be reassessed as more effective combination therapies are evaluated in the very near future.
Acknowledgements
This work was supported in part by the Canadian Foundation for AIDS Research and the Physicians of Ontario (Physicians Services Inc Foundation). B.C. and D.W.C. are Career Scientists of the Ontario Ministry of Health. This work was presented in part at the meeting of the Royal College of Physicians and Surgeons of Canada (1992) and the CIDS/Amplicor Symposium on Clinical Applications of the Polymerase Chain Reaction (1993). The authors wish to thank Ms. C. Nesrallah for expert editorial assistance.
References Andrieu, J.M., Manolikakis, G. and Lu, W. (1991) Clinical application of viral quantitation by measuring the frequency of infectious virus-harboring cells in blood samples of HIV-1-seropositive individuals. In: J.M. Andrieu (Ed), Viral quantitation in HIV infection. John Libbey Eurotext, Montrouge, France. pp. 75-85. Bagasra, D., Hauptman, S.P., Lischner, H.W., Sachs, M. and Pomerantz, R.J. (1992) Detection of human immunodeficiency virus type 1 provirns in mononuclear cells by in situ polymerase chain reaction. N. Engl. J. Med. 326, 1385-1391. Bieniasz, P.D., Ariyoshi, K., Bourelly, M.A.D., Bloor, S., Foxall, R.B., Harwood, EC. and Weber, J.N. (1993) Variable relationship between proviral DNA load and infectious virus titre in the peripheral blood mononuclear cells of HIV-l infected individuals. AIDS 7, 803-806. Collier, A.C., Bozette, S., Coombs, R.W., Causey, D.M., Schoenfeld, D.A., Spector, S.A., Pettinelli, C.B., Davies, G., Richman, D.D., Leedom, J.M., Kidd, P., and Corey, L. ( 1990) A pilot study of low-dose zidovudine in human immunodeficiency virus infection. N. Engl. J. Med. 323, 1015-1021. Connor, RI., Mohri, H., Cao, Y. and Ho, D.D. (1993) Increased viral burdern and cytopathicity correlate temporally with CD4 and lymphocyte decline and clinical progression in human immunodeficiency virus type l-infected individuals. J. Virol. 67, 1772-1777. Conway, B., Adler, K.E., Bechtel, L.J., Kaplan, J.C. and Hirsch, M.S. (1990a). Detection of HIV-l DNA in crude cell lysates of peripheral blood mononuclear cells by the polymerase chain reaction and non-radioactive oligonucleotide probes, J. Acq. Imm. Def. Synd. 3, 1059-1064. Conway, B. (1990b). Detection of HIV-l by PCR on clinical specimens. In: A. Aldovini and B.D. Walker (Eds.). Techniques in HIV research. Stockton Press, New York. pp. 40-46.
