Highly active antiretroviral therapy (HAART) reduces HIV-1 RNA in plasma to <50 copies/mL in most patients but cannot eradicate HIV-1. Virus rebounds rapidly after treatment cessation and stabilizes at levels comparable to those seen before treatment.4,5 Investigations in patients with controlled discontinuation of treatment or during treatment-free intervals of structured treatment interruptions (STIs) have demonstrated that the viral rebound occurs within days.6,7
Although the dynamics of the HIV-1 RNA rebound in plasma are thus well established, little is known about the behavior of viral proteins, among which the p24 antigen (p24) is measured easily. During the past decade, p24 measurement has been highly improved by measures that include heat denaturation of interfering antibodies, signal amplification, and use of an efficient virus disruption buffer.8 These improvements have rendered the test more accurate and more sensitive.9-15 The p24 concentration is as predictive of disease progression in early and late disease as is HIV-1 RNA.16,17 The measurement of p24 has potential for antiretroviral treatment monitoring in adult and pediatric HIV-1 infection.9-14
Of note, extravirally located p24 has been measured in some patients with stably suppressed viremia, and the changes in concentrations of p24 and CD4+ T cells in such patients were inversely correlated.18 Here, we have investigated the dynamics of p24 during 52 weeks of an STI trial.19 Patients interrupted treatment 4 times for a 2-week period, each followed by 8 weeks of retreatment and, finally, for a period of at least 12 weeks, which permitted the virus to rise to a new set point.20-23 We relate the changes in concentrations of p24 to those observed for HIV-1 RNA and CD4+ T cells.
MATERIALS AND METHODS
Patients and Specimens
We investigated patients of the Swiss-Spanish Intermittent Treatment Trial (SSITT).19 They had started HAART during chronic HIV infection, had been taking 2 classes of drugs (nonnucleoside reverse transcriptase inhibitors excluded) for >6 months, and had presented for >6 months with an HIV-1 RNA level <50 copies/mL in plasma and a CD4+ T-cell count of >300 cells/μL. During the SSITT, the patients underwent 4 cycles consisting of a 2-week treatment interruption followed by an 8-week retreatment phase with the identical drug combination used before the trial. At the fifth and final cycle (week 41 onward) treatment was suspended for >12 weeks if no adverse effects occurred. Weeks 1 through 42 of the trial were used for studying the reappearance of p24 during 5 2-week treatment-free intervals spanning weeks 1 through 2, 11 through 12, 21 through 22, 31 through 32, and 41 through 42. Weeks 39 through 52 were used to study associations between p24, HIV-1 RNA, and CD4+ T cells in a situation of prolonged viral rebound up to a new set point. Patients were included in the present investigation if (1) they had been treated at one of the participating Swiss centers, (2) they had completed the first 4 STIs and started the fifth long STI, and (3) frozen plasma was available for p24 testing. Of the total 133 participants of the SSITT, 65 patients (48.9%) met all criteria; they had a follow-up interval of 44 weeks. One patient each dropped out after weeks 44, 48, and 50. Sixty-two of the patients studied here thus completed the study up to week 52.
