In a pilot study of a larger 32-week double-blind placebo-controlled clinical trial, we examined the effect of highly active antiretroviral therapy (HAART) and a HIV-1-specific immune-based therapy compared with HAART alone on peripheral blood mononuclear cell (PBMC)-associated HIV-1 RNA and DNA in CD4 and CD14 positive cells by ultrasensitive fluorescence in situ hybridization (UFISH) or polymerase chain reaction (PCR) in situ hybridization (PISH)[2-8]. PISH has been previously used to quantify the number of HIV-1-infected PBMCs  and CD4 T cells  in antiretroviral-naïve patients. UFISH has been previously used to quantify the number of productively infected monocytes, CD8 cord blood cells, and CD4, CD45RO T lymphocytes in patients on and off antiretroviral therapy. Using UFISH, our laboratory has determined that adoptively transferred anti-HIV CD8 T cells effectively reduce the number of HIV-infected cells in peripheral blood. This technology also allowed us to show persistent gag-pol expression in T lymphocytes from HIV-infected patients with undetectable viral loads on HAART therapy. Previously, treatment with this HIV-1-specific immune-based therapy (inactivated gp120-depleted HIV-1 antigen in incomplete Freund‚s adjuvant (IFA), HIV-1 immunogen, REMUNE®) had shown an augmentation of HIV-1-specific cell-mediated immune response in subjects with chronic asymptomatic HIV-1 infection. For this pilot study, five protease-inhibitor-naive subjects who received HAART (zidovudine, lamivudine, indinavir) plus HIV-1 immunogen were compared with three protease-inhibitor-naive subjects who received HAART plus IFA control.
Materials and methods
Eight HIV-1-seropositive protease-naive individuals enrolled at one clinical site were evaluated using dual immunophenotyping/PCR in situ hybridization for cells containing HIV-1 DNA and dual immunophenotyping/ FISH for cells containing HIV-1 RNA. Informed consent was obtained from all participants before enrollment into this study. PBMCs were isolated from fresh anticoagulated blood layered on a Histopaque 1077 (Sigma, St. Louis, Missouri, USA) discontinuous density gradient and centrifuged at 600 g for 30 min at ambient temperature. The turbid layer was removed, washed twice with three volumes of RPMI and once with phosphate-buffered saline (PBS).
HIV-1 immunogen (REMUNE®; Immune Response Corporation, Carlsbad, CA, USA) is composed of an HIV-1 isolate (HZ321) from serum collected from a patient in Zaire in 1976 which has been recently sequenced and classified as clade A envelope and clade G gag. HIV-1 immunogen is obtained by concentration and purification from the supernatant fluid of HZ321-infected Hut-78 cells. In the preparation of the immunogen, envelope gp120 is depleted during freezing, thawing and the purification process. Antigen preparations were viral inactivated through a sequential application of beta-propiolactone (BPL) and 60Co irradiation. For injection, the antigen (10 units) is emulsified in incomplete Freund‚s Adjuvant (IFA) for intramuscular injection. Injections of HIV-1 immunogen or IFA were given at week 4, 16, and 28 weeks in this study. Over 3000 HIV-1-seropositive individuals have participated in trials of the HIV-1 immunogen with the most common side-effect reported being transient local injection site reactions.
Cells and cell lines
The ACH-2 cell line (AIDS Research and Reagent Program, NIAID, NIH, Bethesda, Maryland, USA), containing a single copy of integrated HIV-1 proviral DNA per cell, was harvested at an early passage number and used as the HIV-1-infected cell copy number control. ACH-2 cells were stimulated to produce virus with 0.1 μg/ml TNF-agr; (Sigma).
PCR-driven in situ hybridization
Cell samples were adjusted to a final concentration of 1×106 cells/l. The supernatant was removed and the cell pellet was resuspended in 90μl of PBS and 10μl of biotinylated anti-CD4 or CD14 (PharMingen, San Diego, California, USA). Cells were again centrifuged at 300 to 600 g for 2 min and the cell pellet was washed in PBS and once in 1×PCR buffer (PE Applied Biosystems, Foster City, California, USA). Cells were fixed and permeabilized by the addition of 50μl of Permeafix (Ortho Diagnostics, Raritan, New Jersey, USA) at ambient temperature for 60 min. HIV-1 DNA was amplified in situ as previously described. Appropriate positive and negative controls amplified with or without the addition of Taq polymerase were simultaneously run with each sample. Following the last wash, the cells were resuspended in 80μl of PBS and 20μl streptavidin-phycoerythrin (PE) and incubated for 30 min at ambient temperature. The cells were then washed in PBS as described above.
Dual immunophenotyping/in situ hybridization
Duplicate samples of cells (1×106) were labeled with optimized concentrations of phycoerythrin-conjugated antibodies specific for the cell types of interest (CD4, CD14) (PharMingen, Inc.) and fixed and permeabilized by the addition of 50 μl of Permeafix (Ortho Diagnostics) per 106 cells at ambient incubation temperature for at least 60 min and up to 18 h. The cells were then washed twice in PBS, pH 7.4 at ambient temperature and once in 2×SSC (0.3M sodium chloride, 0.03M sodium citrate) at ambient temperature. The cells were then resuspended with 50 μl hybridization buffer containing a cocktail of 5-carboxyfluorescein-labeled oligonucleotides specific for HIV gag-pol MRNA (MolPath, Inc., Frankfort, Michigan, USA). The probe was hybridized to target for 30 min at 43ºC in a water bath. The cells were washed for 5 min with 2 × SSC, 0.1% Triton X-100 at 42ºC, and 15 min with 0.1 × SSC, 0.1% Triton X-100 at 42ºC. Multi-parameter analysis of cell surface molecules and HIV RNA was performed on a Coulter XL flow cytometer (Beckman-Coulter, Miami, Florida, USA).
