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1 November 1995 | Volume 123 Issue 9 | Pages 641-648
Objective: To establish human immunodeficiency virus type 1 (HIV-1) messenger RNA (mRNA) expression in peripheral blood mononuclear cells as a marker of risk for progression to the acquired immunodeficiency syndrome (AIDS) in a large cohort of HIV-infected persons followed for a prolonged period.
Design: Retrospective testing of cryopreserved, coded specimens.
Setting: Research laboratories at the New York Blood Center and the Rockefeller University.
Patients: 150 homosexual men infected with HIV-1 who did not have an AIDS diagnosis at the time of testing.
Measurements: Multiply spliced and unspliced HIV-1 mRNAs in total peripheral blood mononuclear cell RNA were quantitated using reverse transcriptase-initiated polymerase chain reaction (PCR) and compared with other laboratory data and clinical outcome during the subsequent 8 years.
Results: Although HIV-1 mRNA expression generally correlated with immunologic status, it was associated with future disease progression independently of CD4+ cell counts or their rate of decrease at the time of sampling. The association of HIV-1 mRNA with disease progression in persons with CD4+ cell counts higher than the median (> 624 cells/mm3) was particularly noteworthy; further variation in the CD4+ cell counts within this group was not prognostically significant.
Conclusions: The expression of HIV-1 mRNA in peripheral blood mononuclear cells is a strong independent marker for future HIV disease progression, even in persons with normal T-cell subsets.
Progression of HIV disease is usually monitored by quantitating circulating CD4+ lymphocytes: A decrease in the CD4+ cell count is a hallmark of the deteriorating immune system and the development of clinical acquired immunodeficiency syndrome (AIDS) [3-7]. During most of the asymptomatic stage of HIV infection, however, the count often remains both relatively constant and within the normal range [8]. Thus, it provides little insight into the activity of pathogenic processes during this period. Substantial biological and methodologic fluctuation in serial CD4+ cell counts also complicate the interpretation of CD4+ cell enumeration [7, 9]. Several cellular and serologic correlates for HIV disease progression, typically serum proteins indicative of immune system activation, have also been reported [3, 6, 10-21]. Although many of these markers seem to have predictive value, changes in them usually occur relatively late in the pathogenesis of HIV disease. The utility of these markers is also limited by the broad overlap in their distribution among persons with AIDS and healthy controls and by their lack of specificity for HIV-1 infection.
To overcome these difficulties, much interest has been directed toward potential virologic markers of HIV disease progression, including p24 antigenemia [22-26], infectious cell or plasma-associated virus [27-32], total plasma virus load [33-35], number of provirus DNA-containing peripheral blood mononuclear cells [29, 36-39], HIV messenger RNA (mRNA) expression in peripheral blood mononuclear cells [40-45] and lymphatic tissue [46, 47], and genotype and in vitro phenotype of the virus [48-53]. Of these, measurements of viral load and replication made by quantitating HIV-specific nucleic acids have shown particular promise. However, because of a lack of comprehensive longitudinal studies, the temporal relation between disease progression and HIV replication is still poorly understood.
The retroviral life cycle of HIV involves reverse transcription of the genomic RNA of the virus into DNA, which integrates into the genome of the infected host cell. The integrated HIV provirus is then transcribed, much like a cellular gene, to produce differentially spliced HIV mRNAs. Multiply spliced HIV mRNAs encode regulatory HIV proteins; unspliced HIV mRNA is used to translate structural viral proteins and serves as the genomic RNA for new virus particles. Unspliced HIV mRNA in cells can therefore result from the presence of virus particles in the specimen, from viral replication, or both, whereas the expression of multiply spliced HIV mRNA indicates replicative activity of the virus.