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Conway, B., Bechtel, L.J., Adler, K.A., D’Aquila, R.T., Kaplan, J.C. and Hirsch, M.S. ( 1992) Comparison of spot-blot and microtitre plate methods for the detection of HIV-I PCR products. Mol. Cell Probes 6, 245-249. Conway, B., Ko, D.S., Shaw, M., Diaz-Mitoma, F. and Wells, G.A. (1994) Dynamic relationship of virologic correlates of HIV disease progression. J. Cell. Biochem. 18B, 129. Coombs, R.W., Collier, A.C. and Corey, L. (1991) Plasma viremia as an endpoint in evaluating the effectiveness of drugs against human immunodeficiency virus type-l (HIV) infection: natural history of plasma viremia and monitoring of antiretroviral therapy. In: J.M. Andrieu (Ed.), Viral quantitation in HIV infection. John Libbey Eurotext, Montrouge, France, pp. 9-19. Coutlte, F., Viscidi, R.P., Saint-Antoine, P., Kessous, A. and Yolken, R.H. (1991) The polymerase chain reaction: a new tool for the understanding and diagnosis of HIV-l infection at the molecular level. Mol. Cell Probes 5, 241-249. Donovan, R.M., Dickover, R.E., Goldstein, E., Huth, R.G. and Carlson, J.R. ( 1991) HIV-l proviral copy number in blood mononuclear cells from AIDS patients on zidovudine therapy. J. Acq. Imm. Def. Synd. 4, 766-769. Hamilton, J.D., Hartigan, P.M., Simberkoff, M.S., Day, P.L., Diamond, G.R., Dickinson, G.M., Drusano, G.L., Egorin, M.J., George, W.L., Gordon, F.M., Hawkes, CA., Jensen, P.C., Klimes, N.G., Labriola, A.M., Lahart, C.J., O’Brien, W.A., Oster, C.N., Weinhold, K.J., Wray, N.P. and Zolla-Pazner, S.B. (1992) A controlled trial of early versus late treatment with zidovudine in symptomatic human immunodeficiency virus infection. N. Engl. J. Med. 326, 437-443. Hammer, S., Crumpacker, C., D’Aquila, R., Jackson, B., Lathey, J., Livnat, D., Reicheldeifer, P., et al. (1993) Use of virologic assays for detection of human immunodeficiency virus in clinical trials: recommendations of the AIDS clinical trials group virology committee. J. Clin. Micro. 31, 2557.-2564. Higuchi, R. (1989) Simple and rapid preparation of samples for PCR. In: H.A. Erlich (Ed). PCR Technology. Stockton Press, New York. pp. 31-38. Ho, D.D., Mougdil, T. and Alam, M. (1989) Quantitation of human immunodeficiency virus type 1 in the blood of infected persons. N. Engl. J. Med. 321, 1621-1625. Ho, D.D. (1992) Virus load and HIV pathogenesis. In: Prevention and Treatment of AIDS, Keystone Symposium on Molecular and Cellular Biology, J. Cell Biochem, Suppl. 16E. Hsia, K. and Spector, S.A. ( 1991) Human immunodeficiency virus DNA is present in a high percentage of CD4 + lymphocytes of seropositive individuals. J. Infect. Dis. 164, 470-475. Innes, M.A. and Gelfand, D.H. (1990) Optimization of PCR. In: M.A. Innes, D.H. Gelfand, J.J. Sninsky, T.J. White (Eds.), PCR Protocols. Academic Press, New York. pp. 3-12. Jurriaans, S., Dekker, J.T. and De Ronde, A. (1992) HIV-l viral DNA load in peripheral blood mononuclear cells from seroconverters and long-term infected individuals. AIDS 6, 635-64 1. Kozal, M.J., Shafer, R.W., Winters, M.A., Katzenstein, D.A. and Merigan, T.C. (1993) A mutation in human immunodeficiency virus reverse transcriptase and decline in CD4 lymphocyte numbers in longterm zidovudine recipients. J. Infect. Dis. 167, 526-532. Michael, N.L., Vahey, M., Burke, D.S. and Redfield, R.R. (1992) Viral DNA and mRNA expression correlate with the stage of human immunodeficiency virus (HIV) type 1 infection in humans: evidence for viral replication in all stages of HIV disease. J. Virol. 66, 310-316. O’Shea, S., Rostron, T., Hamblin, A.S., Palmer, S.J. and Banatvala, J.E. (1991) Quantitation of HIV: Correlation with clinical, virological, and immunological status. J. Med. Virol. 35, 65-69. Piatak, M.. Luk, K.C., McGrath, M., Williams, B. and Lifson, J. (1992) Accurate quantitation of HIV DNA and RNA sequences using quantitative competitive polymerase chain reaction (QC-PCR). Antivir. Res. 17 (suppl l), 64. Piatak, M., Saag, M.S., Yang, L.C., Clark, S.J., Kappes, I.C., Luk, K.C., Hahn, B.H., Shaw, G.M. and Lifson, J.D. (1993) High levels of HIV-l in plasma during all stages of infection determined by competitive PCR. Science 259, 1749-1754. Poznansky, M.C., Walker, B., Haseltine, W.A., Sodroski, J. and Langhoff, E. (1991) A rapid method for quantitating the frequency of peripheral blood cells containing HIV-l DNA. J. Acq. Imm. Def. Synd. 4, 368-373. Semple, M., Loveday, C., Weller, 1. and Tedder, R. (1991) Direct measurement of viraemia in patients
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infected with HIV-1 and its relationship to disease progression and zidovudine therapy. J. Med. Virol. 35, 38-45. Wood, R., Dong, H., Katzenstein, D.A. and Merigan, T.C. (1993) Quantification and comparison of HIV-1 proviral load in peripheral blood mononuclear cells and isolated CD4-positive T cells. J. Acq. Imm Def. Synd. 6, 237-240. Yang, B., Yolken, R. and Viscidi, R. (1993) Quantitative polymerase chain reaction by monitoring enzymatic activity of DNA polymerase. Anal. Biochem. 208, 110-l 16.