CD4+ T cell counts were determined prospectively at weeks 0, 9, 19, 29, 39, 44, 48, and 52. HIV-1 RNA concentration in plasma determined prospectively by the ultrasensitive Amplicor Monitor version 1.5 test, with a quantification limit of 50 copies/mL, was available from weeks 0, 2, 9, 12, 19, 22, 29, 32, 39, 42, 44, 46, 48, 50, and 52. HIV-1 RNA concentration before initiation of HAART was available from the Swiss HIV Cohort Study database. Concentrations of cell-associated unspliced HIV-1 RNA and proviral DNA at week 0 were available from study files and had been determined batchwise as described elsewhere.24,25 The p24 was tested retrospectively at the same time points at which HIV-1 RNA had been tested. All remaining plasma samples, stored at −70°C, were analyzed after heat denaturation by a signal amplification-boosted enzyme-linked immunosorbent assay (ELISA).8 All specimens of a given patient were tested on the same ELISA plate, thus minimizing test-associated variation. The HIV-1 p24 Ag Ultra kit (Perkin Elmer Life Sciences) was used according to the manufacturer's instructions except for the following crucial modification of sample pretreatment: 100 μL of plasma was added to an Eppendorf tube containing 50 μL of a mixture comprising 30 mM of Tris/HCl pH 7.2, 450 mM of NaCl, 1.5% Triton X-100, 1.5% deoxycholic acid (sodium salt), 0.3% sodium dodecylsulfate, and 10 mM of ethylenediamine tetra-acetic acid (EDTA), mixed well and left for 10 minutes at room temperature. After further dilution with 450 μL of the kit's prediluted Dissociation Solution, the sample was boiled for 5 minutes on a Techne dry heat block preheated to 100°C. After cooling down to room temperature, 250 μL of the mixture was tested in duplicate wells according to the manufacturer's instructions. Concentrations were deduced by end point reading from a 3-fold dilution series of standards extending from 170 to 10,000,000 fg/mL. Frozen aliquots of prediluted standards were used in all experiments to reduce intertest variation. Reactivity in the test was related to a quantitative cutoff based on 4 negative controls run on every assay plate. As in earlier studies this cutoff equaled the mean plus 3 standard deviations (SDs) of these negative controls.9,17,18
Evaluation and Statistics
For statistical analysis, the StatView 5.0 program for Macintosh (SAS Institute, Cary, NC) was employed. The Wilcoxon signed-rank test and paired t tests were used for analyzing differences in concentrations. The Spearman rank correlation, Pearson correlation, and multivariate linear regression analysis were used for assessing correlations. All concentrations were evaluated as log10-transformed values. HIV-1 RNA <50 copies/mL was set to 50 copies/mL. The p24 concentrations were entered as calculated based on the standard curve, irrespective of whether reactivity was above or below the cutoff. This is justified because the concentrations directly represent the recorded absorbance readings (ie, experimentally determined values) and because many of the statistical analyses remained significant even when only p24 concentrations below the cutoff were considered, as detailed in the Results section. HIV-1 RNA was always above the limit of quantification when analyzed together with p24.
We investigated 65 patients, 39 men and 26 women, with a median age of 40 years (range: 22-68 years), who had received antiretroviral treatment for 27.0 months (median, range: 8.5-44.6 months) and had presented with <50 copies/mL of plasma viremia for 24.7 months (median, range: 6.2-42.8 months). On entry into the study, these patients interrupted their individual treatment during weeks 1 through 2, 11 through 12, 21 through 22, 31 through 32, and, finally, for a 12-week period beginning with week 41. p24 persists during long-term suppressed viremia. At time point 0 of the study, whereas HIV-1 RNA was <50 copies/mL in all patients, p24 reactivity was above the cutoff in 22 (34%) patients. At time point 0, p24 exhibited no dependence on the pre-HAART viral load, the total duration of HAART or of viremia <50 copies/mL under HAART, the gain in CD4+ cells while receiving HAART, the concentrations of peripheral blood mononuclear cell (PBMC)-associated proviral DNA or HIV-1 RNA at time point 0, age, or gender (all correlations <0.2; P > 0.15). Positive results were found in patients who had maintained <50 copies/mL during a median time of 25.4 months (range: 6.2-38.9 months). The fraction with p24 reactivity above the cutoff remained similar at the end of each retreatment period (ie, at weeks 9, 19, 29, and 39) (Fig. 1).