By quantitating HIV-1 mRNA in serial peripheral blood mononuclear cell samples collected during a longitudinal study from selected asymptomatic HIV-infected men, we found that increased viral replication appeared to precede immunodeficiency by at least 1 to 3 years [45]. High levels of HIV-1 replication in samples from persons who had normal CD4+ cell counts but who later developed AIDS suggested that cellular HIV mRNA expression reflects subclinical progression of the infection and might therefore serve as an early predictor for the development of AIDS. Recently, the results of two other studies examining peripheral blood mononuclear cell HIV mRNA expression in longitudinally collected specimens from HIV-infected persons were reported, confirming the potential predictive value of HIV mRNA expression [54, 55]. We examined the general utility of HIV-1 replication in peripheral blood mononuclear cells as a marker for HIV-1 disease progression by evaluating its association with future disease progression in a large cohort of HIV-infected persons who were subsequently followed for as long as 8 years.
Specimens tested were collected in 1984-1985 from a cohort of homosexual men in New York City [56, 57], many of whom had participated in hepatitis B virus infection and vaccine studies in the late 1970s [58]. Participants were enrolled in the Prospective AIDS Study in 1984 and returned for follow-up visits every 4.5 months for as long as 8 years. At follow-up visits, blood specimens were obtained and tested for various serologic and immunologic markers, and residual peripheral blood mononuclear cells were cryopreserved in liquid nitrogen. During follow-up, information was gathered on the clinical signs and symptoms of AIDS, and documentation of AIDS-defining illnesses [59] was obtained from the participants' physicians. We also searched the National Death Index (1979-1991) to identify deaths among participants. These searches were the only source of information on AIDS diagnoses for 5% of participants.
We detected anti-HIV-1 antibodies in stored and fresh serum specimens using a commercial enzyme-linked immunoassay and Western blot as defined by the Centers for Disease Control and Prevention [60]. ß2-Microglobulin levels were measured on freshly collected serum specimens using enzyme-linked immunoassay (Pharmacia, Piscataway, New Jersey). We detected p24 antigen in stored serum specimens using an enzyme-linked immunoassay without immune complex dissociation (Abbott Laboratories, North Chicago, Illinois).
For our study, cryopreserved specimens were randomly (without reference to the patient's subsequent disease patterns) selected from specimens collected in 1984 or early 1985 from 327 HIV-positive participants in the Prospective AIDS Study who had not developed an AIDS-defining disease by the time the specimens were taken. Men in whom an AIDS-defining disease had already been diagnosed were excluded because diagnosis of an AIDS-defining disease was the primary end point for the study. All study participants had been seropositive for HIV for at least 9 months before testing, which excluded the possibility that the test results might reflect events related to an acute HIV infection. A total of 327 men in the cohort met these criteria. The characteristics of the 150 of these men who had specimens tested were similar to those of the 177 men in the New York Blood Center cohort who did not have specimens tested (Table 1). When the analyzed specimens were collected, none of the participants was receiving or had previously received antiretroviral medication. ARTICLE
HIV-1 Messenger RNA in Peripheral Blood Mononuclear Cells as an Early Marker of Risk for Progression to AIDS
A characteristic feature of human immunodeficiency virus type 1 (HIV-1) infection is a highly variable latent period of disease that averages 9 to 10 years in length [1, 2]. The variation poses fundamental questions about the molecular basis of the pathogenesis of HIV disease and creates dilemmas about the clinical care of HIV-infected persons. To address these challenges, underlying host and viral factors that contribute to variation in disease progression need to be identified.
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Patients and Follow-up
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Quantitation of Cellular HIV-1 mRNA
One µ g of each total peripheral blood mononuclear cell RNA preparation [61] was reverse transcribed into complementary DNA (cDNA) using random hexamer primers, as described previously [62]. One tenth of each cDNA preparation was used as a template for radiolabeled quantitative polymerase chain reaction (PCR) reactions specific for multiply spliced HIV-1 mRNA, unspliced HIV-1 mRNA, or a cellular reference mRNA [45, 62]. Incorporation of radioactive deoxycytidine into the specific amplification products analyzed by gel electrophoresis was quantitated by a PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, California) for calculation of the copy numbers of multiply spliced and unspliced HIV mRNA molecules per µ g of total cellular RNA. For this purpose, an identical area of radioactivity above the specific amplification signal in each lane was subtracted. Standards of in vitro transcribed target RNAs diluted into RNA from HIV-negative donors indicated that the threshold of detection of the reverse transcriptase-initiated PCR assays for multiply spliced and unspliced HIV-1 mRNA was approximately 100 copies per µ g of peripheral blood mononuclear cell RNA. Quantitation of increasing amounts of target RNAs was linear over a range greater than two orders of magnitude. All specimens were tested under code, and the investigators doing the testing had no knowledge of the clinical or immunologic status of the study participants.
