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. 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. Methods Patients and Follow-up 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]. 2-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. Table 1. Cohort of Homosexual Men in New York City: Comparison of Anti-HIV-1-Positive Men Who Were or Were Not Tested for HIV-1 Messenger RNA Expression* Quantitation of Cellular HIV-1 mRNA One 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 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 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 C