ELSEVIER
Virology
Quantitative PCR for the measurement of circulating proviral load in HIV-infected individuals Brian Conway *, Doreen Shui-Wah Ko, D. William Cameron Department of Medicine, Ottawa General Hospital, Department ofMicrobiologyand Immunology, University of Ottawa, University of Ottawa AIDS Research Group, Ottawa, Ont. KIH8L6, Canada Received
22 October
1993; revision received 20 April 1994; accepted
26 April 1994
Abstract Background: PCR has been applied extensively as a tool for the detection of HIV. We have previously developed a semi-quantitative microtiter plate assay for the specific detection of HIV PCR products. Objective: To develop and evaluate a novel quantitative assay for the measurement of circulating proviral load in HIV-infected individuals. Study design: We evaluated 70 consecutive, unselected HIV-infected patients, divided into 3 groups, according to their CD4 cell count: greater than 500 cells/u1 (10 subjects); 200-500 cells/pi (31 subjects); less than 200 cells/$ (29 subjects). Peripheral blood mononuclear cell lysates were amplified, and a portion of the product was added to streptavidin-coated wells with a biotinylated capture probe and a horseradish peroxidase-linked reporter probe, complementary to separate regions of the amplified product. Following incubation, readings were taken with an automated plate reader. Products were quantitated by interpolation into a standard curve of serial dilutions of an HIV-containing plasmid, included in each assay. Results: HIV sequences were detected in all 70 clinical samples. Within each patient category, a wide range of proviral loads were observed. However, proviral load/lo6 CD4 cells was associated with disease progression when the patient groups were considered (up to 9.6% infected cells in subjects with CD4 cell counts below 2OO/ul). Conclusions: This quantitative PCR assay will allow the measurement of proviral load in clinical samples. It may be useful in the managment of HIV-infected individuals and the evaluation of the efficacy of antiretroviral therapy. Keywords;
HIV; AIDS; PCR; Proviral load
1. Introduction
The polymerase chain reaction (PCR) has been applied extensively in clinical and research settings as a tool for the detection of HIV (CoutlCe et al., 1991). We have *Corresponding
author.
Fax:
+ 1 (613) 737-8141
092%0197/95/$9.50 0 1995 Elsevier Science B.V. All rights reserved SSDI 0928-0197(94)00026-Q
96
B. Conway et al.IClinical and Diagnostic Virology 3 (1995) 95-104
described a method for the detection of HIV DNA in crude cell lysates using nonradioactive oligonucleotide probes (Conway et al., 1990a). We have further developed a rapid, semi-quantitative microtitre plate assay for the specific detection of HIV PCR products (Conway et al., 1992). In this study, we describe the development of a novel quantitative assay based on this methodology, using a more efficient cell lysis protocol and product detection system. This technique was applied to the evaluation of circulating proviral load in HIV-infected individuals, at various stages of disease.