In cell analysis of specific cell populations was performed as previously described[2,6]. Plasma viral load was measured by the Hoffman La Roche Ultrasensitive assay (Hoffman La Roche, Nutley, New Jersey, USA).
The 95% confidence intervals are presented for the two treatment groups at baseline and week 32. For comparison at baseline between the groups the Mann-Whitney U test was performed. For analysis of plasma RNA between the groups of plasma RNA at week 32, as all subjects reached the lower limit, means were calculated and a one-sample t test was performed (Ho: μ=40). Spearman rank correlation was performed to examine associations.
Effects of immunogen on plasma viral load and CD4 cell counts
To determine the effects of immunogen on routine parameters of HIV-1 disease progression, we performed CD4 cell counts and plasma viral load by quantitative RNA (data not shown). Subjects in both arms of the study had similar CD4 cell counts and plasma viral load at baseline (day 1). HAART suppressed plasma viral load in both the immunogen plus HAART group (P = 0.03) and IFA plus HAART groups (P = 0.13).
Effects of immunogen on HIV-1-infected lymphocytes
To determine the effects of immunogen on productive HIV-1 infection in lymphocytes, we simultaneously labeled lymphocytes with CD4 and detected gag-pol messenger (m)RNA with ultrasensitive fluorescence in situ hybridization[2,7,8]. Using flow cytometry, lymphocytes were analyzed on the basis of light scatter characteristics and CD4 staining including in the scatter gates large, atypical lymphocytes previously determined to be the morphology of infected lymphocytes. Cell-associated RNA in CD4 cells correlated with plasma RNA overall (r=0.66, P=0.008). There was no significant difference in CD4 cells expressing HIV-1 RNA between the two groups at baseline (day 1, P > 0.05). Cell-associated HIV-1 RNA in CD4 cells decreased in the immunogen plus HAART group (P < 0.01; baseline 95% confidence interval (CI) =-1.1 to 5.2; week 32 95% CI=0.05 to 0.27) compared with the IFA plus HAART group (P=0.2; baseline 95% CI=-1.1 to 7.5; week 32 95% CI=0.78 to 2.7) as shown in Fig. 1a. Cell-associated HIV-1 DNA tended to decrease in CD4 cells in the immunogen plus HAART group (P=0.15; baseline 95% CI=-7.4 to 43.6; week 32 95% CI= 0.53 to 12.2) but not in the IFA plus HAART group (P=1.0; baseline 95% CI=2.8 to 11.4, week 32 95% CI=4.5 to 9.5) as shown in Fig. 1b.
Effects of immunogen on HIV-1-infected monocytes
We have previously described productive infection of monocytes in patients that were infected by HIV-1. To determine the effects of immunogen plus HAART on productively infected monocytes, we stained peripheral blood monocytes with CD14 and detected gag-pol mRNA as described. There was no significant difference between the treatment groups in terms of CD14 HIV-1 RNA positive at baseline (P=0.4, day 1). HIV-1 gag-pol mRNA+ monocytes tended to decrease in the immunogen plus HAART group (P=0.15; baseline 95% CI=2.5 to 89.9, week 32 95% CI=-0.6 to 25) but not in the IFA plus HAART group (P=0.7; baseline 95% CI=-10.9 to 45.7, week 32 95% CI=2.9 to 19.4) as shown in Fig. 2a. There was a difference at baseline in the number of CD14 positive cells expressing HIV-1 DNA with lower numbers of cells in the IFA plus HAART group (P=0.04). Decreases in HIV-1 DNA in monocytes approached statistical significance in the immunogen plus HAART group (P=0.095; baseline 95% CI=-12.3 to 71.9, week 32 95% CI=3.3 to 17.9) but not in the IFA plus HAART group (P=0.4; baseline 95% CI=-2.5 to 12.7, week 32 95% CI=0.27 to 13.7) as shown inFig. 2b.
Highly active antiretroviral therapy has been effective in lowering plasma viral load to undetectable levels in patients are infected by HIV-1. Several studies indicate, however, that cellular reservoirs of HIV-1 exist following long-term therapy with HAART[15-18]. Previously, we showed that despite undetectable plasma viral load, cells containing HIV-1 DNA remained in tonsil biopsies following 24 weeks of HAART. These studies emphasized the need for therapies not only directed at decreasing viral production in cells but also eliminating cells containing HIV-1 DNA that can cause virus rebound following a treatment failure.
In the present study, we have shown that HIV-1-specific immune-based therapy in combination with HAART lowers plasma viral load levels. HIV-1-specific immune-based therapy plus HAART, however, was observed to be more effective than IFA plus HAART in reducing the number of productively HIV-1-infected cells. This effect mimics the results seen in another recent study from our laboratory determining the effects of combined HAART and HIV-1-specific cytotoxic T lymphocyte infusions.
The effects observed in the present study were most pronounced in lymphocytes, but are also seen in monocytes. Immunogen plus HAART also tended to be more effective than IFA plus HAART in eliminating reservoirs of cells containing HIV-1 DNA. Taken together, these data indicate that the combination of an immune-based therapy with highly active antiretroviral therapy may decrease both viral production from cells that are infected by HIV-1 and the number of cells containing HIV-1. These two activities are essential for any chance of viral eradication. This preliminary observation supports further study with more patients to examine the effects of immune-based therapies and HAART on the reservoirs of viral burden in HIV-1 infection.
The authors acknowledge the assistance of G. Theofan in the monitoring of this study and M. Harden for manuscript preparation.
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