Reduction of the HIV-1 reservoir in resting CD4⁺ T-lymphocytes by high dosage intravenous immunoglobulin treatment: a proof-of-concept study

Nine patients followed at the Department of Infectious Diseases, Sahlgrenska University Hospital, Gothenburg, Sweden, with continuous ART for ≥ 2 years and plasma HIV-1 RNA levels < 50 copies/mL ≥ 1.5 years, were included in the study. Written informed consent was obtained and the study was approved by the Research Ethics Committee at the University of Gothenburg and the Medical Products Agency of Sweden. Patient characteristics are summarized in Table 1. Seven subjects had detectable levels of replication-competent virus in the latent pool and were thus possible to evaluate regarding changes of the latent reservoir. Two patients had undetectable virus both before and after intervention with IVIG (subjects 1 and 5). All subjects received 30 g IVIG per day as intravenous infusions for five consecutive days (Kiovig^®, Baxter Healthcare Corporation, Chicago, IL, USA). ART was continued throughout the study period.

Quantification of HIV-1 in resting memory T-cells was performed before, and 8–12 weeks after initiation of IVIG. Resting CD4⁺ T-cells were isolated from peripheral blood mononuclear cells (PBMC) by negative selection. PBMCs were obtained by Ficoll-Hypaque density gradient centrifugation from 180 mL of peripheral blood. The PBMCs were washed twice with PBS Buffer (pH 7.2 to 7.4) that contained 0.1% BSA and 2 mM EDTA. After the first wash, the cells were resuspended in PBS Buffer, and after the second wash in 30 mL of culture medium (RPMI+L-glut, 10% FCS and PenStrept), then transferred to a tissue culture flask and placed flat overnight at 37°C in a 5% CO₂ incubator to remove monocytes by adherence. The following day the monocytes-depleted PBMCs were further purified using a Dynal CD4 Negative Isolation Kit (Invitrogen, Carlsbad, CA, USA). The kit contains magnetic beads and a monoclonal antibody mix directed towards CD8, CD14, CD16a,b, CD56, CDw123, Glycophorin A, and HLA Class II DR/DP. Additional monoclonal antibodies, Mouse-anti-human CD8 and CD25 (AbD Serotec, Kidlington, Oxford, England), were added to the monoclonals in the Dynal kit and incubated at 4°C for 20 min. During the incubation period, 100 uL beads/10⁷ cells were washed in PBS Buffer. The PBMCs were washed and magnetic beads were added for another incubation period of 15 min in RT. The bead and cell mix were put into a magnetic rack and the supernatant containing purified resting CD4⁺ T-cells was collected. The purity of the cell supernatant was checked by flow cytometry.

Detection and quantification of latently-infected resting CD4⁺ T-cells were performed by a limiting dilution culture assay, as previously described [5]. Serial five-fold dilutions of cells (10⁶, 200,000, 40,000, 8,000, 1,600, and 320) were set up in duplicates and induced to express replication-competent HIV-1 by exposure to 0.5 (-2.0) ug/mL of the mitogen phytohemagglutinin (PHA), 10 (-100) U/mL interleukin 2 (IL-2), and allogeneic irradiated PBMCs, ratio 1:10. The day after activation, the culture supernatants were removed and fresh culture medium was added. To allow detection of virus growth, the resting CD4⁺ T-cells were also co-cultured with CD8⁺ depleted CD4⁺ lymphoblasts from healthy donors. These blasts had been prepared by stimulation with 0.5 ug/mL PHA two days before being added to the cultures. On the seventh day after activation, the cells were split and a new set of blasts were added to the cultures. The cultures were then incubated at 37°C in a humidified incubator with 5% CO₂ and were fed and split as needed. Supernatants were collected on a weekly basis and tested for the presence of HIV-1 p24 antigen with Architect i2000 HIV-1 Ag/Ab Combo Detection System (Abbott Diagnostics, Abbott Park, IL, USA).

Statistical analysis by the maximum likelihood method provided estimates of the infected cell frequencies expressed as infectious units per million (IUPM) resting CD4⁺ T-cells [6]. Samples with undetectable growth were estimated to 0.25 IUPM cells, i.e. half the concentration of the lowest possible estimate (0.5 IUPM) of detectable growth. The estimate 0.5 IUPM is the maximum likelihood estimate when only one of the two replicates at the highest concentration reveals presence of infected cells and all other dilutions are negative

HIV-1 RNA was analyzed in cell-free plasma using a previously described [7] modified version of the Roche Amplicor Monitor Test (Version 1.5, Roche Diagnostic Systems, Hoffman-La Roche, Basel, Switzerland). In order to yield a detection limit of 2 copies/mL, 8.25 mL of plasma were ultracentrifuged at 180,000 G in 4°C for 30 min prior to quantification [7].