2. Materials and methods 2.1. Patientpopulation We have studied blood specimens collected from HIV-infected individuals attending the Immunodeficiency Clinic at the Ottawa General Hospital. A total of 70 consecutive, unselected patients were enrolled in this study. CD4 cell counts had been evaluated within one month of study entry by flow cytometry with commercially available monoclonal antibodies. The study subjects were divided into 3 groups according to their circulating CD4 cell count: greater than 500 cells/u1 (10 subjects); 200-500 cells/u1 (3 1 subjects); less than 200 cells/u1 (29 subjects). 2.2. Polymerase chain reaction In preparing specimens for amplification, peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Hypaque centrifugation of non-heparinized anticoagulated blood. An aliquot of 3 x lo6 cells was washed twice in PBS, then lysed in 100 ul of lysis buffer consisting of 10 mM Tris-HCl (pH 7), 2.5 mM MgC12, 0.1% Tween-20 and 0.1% NP-40. Proteinase K (1 mg/ml) was added, and the lysate was incubated at 56°C for 45 min and heated to 94°C for 15 min to inactivate the enzyme. After brief centrifugation, the supernatant was transfered to a fresh tube and stored at 4°C prior to amplification. An aliquot of 10 ul of the cell lysate (equivalent of 0.3 x lo6 cells) was used as a template for amplification. The PCR reaction was carried out in duplicate in a total volume of 50 ul using standard reagents (GeneAmpKit, Perkin-Elmer-Cetus, Norwalk, USA), with 2.0 mM MgC12. Primers designated POL-l/2 (Conway, 1990b), chosen to amplify a 323 base pair segment in the pol region of the HIV genome, were added in a concentration of 0.5 uM each. The amplification protocol consisted of 30 cycles, each with a 1-min denaturation step at 94°C a 1-min annealing step at 45°C and a 1-min extension step at 72°C. Positive amplification controls, included in each assay, consisted of HIV-BHlO cloned into a plasmid vector (5-500 copies). The plasmid DNA was diluted in HIV-seronegative PBMC lysate to approximate the total DNA content of other samples. Negative controls consisted of lysates of cells isolated from the blood of a seronegative volunteer, as well as an aliquot of PCR reaction mixture amplified without sample addition.
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Following PCR, the reaction mixture was denatured by heating to 94°C for 15 min and placed on ice to prevent renaturation. An aliquot of 10 yl of each sample was then added to a streptavidin-coated microtitre plate well (Catalogue number FP-155, DuPont NEN Research Products, Boston, USA), with one well serving as a substrate blank. Sample wells were overlaid with 100 ul hybridization buffer (6 x SSC, 1% Triton X-100, 0.1% BSA), containing a biotinylated oligonucleotide capture probe (10 uM) and a horseradish-peroxidase-linked oligonucleotide reporter probe (5 PM), both of which were complementary to separate regions of the amplified product (DuPont NEN, Research Products, Boston, USA). Following incubation in the dark at 37°C for 30 min, the plate was washed 6 times with 1% Triton X-100 in PBS and blotted dry. A chromogenic substrate for horseradish peroxidase was added to each well (TM Blue, Trans Genie Science Center for Diagnostic Products, Milford, USA). The plate was incubated in the dark at 37°C for 60 min and color development was stopped with 4 N H2S04. Readings were taken using an automated plate reader (Bio-Rad Laboratories, Chemical Division, Richmond, USA) at 405 nm. A sample was considered positive if the optical density was greater than twice the optical density of the negative controls. An assay was considered valid if the reaction mixture control and seronegative cell lysate control were negative and the plasmid control containing 5 HIV copies was positive. If these conditions were not fulfilled, test sample results were not interpreted, and the PCR reaction was repeated. The results of the plasmid control samples were used to generate a standard curve for the quantitation of clinical samples over a range of 5-500 proviral copies/PCR reaction. To assess the accuracy of quantitation, periodic PCR reactions included a clinical sample previously quantitated at 200-250 proviral copies/lo6 PBMCs. Quantitation was considered accurate if the repeat evaluation remained within this range.