p24 Increases Little During Structured Treatment Interruptions
During all 5 STIs, the concentrations of p24 rose little compared with HIV-1 RNA (Fig. 2). During the first STI, mean viral RNA rose from <50 copies/mL or <1.7 log10 to 2.36 log10 (ie, by >0.66 log10). In contrast, mean p24 increased by only 0.19 ± 0.37 log10. For weeks 12, 22, 32, and 42, the respective mean increases of p24 were 0.11 ± 0.36, 0.19 ± 0.31, 0.17 ± 0.26, and 0.08 ± 0.30 log10. The resulting ratios by which the increases of HIV-1 RNA exceeded those of p24 were >5.2, >5.6, >4.7, and >4.8 (average: >4.6 ± 1.0). Despite their low magnitude, the p24 increases during successive 2-week STIs were statistically significant in cycles 1, 3, and 4 (Wilcoxon test, P < 0.0001 for all 3 cycles) and weakly significant for the first 2 weeks of cycle 5 (P = 0.042). The p24 increase during the first STI but not the subsequent short STI correlated significantly with the corresponding HIV-1 RNA peak (Spearman ρ: 0.393; P = 0.005). Concentrations of p24 at many time points off therapy, namely, at weeks 12, 22, 32, 44, 46, 48, and 50, correlated significantly with those of HIV-1 RNA before initiation of HAART (Spearman ρ: 0.26-0.48; P = 0.0003 to P = 0.05). Multivariate linear regression analysis indicated that p24 at time point 0 and pre-HAART viral load were independent predictors of p24 levels at weeks 2, 12, 22, and 32 (Table 1A). The influence of p24 at time point 0 was always stronger than that of the pre-HAART viral load.
The p24 also increased less during the long STI than did the HIV-1 RNA (see Fig. 2). Mean HIV-1 RNA rose rapidly from <50 copies/mL (<1.7 log10) at week 39 and reached a peak of 3.6 log10 at weeks 46 through 48. The mean increase amounted to >1.9 log10 (ie, >80-fold). For p24, the overall mean increase was approximately 0.5 log10 (3.2-fold). The relative increase of p24 was at least 25-fold lower than that of HIV-1 RNA. Nevertheless, p24 and HIV-1 RNA concentrations correlated significantly at all time points (Spearman ρ: 0.33-0.61; P = 0.001 to P = 0.05). The p24 levels during this period were, however, also markedly influenced by the p24 concentration at time point 0. Indeed, p24 at time point 0 was correlated significantly or at least showed a trend of correlation with all subsequently measured p24 concentrations up to and including week 52 (Spearman ρ: 0.27-0.53; P < 0.001 to P < 0.07). This even remained the case if only patients with a p24 level below the cutoff at time point 0 were analyzed, indicating that even p24 levels below the cutoff represented true concentrations rather than chance values.
We investigated by multivariate linear regression analysis how p24 concentration at different time points during the final long STI was influenced by the concomitant concentration of HIV-1 RNA and p24 at time point 0 (see Table 1B). All models were adjusted for age, gender, and other parameters listed at the bottom of the table. The p24 at time point 0 was a stronger correlate of p24 during viral rebound than was the concomitant HIV-1 RNA, which was also independently significant at all time points however. Pre-HAART viral load had no significance.
The Rebound of p24 After Treatment Stop Correlates with CD4+ T Cell Loss
As seen from Figure 2, there was a noticeable drop in CD4+ cell counts during weeks 39 through 44 (−0.094 log10; Wilcoxon signed-rank test, P < 0.0001). Subsequent changes lacked significance despite considerable individual change. The pattern of CD4+ cell count changes in the patients studied here was in accordance with that seen in the whole SSITT.19
We investigated whether CD4+ cell counts or their changes during the final STI correlated with the respective concentrations or changes of the 2 viral markers. CD4+ cell counts at weeks 44, 48, or 52 did not correlate with HIV-1 RNA at any time point. There was also no correlation of the CD4+ cell count changes between any of these time points with the concomitant or preceding HIV-1 RNA changes (not shown). In contrast, there were multiple correlations between CD4+ cell counts and p24 at different time points (Table 2).