Statistical Analyses
In the case of HIV mRNA, data analyses related to the prediction of progression to an AIDS-defining disease from the time of collection of the specimen tested for HIV mRNA expression were based only on this specimen, whereas analyses of other markers of disease progression were based on the average of results from the specimen tested for HIV mRNA and from preceding and subsequent specimens (mean collection interval, ± 4.5 months) to minimize their inherent methodologic variability. To estimate the rate of decrease in CD4+ T-lymphocyte count at the time of testing, we calculated the percentage change from the original average CD4+ cell count to the value obtained approximately 1 year later (mean, 391 ± 68 days).
The significance of differences between groups in categorical variables was estimated using the chi-square or the Fisher exact test (two-tailed). Incidence of AIDS was calculated from the time of testing to the date of the first AIDS diagnosis (if no AIDS-defining disease was identified, to the last follow-up visit) using the Kaplan-Meier estimate of survival and the log-rank test to estimate the significance of differences in incidence between groups (two-tailed). In analyses of the risk for progression to AIDS, the Cox regression model for continuous variables was used. In addition, continuous variables were divided into categories of known normal or abnormal values (leukocyte count, p24 antigen level) or arbitrarily divided into groups of equal size (CD4+ cell count), or a combination of these strategies was used (HIV mRNA, percentage change in CD4+ cells, ß2-µglobulin). Relative risk (with the 95% CI) for AIDS from the time of testing was estimated using univariate and multivariate Cox proportional-hazards regression models (two-tailed) [63]. Statistical analyses were done using SPS software (Statistical Package for Social Sciences, SPS, Inc., Chicago, Illinois).
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The amounts of unspliced and multiply spliced HIV-1 mRNAs measured from the peripheral blood mononuclear cells of 150 HIV-infected persons showed a strong intrasample correlation with each other (0.71; P < 0.001). No prognostic significance for the remaining variance in their expression levels was noted, and almost identical conclusions could be reached by analyzing the data on either of these mRNAs. Because multiply spliced HIV-1 mRNA is a more unambiguous measure of HIV-1 replication, our analyses are based on quantitation of that (hereafter referred to as HIV mRNA).
Although none of the 150 men studied had had a diagnosed AIDS-defining disease when the analyzed specimens were collected, these men had a wide range of immunodeficiency states, as evidenced by the broad distribution of their CD4+ T-lymphocyte counts (range, 68 to 1923 cells/mm3). Only 6 men had a confirmed CD4+ T-cell count of less than 200 cells/mm3, which would merit the current definition for AIDS. For analysis, our study sample was arbitrarily divided into four evenly sized groups on the basis of their original average CD4+ cell counts (< 444, 444 to 624, 625 to 820, and more than 820 cells/mm3). We also separated the sample into subgroups on the basis of HIV-1 mRNA expression. Group A was composed of men whose cells showed no detectable HIV mRNA (n = 17) and who were considered to potentially be a biologically distinct group. The rest of the participants were arbitrarily assigned to four groups of equal size that had different levels of HIV mRNA expression (groups B, C, D, and E; see Figure 1).
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HIV mRNA and Development of AIDS
To determine the relation between HIV mRNA expression and subsequent clinical course, we examined the incidence of AIDS during follow-up in the different HIV RNA expression categories (A to E). In Figure 2, a Kaplan-Meier plot illustrates the cumulative incidence of AIDS among these persons from the time the tested specimen was collected, showing the capacity of HIV mRNA expression to predict the risk for progression to AIDS. By 3 years, AIDS had developed in 69% of men in the highest HIV mRNA expression category (group E), 34% of men in the next highest category (group D), only 6% of men overall in the two low-expressor categories (groups B and C), and none in the nonexpressor category (group A). After 7 years of follow-up, only 9% of persons with more than 4500 copies of HIV mRNA per µ g of cellular RNA (groups D and E) were alive and free of AIDS compared with 44% of the men who expressed lower but detectable levels of HIV mRNA (groups B and C).