3. Results The plasmid control samples, taken from amplification reactions run on different days, consistently generated a linear relationship, with a standard curve r-value between 0.986-0.996 (Table 1 and Fig. 1). With respect to the interpretation of microtitre plate hybridization results, the range of optical densities of the substrate blank was 0.003-0.005 and the negative controls 0.010-0.013. As shown, samples containing five HIV plasmid copies yielded optical densities of 0.026-0.033. HIV sequences were detected in all 70 clinical samples. Quantitation was possible in all cases within the standard curve (highest value 483 copies/PCR reaction). Samples with optical density values more than twice the mean of the negative controls but less than the five copy standard were judged to contain a single proviral copy, for purposes of analysis. Patients were evaluated in three separate categories according to their circulating CD4 cell count. Proviral load is expressed as copies/lo6 PBMCs and copies/lo6 CD4 cells (Table 2 and Fig. 2). The latter value was calculated based on recent T-lymphocyte population assays performed in a given patient.
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B. Conway et al.IClinical and Diagnostic
Virology 3 (1995) 95-104
Table 1 Results of amplification of serial dilutions of an HIV-containing plasmid (5-500 copies) following three representative PCR reactions with products detected using the microtitre plate assay HIV Proviral copies
Optical densities Assay 1
Assay 2
Assay 3
0.724 0.285
0.999 0.299
50
0.924 0.299 0.190
10 5
0.050 0.026
0.100 0.042 0.033
0.146 0.064 0.033
500
100
1
0.8 c fz Q) 0.6 m z 0.4 E
0
0.2 0 0
100
200
300
400
500
HIV proviral copies - Series 1 + Series 2 - Series 3 Fig. 1. Curves generated following amplification of 5-500 copies of an HIV-containing plasmid (curve r-values 0.986-0.996).
Within each patient category, a wide range of proviral loads were observed, accounting for the very large standard deviations around the means. When proviral load/lo6 PBMCs was considered, no clear relationship was observed between advancing immune disease and increasing proviral load. In fact, there was a slight decrease in mean and median proviral load when comparing patients with CD4 cell counts below 2OO/ul with those having less advanced immune disease (counts 200-500 ~1). In contrast, we observed a trend towards increasing proviral load/lo6 CD4 cells with progressive immune disease when all three patient groups were considered.
4. Discussion PCR is a powerful tool for the detection of HIV. We have described a rapid assay for the detection of HIV PCR products (Conway et al., 1992). The attenuation in
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99
Table 2 Circulating proviral load in peripheral blood mononuclear cells (PBMCs) and CD4 cells in HIV-infected individuals, expressed as a function of the level of CD4 cell depletion CD4 cell count (cells/u1 ) > 500 (n=lO)
200-500
< 200
(n=31)
(n=29)
Proviral copiesll0’ PBMC.s
Mean Median Range
159+283 86.5 3-907
376+383 279 331613
250 & 309 105 3-1306
721+1651 231 3-5335
2507 & 3950 1391 3-17922
10453-18975 5050 9995700
Proviral copies/IO6 CD4 cells
Mean Median Range
,E5
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200-500 N=29
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>500 N=lO
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Fig. 2. Individual results of circulating proviral load measurements expressed as a function of the level of CD4 cell depletion, in three discrete categories.