Multivariate linear regression analysis was used to investigate the factors that determined the CD4+ cell count changes in the final STI (Table 3). The models investigate how the CD4+ cell count changes during 3 time intervals were influenced by the concentrations of CD4+ cells and p24 at the beginning of the respective interval and by the p24 changes during the interval. We also investigated a reported effect of the gain in CD4+ cells during uninterrupted HAART preceding the study on the CD4+ cell loss after treatment cessation.26 All models were also adjusted for gender, age, and time of HIV-1 RNA <50 copies/mL during uninterrupted HAART. The CD4+ cell count drop observed during weeks 39 through 44 was independently associated with the CD4+ count at week 39 and the p24 increase during weeks 42 through 44 (model 1). Further changes in CD4+ cell counts during weeks 44 through 48 were associated with CD4+ cell counts at week 44 and with p24 at week 44 and its change during weeks 44 through 48 (model 2). These factors remained significant when the entire interval spanning weeks 39 through 48 was examined (model 3). No significance was found for the CD4+ cell count gained during uninterrupted HAART or for any variable listed at the bottom of the table. Similar findings were seen for the change in nontransformed CD4+ cell numbers (not shown). None of the parameters was significant regarding the CD4+ cell count change in the interval during weeks 48 through 52. Thus, during the first 8 weeks of the final STI, p24 was a significant correlate of CD4+ count change.
We used an STI trial to study the dynamics of HIV-1 p24 on treatment withdrawal. We found that (1) p24 in plasma persisted in one third of patients after a median of 2 years of stably suppressed viremia (see Fig. 1), (2) levels of persisting p24 were affected little during short STIs (see Fig. 2), (3) the concentrations of the persisting p24 and rebounding RNA determined the concentration of the p24 rebound after final treatment cessation (see Table 1), and (4) the rebounding p24 correlated inversely with the CD4+ cell count changes seen during the first 8 weeks of the final STI (see Tables 2, 3).
STI was originally proposed as an “autovaccination” concept by which the immune system of patients with suppressed virus replication under HAART would be boosted by brief intervals of virus replication. Results of STI studies have shown, however, that virus control in the absence of HAART is rare.19 The autovaccination concept implies that patients successfully treated by HAART lack viral antigen and that sufficient viral immunogen is produced during STI. Our findings suggest that neither expectation was met in the SSITT. At least 1 important protein, p24, remained measurable in many patients (see Fig. 1), and it is likely that other viral proteins persisted as well. The p24 concentrations at time point 0 clearly did not represent random values or technical artifacts because they were firmly correlated with p24 at multiple other time points (see Table 1). The persistence of p24 in patients with HIV-1 RNA <50 copies/mL under HAART has recently been confirmed by preliminary results of studies using immuno-polymerase chain reaction (PCR)27 or new ultrasensitive nanoparticle technology.28 The latter study also identified persisting nucleocapsid p7, which is cleaved from the same precursor Pr55Gag as p24.
Although more sensitive tests may detect residual HIV-1 RNA in a similarly high proportion,29-32 the p24 detected in our patients cannot be the structural p24 contained in the few virus particles present in plasma.33 Based on an estimate of 2000 to 4000 p24 molecules contained in 1 virus particle,34 6150 to 12,500 particles (or 12,500-25,000 copies of viral RNA) are needed for 1 pg of particle-associated p24. The mean concentration of p24 at week 0, approximately 1 pg, exceeded the maximum of particle-associated p24 present in a plasma with <50 copies/mL by 3 orders of magnitude.