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Most strikingly, with one exception, none of the men with undetectable HIV-1 replication in their peripheral blood mononuclear cells progressed to AIDS during 6 to 7 years of follow-up. In the single disparate case, two additional specimens, collected 4 months before and after the first analyzed sample, were tested. Both showed readily measurable HIV mRNA expression, strongly suggesting that the original result was artifactual. Because other persons were not similarly retested, however, statistical analyses in this case were based on the original negative result.
Although the rate of progression in HIV mRNA subgroups B and C differed from the rest of the categories, they did not differ from each other. The P values for the differences in the incidence of AIDS between the HIV mRNA expression groups shown in Figure 2 were as follows: A compared with B, P = 0.076; A compared with C, P = 0.019; A compared with D, P < 0.001, A compared with E, P < 0.001; B compared with C, P = 0.202; B compared with D, P < 0.001; B compared with E, P < 0.001; C compared with D, P = 0.006; C compared with E, P < 0.001; and D compared with E, P = 0.131. The lack of difference in progression between the subgroups B and C suggests that technically the assay may not be able to distinguish between the relatively small differences in HIV mRNA levels defining these categories or that this variation has no biological significance, or both.
It is worth noting, however, that when we excluded the participants with AIDS in whom Kaposi sarcoma was the only AIDS-defining illness during the follow-up period from the statistical analyses, the AIDS-free survival curves of the low-expressor groups (groups B and C) were distinctly separated from each other (data not shown). This is probably because men with no AIDS-defining illness other than Kaposi sarcoma were statistically significantly over-represented (P = 0.03) among the participants with AIDS in the low-expressor groups (groups B and C; 9 of 33 [27.3%]) compared with the high-expression groups (groups D and E; 5 of 53 [9.4%]). Thus, cellular HIV mRNA expression may reflect the risk for developing Kaposi sarcoma less well than it reflects the risk for developing other AIDS-defining conditions.
Human Immunodeficiency Virus mRNA and Other Markers of Disease Progression
Because CD4+ lymphocyte counts are known to correlate with the risk for AIDS, we also examined the incidence of AIDS in the four CD4+ cell count quartiles of the study cohort (< 444, 444 to 624, 625 to 820, and more than 820 cells/mm3; Figure 3). As expected, CD4 count correlated with the risk for progression to AIDS. However, in contrast to the similar analysis based on HIV mRNA Figure 2, CD4+ cell count did not identify a distinct subsample of long-term nonprogressors. Notably, the incidence of AIDS in the men in the two highest CD4+ cell count quartiles (625 to 820 and more than 820 cells/mm3) did not differ. Moreover, analysis of smaller subgroups within the highest CD4+ cell count category showed no prognostic differences (data not shown).
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The independent association of peripheral blood mononuclear cell HIV mRNA expression with future disease progression is further manifested by the statistical analyses shown in Table 2. This table shows relative risk values for the development of AIDS calculated by univariate and multivariate Cox regression analyses on the basis of HIV mRNA expression and a panel of known markers for HIV disease progression.
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The relatively large magnitude of the prognostic value of cellular HIV mRNA is shown by the univariate analysis. Notably, when the other five variables were considered in a multivariate analysis, HIV mRNA expression remained a strong independent marker for risk for AIDS. Because the number of men with undetectable HIV mRNA, used as the reference category, was relatively small and their incidence of AIDS was extremely low, the 95% CIs for the relative risks in the HIV mRNA expression categories are wide. However, when the multivariate analysis was repeated, excluding those in the nonexpressor category and using the HIV mRNA category 100 to 4500 copies/mu g as the reference group, the same variables as in Table 2 (including HIV mRNA more than 4500 copies/mu g) remained significant markers of risk for AIDS. Additionally, when HIV mRNA expression and CD4+ cell counts (transformed into log values because of their wide range) were analyzed in a Cox regression model as continuous variables instead of as categories of values, HIV mRNA and CD4+ cell count were each independently associated with progression to AIDS. In such an analysis, the relative risk for a 10-fold increase in HIV mRNA expression adjusted for variation in CD4+ cell count was 8.9 (95% CI, 4.9 to 20.4).