the exponential rate of product accumulation that occurs during late PCR cycles (Innes and Gelfand, 1990) precluded true quantitation over the entire template range studied in our experiments. We reasoned that with a more efficient cell lysis protocol and product detection system, the sensitivity of the assay could be improved. With this in mind, we have modified our DNA extraction procedure to include an initial lysis step with non-ionic detergents in Mg-containing buffer, followed by protein digestion (Higuchi, 1989). This method is widely used in the processing of clinical samples. We have also experimented with alternative non-radioactive probes for product detection. In labelling our reporter probe with horseradish peroxidase,
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B. Conway et al.IClinical and Diagnostic Virology 3 (1995) 95-104
we have been able to reduce the non-specific binding to the streptavidin plate by 40% as compared with our previously described alkaline phosphatase-linked probes (Conway et al., 1992). With this decreased level of background readings, we were able to increase the hybridization time from 30 to 60 min without affecting the interpretability of our assay results. In this way, with 30 cycles of amplification, we were able to design an assay providing quantitative results with reproducible sensitivity with five proviral copies in the reaction mixture. With this decreased cycle number, the plateau effect was reduced allowing quantitation over a significant range of proviral loads (55500 copies/PCR sample or 17-1667 copies/lo6 PBMCs, given 0.3 x lo6 cells/PCR sample). In our study, all samples could be accurately quantitated within this range. Using the plate reader, objective numerical results were obtained, easily amenable to statistical analysis. This is a distinct advantage compared with other methods relying on comparative intensities of bands on autoradiographs. Although we can accurately distinguish between samples carrying 5, 10 and 50 proviral copies, our methodology is not, strictly-speaking, quantitative. Some have advocated alternative techniques such as the use of competitive templates (Piatak et al., 1992) to achieve a more precise degree of quantitation. It is reasoned that this is necessary as the quantitative nature of PCR is obscured by unpredictable variations in reaction conditions and by inhibitory and/or stimulatory substances in sample preparations (Yang et al., 1993). However, these assays are technically more difficult and involve multiple detection reactions for the same amplified product. It is likely that the degree of quantitation achieved in our protocol is sufficient for the majority of clinical applications, for DNA-based assays. In fact, from the perspective of HIV disease progression, it may be that circulating proviral load behaves as a threshold phenomenon, with loads above lo3 copies/106 CD4 cells associated with clinical deterioration (Conway et al., 1994). Our assay would be entirely appropriate in this context. Further work is underway in our facility to determine the variability of proviral load in clinically stable patients over time as well as the coefficient of variation for multiple repeat amplifications and detections of the same lysed sample. This information will allow us to interpret our future results with more insight and enhance their clinical relevance. For RNA-based methods, an initial reverse transcriptase step is required prior to PCR. As this initial step is essentially non-quantitative, use of competitive templates in the same reaction mixture will be required to approach quantitation (Piatak et al., 1993). Current assays based on this methodology are felt to be accurate within one loglO. Technical improvements may allow for more precise quantitation in the near future. Some groups have suggested that HIV genomes are present in at least 10% of CD4 cells in symptomatic patients (Hsia and Spector, 1991; Michael et al., 1992). However, most groups, using culture (Andrieu et al., 1991; O’Shea et al., 1991; Ho et al., 1992) or PCR (Poznansky et al., 1991) methodologies, have continued to demonstrate infection in 0.01-l% of CD4 cells, depending on disease staging. These latter results are in keeping with those generated in our current study. A recent publication has reported similar results, with O.Ol-10% CD4 cells carrying HIV sequences, depending on the severity of immune disease (Wood et al., 1993). This
