Mechanisms of HIV Non-progression; Robust and Sustained CD4+ T-cell Proliferative Responses to P24 Antigen Correlate With Control of Viraemia and Lack of Disease Progression After Long-term Transfusion-acquired HIV-1 Infection

Wayne B. Dyer; John J. Zaunders; Fang Fang Yuan; Bin Wang; Jennifer C. Learmont; Andrew F. Geczy; Nitin K. Saksena; Dale A. McPhee; Paul R. Gorry; John S. Sullivan

Disclosures

Retrovirology 

In This Article

Materials and Methods

Definitions of Non-progression and Disease Progression

When this prospective study began in 1994, 13 LTNP were identified in the NSW TAHIV cohort according to the original guidelines for classifying LTNP: at least 10 years infection, stable CD4 T cell counts >500 cells/µl, and no history of ART.[27,28] Subsequently, loss of LTNP status was defined by any of the following events: a consistent decline in CD4 T cell counts below 500/µl, commencement of ART, and after viral load testing became routine, plasma viraemia > 5000 copies/ml. Elite non-progressors were also defined by viraemia suppressed to < 50 copies/ml in addition to the above criteria. Disease progression was defined by a CD4 T cell count of < 200 and/or plasma viraemia > 100,000 copies/ml.

Patient Details

The two non-progressor groups in this study included the SBBC, consisting of 6 recipients of HIV-infected blood from a common donor, and the other (Cohort 2) consisting of 7 recipients infected by blood from different donors. Clinical data from these LTNP were collected prospectively since the late 1980s. T cell counts and viral load tests were performed as part of routine clinical care. Blood samples and clinical histories were provided after informed consent was granted in accordance with guidelines from the ARCBS institutional Human Research Ethics Committee.

T Cell Functional Analyses

Anti-HIV T cell function assays were performed as previously described.[15,29] Briefly, the proliferative response to HIV-1 p24 was determined by 6 day culture of PBMC (1 × 105 cells/well) in RPMI medium with 15% pooled human serum in round bottom microtitre plates, with 2 µg/ml HIV-1SF2 p24 (Chiron, Emeryville, CA, USA), or medium alone for control. After 6 days, proliferative responses were determined by 3H-thymidine incorporation during a further 6 hours culture, followed by cell harvest and reading in a liquid scintillation counter. Results were expressed as stimulation index (SI; mean counts antigen wells/mean counts control wells), and a SI >3 was considered a positive response.

The response of CD8+ T cells to HIV antigen was measured by IFNγ ELISPOT, using pre-coated ELISPOT kits according to the manufacture's protocol (Mabtech, Mosman, Australia). Firstly, the response to whole HIV proteins was determined in response to antigen presented by autologous B lymphoblastoid cell lines infected for 18 hours with 5 pfu/cell recombinant Vaccinia expressing the HIV-1IIIB env, gag, pol, or nef genes (Therion Biologics, Cambridge, MA, USA), or E. coli lacZ as a control. Gag responses were further characterised using overlapping Gag peptides, firstly using a matrix of peptide pools, and then individual peptides for confirmation (full Gag peptide set; kindly provided by the NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH).

Provirus Sequencing

DNA from PBMC was isolated using a QIAamp DNA mini kit (Qiagen, Valencia, CA) according to the manufacturer's protocol. A nested polymerase chain reaction (PCR) was used to amplify ~1.5 kb of the HIV gag gene using the following primers:

5'-TCTCGACGCAGGACTCGGCTTGCTGA-3' (outer, sense),

5'-TACTGTATCATCTGCTCCTGTAT-3' (outer, antisense),

5_-GACAAGGAACTGTATCCTTTAGCTTC-3 (inner, sense),

And 5'-TCTGCTCCTGTATCTAATAGAGCTT-3' (inner, antisense).

Both primary and secondary PCR reactions contained 2 units of Taq DNA polymerase (Promega, Madison, WI), 1× PCR buffer (Promega: 1 mM Tris- HCl, 5 mM KCl, 0.1% Triton X-100), 2.5 mM MgCl2, 200 nM of each dNTP, and 0.4 nM of each primer in a total volume of 50 ul. Thermocycling conditions were as follows: 95ºC for 2 min and then 35 cycles of 94ºC for 30 s, 55ºC for 30 s, 72ºC for 2 min and a final a single cycle of 72ºC for 7 min.

RNA was isolated from plasma using the QIAamp Viral RNA Mini Kit (Qiagen, Valencia, CA) according to the manufacturer's protocol. Gag gene was amplified using the QIAGEN OneStep RT-PCR Kit using the outer primer pairs mentioned above. Second round PCR reactions were performed using the inner primer pair under the same conditions.

PCR products were purified using a Millipore PCR purification plate (Millipore, Billerica, MA, USA) and sequenced by the ABI PRISM BigDye Terminator V3.1 Ready Reaction Cycle Sequencing kit (Applied Biosystems, Foster City, CA, USA) on an ABI 377 automated sequencer. Multiple sequences derived from each patient were analysed using Sequencher 3.11 software (Gene Codes Corp., Ann Arbor, MI, USA). Chromatograms derived from both forward and reverse primers were aligned with the reference strain HIV-1 HXB2.

Host Genetic Typing

Methods for HLA and chemokine receptor polymorphisms[30] and toll-like receptor (TLR) and FcγRIIA polymorphisms[31,32,33] have been described elsewhere.

Statistical Analysis

The Fishers Exact test was used to associate genetic and immune factors with viraemia and non-progressor status.

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