Ultracentrifugation experiments have shown that in contrast to HIV-1 RNA and reverse transcriptase, most of the p24 present in chronically infected patients cannot be pelleted and is located outside virus particles as immune-complexed protein or free protein of essentially unknown origin.35 The absence of a correlation between p24 at time point 0 and the pre-HAART viral load, the duration of HAART, or the duration of viremia <50 copies/mL in this study suggests that this p24 does not represent protein remaining from the time before HAART. Lack of correlation with PBMC-associated HIV-1 RNA or DNA at time point 0 also suggests that PBMCs are not a major source. The p24 may instead be derived from ongoing virus replication in reservoirs not represented by HIV-1 RNA in plasma. Persisting viral protein may also have implications for the concept of therapeutic vaccination in such patients. The persistence of viral protein after prolonged HAART is in agreement with the increased CD8+ T-cell activation state of such patients compared with controls negative for HIV-1.36,37 It is tempting to speculate that this residual immune activation is attributable to p24 and other persisting viral antigens. This hypothesis is testable but exceeds the scope of this paper.
With regard to the dose aspect of the autovaccination concept, it is evident that the viral rebounds during short STIs, although leading to sizable increases in HIV-1 RNA,7 had a limited additive effect, with average p24 increases of only 0.08 to 0.2 log10 (ie, 20%-60%) (see Fig. 2), thus rendering a pronounced strengthening of the antiviral immune response unlikely. Indeed, during the 4 short STIs, the CD8+ T-cell responses in half of the patients of the SSITT appeared with a delay or not at all, although many patients exhibited stronger responses after the final STI, when more virus was produced. Thus, the short STI may indeed have produced a suboptimal dose of viral immunogen.38 It would be of interest to analyze the CD8+ T-cell responses individually with respect to the p24 concentrations present; however, again, this exceeds the scope of our study. We finally found that the changes in CD4+ cells observed during the first 8 weeks after the final treatment stop correlated inversely with the respective changes in p24 concentration. This is in agreement with an evaluation of 115 patients from 3 prospective STI trials in which the increase in HIV-1 RNA during the first 4 weeks of the final STI was also found to be inversely correlated with the early decline in CD4+ cells,39 although such a correlation was not found in the present small study. Our findings contrast sharply with those of another STI study, which found a lack of longitudinal intrapatient correlation between p24 and HIV-1 RNA; it did not find a significant increase of p24 during short or long STIs, which lasted for up to 12 months.40 The discrepancies are likely attributable to the fact that we used an external virus disruption buffer for sample pretreatment, whereas Prado et al40 used the kit's 0.5% Triton X-100 buffer. Previous work has demonstrated the effectiveness of our new sample pretreatment procedure.18,41,42
In conclusion, the results of this study suggest that p24 is a viral marker that has distinct properties, although it correlates with HIV-1 RNA. The presently unclear meaning of p24 persistence under successful long-term HAART should be elucidated and eventually assessed for its relevance to treatment success. The persistence of viral protein would also seem to be of relevance to therapeutic vaccination studies. The possibility of an influence of persisting viral protein on the outcome of such studies should be assessed.
The authors thank their patients for their commitment, C. Schneider and R. Hafner for excellent patient care, and F. Burgener and H. Kuster for laboratory support.
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The members of the Swiss HIV Cohort Study are M. Battegay, E. Bernasconi, J. Böni, H. Bucher, Ph. Bürgisser, S. Cattacin, M. Cavassini, R. Dubs, M. Egger, L. Elzi, P. Erb, K. Fantelli, M. Fischer, M. Flepp, A. Fontana, P. Francioli (President of the Swiss HIV Cohort Study, Centre Hospitalier Universitaire Vaudois, Lausanne), H. Furrer (Chairman of the Clinical and Laboratory Committee), M. Gorgievski, H. Günthard, B. Hirschel, L. Kaiser, C. Kind, Th. Klimkait, B. Ledergerber, U. Lauper, D. Nadal, M. Opravil, F. Paccaud, G. Pantaleo, L. Perrin, J.-C. Piffaretti, M. Rickenbach (Head of Data Center), C. Rudin (Chairman of the Mother and Child Substudy), P. Schmid, J. Schüpbach, R. Speck, A. Telenti, A. Trkola, P. Vernazza (Chairman of the Scientific Board), R. Weber, and S. Yerly.