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group has also shown that the viral load carried by CD4 cells can be accurately estimated from the load in the total mononuclear cells and the percentage of circulating CD4 cells, as we have done. Other recent studies have shown that, in general terms, 0.01-l% CD4 cells in the circulation are infected (as assessed by quantitative PCR), with this number occasionally reaching 10% in advanced AIDS (Bieniasz et al., 1993; Kozal et al., 1993). Reports using in situ PCR suggest that as many as 13”/0of circulating PBMCs may be infected with HIV in some patients with advanced AIDS (Bagasra et al., 1992). These findings await independent confirmation, using more conventional assays. Viral load is clearly associated with the severity of HIV-related immune disease (Ho et al., 1989), and disease progression in a given individual is associated with an increasing viral load ( Andrieu et al., 199 1). The CD4 cell count is widely accepted as a surrogate marker for immune disease (Hamilton et al., 1992). In a recent publication, four patients were followed longitudinally over 668 years, with serial determination of viral load in the circulation by quantitative culture and PCR (Connor et al., 1993). A clear relationship between changing proviral load and deterioration in clinical disease status was observed. In these initially healthy individuals, only 0.01-o. 1% CD4 cells were infected. Only in one individual were 5~10% CD4 cells infected, in association with rapid progression of immune disease. It has been suggested that, especially when circulating viral titres (by quantitative culture) are low, the relationship between proviral DNA load and infectious virus titre may be variable, and that one may not necessarily be able to deduce one value having measured the other (Bieniasz et al., 1993). However, in studies such as the one mentioned above, both appear to correlate with disease progression. A difference between the two measures, if present, likely reflects the presence of non-viable virions in the circulation, detected by PCR but not by culture. The significance of such a phenomenon remains to be elucidated. In our own work, we have clearly demonstrated an association between CD4 cell counts and proviral load/O6 CD4 cells. We observed no such association between these counts and proviral load/lo6 PBMCs. It is likely that in these patient groups, an increased proviral load/IO6 CD4 cells is balanced by a decreased proportion of CD4 cells in the circulating mononuclear cell pool. As with other studies, we have shown a great inter-patient variability between individuals with comparable or dissimilar CD4 cell counts (Jurrians et al., 1992). This precludes the use of this measurement as an individual marker of the severity of immune disease. However, serial measurements of viral and proviral load have been proposed as virologic markers that may be useful as endpoints in therapeutic trials of antiviral agents (Coombs et al., 1991). To date, studies have failed to demonstrate a reduction in proviral load in patients on zidovudine therapy (Ho et al., 1989; Donovan et al., 1991). In our cross-sectional study, the effect of intercurrent antiretroviral therapy was not taken into account. Thus, we cannot comment on the impact it may have had on our results. Potentially, zidovudine use in patients with CD4 cell counts below 5OO/ul (as is practiced in our centre) could have decreased our ability to differentiate proviral load levels between individuals with very mild immune disease and all others. Alternatively, it could have had no effect on our results, if proviral load is not modified by currently available therapy.
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Reduction in viral load in treated patients has been shown by some groups (Collier et al., 1990; Semple et all, 1991), but not others (Coombs et al., 1991). Impressive decreases in circulating viral load following initiation of antiviral therapy have recently been demonstrated using novel RNA PCR assays with competitive templates (Piatak et al., 1993). It is hoped that with the development of rapid, simple, sensitive tests such as the quantitative PCR assay we have described, the precise measurement of viral and proviral load in clinical samples will be possible. This may be useful in the management of HIV-infected individuals and the evaluation of the efficacy of antiretroviral therapy. At the present time, quantitative proviral load PCR assays are not universally recommended in the assessment of the clinical response to many nucleoside analogues (Hammer et al., 1993). This will certainly need to be reassessed as more effective combination therapies are evaluated in the very near future.
Acknowledgements
This work was supported in part by the Canadian Foundation for AIDS Research and the Physicians of Ontario (Physicians Services Inc Foundation). B.C. and D.W.C. are Career Scientists of the Ontario Ministry of Health. This work was presented in part at the meeting of the Royal College of Physicians and Surgeons of Canada (1992) and the CIDS/Amplicor Symposium on Clinical Applications of the Polymerase Chain Reaction (1993). The authors wish to thank Ms. C. Nesrallah for expert editorial assistance.
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infected with HIV-1 and its relationship to disease progression and zidovudine therapy. J. Med. Virol. 35, 38-45. Wood, R., Dong, H., Katzenstein, D.A. and Merigan, T.C. (1993) Quantification and comparison of HIV-1 proviral load in peripheral blood mononuclear cells and isolated CD4-positive T cells. J. Acq. Imm Def. Synd. 6, 237-240. Yang, B., Yolken, R. and Viscidi, R. (1993) Quantitative polymerase chain reaction by monitoring enzymatic activity of DNA polymerase. Anal. Biochem. 208, 110-l 16.