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Men whose peripheral blood mononuclear cells were confirmed to show no detectable sign of HIV replication constituted an interesting subgroup of the study cohort (16 of 150 [10.7%]). Because none of them developed AIDS during the next 6 to 7 years, lack of viral replication appears to indicate a benign prognosis. Moreover, it may be a characteristic feature of a subsample whose virus-host relation is biologically distinct from that of most HIV-infected persons. It is likely, however, that this subgroup is heterogeneous and consists of persons in whom disease is not progressing (long-term nonprogressors of HIV infection) as well as persons whose disease is progressing relatively slowly. Examination of HIV mRNA expression in more recent specimens from these persons could provide interesting insights into this issue. Identifying the relative contributions of factors specific to the virus and to the host in determining long-term nonprogression of HIV infection poses an important challenge for further investigations. Interestingly, evidence indicates that, at least in some cases, this may be due to the virus [64, 65].
The HIV load in plasma and peripheral blood mononuclear cell proviral DNA load have been shown to correlate with cellular HIV mRNA expression [41, 42, 44, 66, 67]. However, it should be noted that these three different virologic indices probably reflect the activity of HIV infection in distinct ways, and temporal changes in their relative levels during the course of HIV disease progression may not occur in concert. It has been reported that the number of provirus-carrying cells coincides with or shortly precedes the decrease in CD4+ cell count [29, 36, 37]. However, some recent studies [44, 68] have suggested that the increase in proviral load associated with disease progression is much less pronounced than was previously thought. Lee and coworkers [68] have suggested that proviral load stabilizes soon after seroconversion and may correlate with the severity of subsequent disease course.
Recently, the plasma HIV RNA load after seroconversion was reported to correlate with the clinical outcome of HIV infection [67, 69]. The prognostic value of plasma HIV RNA is of clinical importance, because its measurement is technically less demanding than quantitation of cellular HIV mRNA. However, despite the overall correlation between the amounts of cellular and cell-free HIV RNA in the peripheral blood, their relative levels in specimens from asymptomatic HIV-infected persons vary by as much as two orders of magnitude [42, 66, 67, 70]. Also, in contrast to cellular HIV mRNA [45], Hogervorst and colleagues [69] and Mellors and colleagues [67] either did not observe disease progression-associated increases in plasma RNA or observed them only after the patients had already developed severe CD4 lymphocytopenia. Thus, further studies are still needed to determine the predictive significance of plasma HIV RNA load during various stages of HIV infection and how this compares with the prognostic value of HIV mRNA expression.
The information on the future clinical course of the disease provided by quantitation of HIV-1 mRNA in peripheral blood mononuclear cells of asymptomatic persons is striking when one considers that much of HIV replication during the early part of the disease appears to take place in the lymphatic tissues [46, 47]. It has been suggested that active HIV replication in peripheral blood cells indicates that substantial destruction in the histologic architecture of the lymphatic tissues has already occurred [1]. Nevertheless, our results indicate that the rate of HIV replication measured in cells in the periphery generally gives a good estimate of the state of disease progression in asymptomatic persons.
In conclusion, the specificity and early predictive value of peripheral blood mononuclear cell HIV mRNA suggests that it may be a useful clinical marker for monitoring HIV disease progression throughout the course of infection. On the basis of our results, it can be suggested that HIV mRNA quantitation should be considered as a diagnostic tool in clinical studies on the pathophysiology and treatment of HIV infection, for example, in the prognostic evaluation of persons participating in trials of antiretroviral compounds. Although the technical and biological aspects of quantitating HIV mRNA in peripheral blood mononuclear cells need further attention, valuable uses for this approach can also be envisioned in more general applications in patient care and counseling.
Drs. Stevens, Taylor, and Rubinstein: The New York Blood Center, 310 East 67th Street, New York, NY 10021.
Dr. Baltimore: Massachusetts Institute of Technology, Building 68, Room 380, 77 Massachusetts Avenue, Cambridge, MA 02139.
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