Selective regulation of human immunodeficiency virus-infected CD4(+) lymphocytes by a synthetic immunomodulator leads to potent virus suppression in vitro and in hu-PBL-SCID mice

Abstract

We have previously observed that the synthetic immunomodulator Murabutide inhibits human immunodeficiency virus type 1 (HIV-1) replication at multiple levels in macrophages and dendritic cells. The present study was designed to profile the activity of Murabutide on CD8-depleted phytohemagglutinin-activated lymphocytes from HIV-1-infected subjects and on the outcome of HIV-1 infection in severe combined immunodeficiency mice reconstituted with human peripheral blood leukocytes (hu-PBL-SCID mice). Maintaining cultures of CD8-depleted blasts from 36 patients in the presence of Murabutide produced dramatically reduced levels of viral p24 protein in the supernatants. This activity correlated with reduced viral transcripts and proviral DNA, was evident in cultures harboring R5, X4-R5, or X4 HIV-1 isolates, was not linked to inhibition of cellular DNA synthesis, and did not correlate with beta-chemokine release. Moreover, c-myc mRNA expression was down-regulated in Murabutide-treated cells, suggesting potential interference of the immunomodulator with the nuclear transport of viral preintegration complexes. On the other hand, daily treatment of HIV-1-infected hu-PBL-SCID mice with Murabutide significantly reduced the viral loads in plasma and the proviral DNA content in human peritoneal cells. These results are the first to demonstrate that a clinically acceptable synthetic immunomodulator with an ability to enhance the host's nonspecific immune defense mechanisms against infections can directly regulate cellular factors in infected lymphocytes, leading to controlled HIV-1 replication.

FIG. 1

Murabutide suppresses HIV-1 replication in CD8-depleted lymphocytes from HIV-1 patients. CD8-depleted PBMCs from 36 patients were activated with PHA for 2 to 3 days, and nonadherent lymphocytes were then collected, washed, and cultured in medium containing 10 U of IL-2/ml in the absence or presence of Murabutide. Culture supernatants were harvested over a 2-week period and were tested for p24 Ag secretion by ELISA. (A) Cultures were left unstimulated or were stimulated with 0.1 to 100 μg of Murabutide/ml, and supernatants were tested 10 days later for p24 content. Results are means ± standard errors of the mean of values of samples from four different patients. (B) Levels of p24 release in 8- to 10-day cultures from 10 asymptomatics, 16 symptomatics, and 6 AIDS patients are shown as median values. (C) Kinetics of p24 release over a 14-day culture period was evaluated in unstimulated (white symbols) and Murabutide-stimulated (black symbols) cultures from three different patients. (D) The mean percent inhibition (± standard errors of the mean) by Murabutide (10 μg/ml) is presented for p24 release in 8- to 10-day cultures harboring X4, X4-R5, and R5 HIV-1 isolates. The recovery and analysis of coreceptor specificity for each of the 23 tested isolates are explained in Materials and Methods. ∗, significantly lower levels (P < 0.05; Wilcoxon matched-pair test) for treated cultures than for untreated (Medium) cultures.

FIG. 2

Murabutide enhances cell viability and cellular proliferation. CD8-depleted PHA-activated lymphocytes from 13 HIV-1 patients were cultured at 5 × 10⁵ cells/ml in the absence or presence of Murabutide (10 μg/ml). The percent (A) and the number (B) of viable cells were evaluated by trypan blue exclusion 7 and 14 days after culture initiation. (C) CD8-depleted blasts were cultured at 10⁵ cells/well and were maintained for a period of 4 days. During the last 16 h prior to harvesting, cultures were pulsed with 0.5 μCi of [³H]thymidine/well, and the amount of radioactivity incorporated into the DNA of dividing cells was measured using a β-counter. Results are means ± standard errors of the mean. ∗, significantly higher levels (P < 0.05; Wilcoxon matched-pair test) for treated cultures than for untreated (Medium) cultures.

FIG. 3

Murabutide inhibits HIV-1 gene expression and viral DNA content in endogenously infected CD8-depleted blasts. Following PHA activation, CD8-depleted blasts from four HIV-1 patients were cultured in the absence or presence of 10 μg of Murabutide/ml. (A) Total RNA was extracted after a 3-day culture period, and samples (33, 100, and 300 ng) were subjected to RT-PCR amplification with primer pair GAG06-GAG04 to detect unspliced Gag or Pol mRNA and with primer pair BSS-KPNA to detect intermediate-size singly spliced viral transcripts. These mRNAs were named on the basis of the exons they contain and the proteins they produce (41). Constitutively expressed β-actin mRNA in the same samples was also amplified. An equivalent amount of RNA from the 8E5 cell line was used as positive control for RT-PCR amplification. The HIV-1 isolates in cultures from patients 1 and 2 were X4 and R5 tropic, respectively. (B) Total DNA was extracted after 5 days of culture in the absence or presence of Murabutide, and various concentrations (5.6, 28, 140, and 700 ng) were subjected to PCR amplification with primer pair GAG06-GAG04 to detect the HIV-1 gag gene. Cell equivalence was determined by amplification of the β-globin housekeeping gene. HIV-1 isolates from cultures of patients 3 and 4 presented X4 and R5 tropism, respectively.