cDNA synthesis was performed using the ThermoScript RT-PCR System (Invitrogen) with gene-specific primer 5'-TCTTTCATTTGRTGTCCTTC-3' (HXB2 nt position 2063–2044) (0.1 uM). To obtain PCR products derived from single cDNA molecules, a modified version of a previously described method was used [8]. Designed subtype B-specific primers were selected to amplify the HIV-1 p24 region of gag, using a nested PCR with Platinum Taq DNA Polymerase (Invitrogen). First round PCRs used forward primer 5'-CATMTAGTATGGGCAAGCAG-3' (HXB2 nt position 886–905) and reverse primer (described above). This was followed by a nested PCR, where each PCR product was subsequently used as a template, with forward primer 5'-GTCAGCCAAAATTACCCTA-3' (HXB2 nt position 1171–1189) and reverse primer 5'-GTCAGCCAAAATTACCCTA-3' (HXB2 nt position 2048–2030). To obtain PCR products for SGS, the cDNA was diluted until approximately 30% of the PCR reactions yielded DNA product [9]. Positive nested PCRs were identified by agarose gel electrophoresis, using E-Gel 96 1% agarose (Invitrogen).

Sequences were aligned using BioEdit Sequence Alignment Editor (Citeline, New York, NY, USA), with reference sequences from the HIV sequence database http://​hiv-web.​lanl.​gov to exclude the possibility of contamination. Phylogenetic trees were constructed using MEGA4.0 software (Center for Evolutionary Functional Genomics, The Biodesign Institute, Tempe, AZ, USA). Bootstrap testing (500 replicates) of phylogeny was performed using neighbor-joining, implementing pairwise deletion of gaps and gamma distribution (0.5) among sites. The sequences have been submitted to GenBank [J496870-FJ497003].

Peripheral blood CD4⁺ and CD8⁺ T-cell counts were measured by direct immunofluorescence in a flow cytometer.

The following mAbs were used: anti-CD3 PE-Cy7, anti-CD4 FITC, anti-CD8 PerCP, anti-CD25 PE, anti-CD38 FITC PE-Cy7, anti-CD127 Alexa647, anti-HLA-DR APC-Cy7, anti-IFNγ FITC, anti-MIP-1β PE, and anti-IL-2 APC, all from BD Biosciences (San Diego, CA, USA). TNFα Pacific Blue from eBioscience, Anti-CD3 Pacific Blue from Dako (Copenhagen, Denmark), and Aqua Live/Dead cell exclusion marker from Invitrogen were used. For each sample 7 × 10⁵ freshly isolated PBMC were stained in a 96-well v-bottomed plate on ice for 30 min, washed three times, and resuspended in CellFix solution (BD Biosciences); all washes were done in PBS with 5% FCS. The HIV-Gag p55 peptide pool (JPT Peptide Technologies, Berlin, Germany) and the HIV-Nef peptide pool (NIH, Germantown, MD, USA) were used to study the HIV-1-specific responses, and a CMV, EBV, and Flu (CEF) control peptide pool, as well as Staphylococcal Enterotoxin B (SEB) (SIGMA-Aldrich Logistic GmbH, Schnelldorf, Germany), were added as positive controls. The PBMCs were plated at a concentration of 1 × 10⁶ cells/well, along with peptides at a final concentration of 2 ug/mL per peptide in the pool, and incubated at 37°C for 12 hrs. As a negative control, cells were incubated with medium only to determine the background responses for each patient. For intracellular staining of cytokines, cells were stained for surface markers before permeabilization with Perm/Fix solution (BD Biosciences) at 4°C for 20 min. Cells were then washed with Perm/Wash solution and stained for intracellular IFNγ, MIP-1β, IL-2, and TNFα for 30 min, washed three times, and resuspended in CellFix solution. Multicolor flow cytometry data was acquired on a CyAn ADP instrument (Dako) [10]. Data were analyzed using FlowJo software (Tree Star, Ashland, OR, USA).

Plasma samples from all patients were analyzed for the presence of IL-2 and IL-7 cytokines on a Luminex 100™ System (Luminex Corp, Austin, TX, USA). The procedure is described in a protocol supplied with the IL-2 and IL-7 Human Singleplex Bead Kits (Invitrogen). Abs from the two kits were combined, and undiluted plasma samples were thoroughly mixed, centrifuged, and filtered prior to analysis.

Wilcoxon's Signed Rank Test was used for pairwise comparisons, the Mann-Whitney U-test for comparisons between two independent groups, and Spearman's Rank Correlation Coefficient for evaluations of correlations.