FIG. 4

Murabutide suppresses c-Myc mRNA accumulation. PHA-activated CD8-depleted lymphocytes from four HIV-1 patients were cultured in the absence or presence of Murabutide (10 μg/ml). After 2, 6, and 24 h, total RNA was extracted and various concentrations (20, 100, and 500 ng) were subjected to RT-PCR amplification using c-Myc- or GAPDH-specific primer pairs. (A) The intensity of the PCR products revealed by ethidium bromide staining is shown for a sample from one representative patient after a 6-h stimulation period. (B) The percent inhibition by Murabutide of c-Myc expression in cultures from four patients is presented at the different time points tested. Horizontal bars reflect the mean percent inhibition.

FIG. 5

HIV-1 coreceptor expression and cytokine release are regulated by Murabutide in CD8-depleted lymphocyte cultures. PHA-activated CD8-depleted lymphocytes from 13 HIV-1 patients were cultured for 4 days in the absence or presence of Murabutide (10 μg/ml). Cells were then washed, and the CD4⁺/CD3⁺ population was analyzed by three-color flow cytometry for the level of expression of either CCR5, CXCR4, or CD25. Results are shown as means (± standard errors of the mean) of the percent of positive cells (A) and MFI (B). ∗, significantly lower (P < 0.05; Wilcoxon matched-pair test) receptor expression for treated cultures than for untreated (Medium) cultures. (C) CD8-depleted cultures from 26 HIV-1 patients were maintained for 2 days with or without 10 μg of Murabutide/ml. Supernatants were then analyzed for the content of the indicated cytokines and chemokines by using commercially available ELISA kits. Levels of secreted IFN-γ and IL-16 were evaluated only on samples from 11 and 17 patients, respectively. Values shown are means ± standard errors of the means. ∗, significantly higher (P < 0.05, Wilcoxon matched-pair test) cytokine levels for treated cultures than for untreated (Medium) cultures. (D and E) Untreated and Murabutide-treated CD8-depleted cultures from HIV-1 patients harboring R5 isolates were maintained in the absence or presence of 150 μg of normal goat IgG/ml or of 50 μg of each of the three neutralizing goat IgG Abs/ml against MIP-1α, MIP-1β, and RANTES. The sums of the levels of the three β-chemokines in the supernatants (D) and of viral p24 protein (E) were analyzed by ELISA kits after a 3- and 7-day culture period, respectively. Results are representative of those of three patients.

FIG. 6

Treatment with Murabutide suppresses plasma viral loads and viral DNA levels in HIV-1-infected hu-PBL-SCID mice. Two weeks after repopulating SCID mice with hu-PBL, HIV-1_Ba-L was administered intraperitoneally at a dose equivalent to 50 kcpm of viral RT activity. Starting 2 h after infection and for the following 13 days, groups of mice were treated daily with Murabutide (10 mg/kg of body weight) or with the excipient (PBS) by injecting a volume of 400 μl intraperitoneally. (A) Viral loads were quantified in plasma samples 24 h after the last administration by using Amplicor Monitor HIV-1 kits. (B) The levels of human IgG (B) in the same samples were evaluated by ELISA. Results are values for individual mice (four to six per group) from six independent experiments and are represented by a different symbol for each experiment. Horizontal bars reflect the median values of pooled data from all experiments. (C) Peritoneal cells from PBS- or Murabutide-treated mice were collected 24 h after the last injection, and an equivalent number of cells were pooled from all mice in each group. Total DNA was extracted, and various concentrations (2, 10, 50, and 250 ng) were subjected to PCR amplification with primer pair GAG06-GAG04 to detect the HIV-1 gag gene. Cell equivalence was determined by amplification of the human β-globin housekeeping gene, and DNA extracts from 8E5 cells were used as PCR standards. Since the 8E5 cells contain one integrated provirus copy per cell, the limit of detection of our PCR was 40 copies (or 40 cells) in 0.26 ng of human DNA. Results are representative of four experiments.