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Immune correlates of unusual control of viral replication after cessation of HAART
Ellen Van Gulck1, Leo Heyndrickx1, Céline Merlin1, Sandra Coppens1, Derek Atkinson1, Eric Florence2, Anne Buvé3, Guido Vanham1,4. 1Virology unit, Department of Microbiology, Institute of Tropical Medicine, Antwerp (ITMA), Antwerp, Belgium. 2HIV/STD unit, Department of Clinical Sciences, ITMA, Antwerp, Belgium. 3STI/HIV Epidemiology and Control Unit, Department of Microbiology, ITMA, Antwerp, Belgium4Department of Medicinal Chemistry, University of Antwerp (UA); Faculty of Medicine and Pharmacy, Free University of Brussels (VUB) and Department of Gynecology and Obstetrics, Catholic University of Leuven (KUL), Belgium.
Institute of Tropical Medicine, Antwerp
Nationalestraat 155, B-2000 Antwerp, BelgiumTel. +32-3-247.65.21Fax. +32-3-216.14.31
Abstract
Background: So far highly active antiretroviral treatment (HAART) has not resulted in “cure” of HIV infection mainly because of latent
virus reservoirs. Cessation of HAART typically results in viral rebound within days or weeks after treatment interruption. We have
defined 4 patients who were treated for progressive HIV infection, but spontaneously controlled their VL for at least 6 months after
cessation of HAART. We labeled these patients “secondary controllers” (SC). We hypothesized that the combined selection pressure
of HAART and immunity had resulted in the induction of T-cell responses that prevent the archived provirus from growing out after
treatment interruption. Here we present immune characteristics of these patients.
Methods: We compared the SC with 4 control groups (of 4 patients each) including elite controllers (EC); patients under HAART;
treatment naïve patients; and non-controllers. All patients were matched on % CD4 T-cells. Proviral load, intracellular unspliced and
multiple spliced HIV mRNA in CD4 T-cells was measured by real-time PCR. Magnitude of T cell response against HIV was measured
using IFN-ELISPOT. Neutralizing activity of IgG was measured using TZMbl assay. The capacity to prevent infection by CD8+ T-
cells was measured using a viral inhibition assay. Purified CD4+ T-cells were infected at MOI 10-3 with labstrains or autologous virus.
Four hours after infection autologous CD8+ T-cells were added. P24 was monitored every 3 days, for 14 days.
Results: Proviral load in SC and EC was much lower compared to the other groups and correlated with the amount of unspliced and
multiple spliced mRNA. There was no difference between the groups in the magnitude of induced HIV-specific responses. However,
there was a significant difference in the capacity of CD8+ T-cells to inhibit infection. CD8+ T-cells from all EC and SC could inhibit
infection of CD4+ T-cells while those from others could not. Broad cross neutralizing antibodies were lacking in all patients. However,
in contrast to the control patients, SC showed neutralization against autologous virus.
Conclusion: SC had a normal disease progression before starting HAART but behave like EC after cessation of HAART. Low
intracellular HIV DNA and RNA, together with strong virus inhibitory capacity are the most discriminating parameter of controllers
versus non-controllers. These findings should be taken into account when designing and evaluating new immuno-therapeutic
strategies.
Patient Characteristics at enrolment of study
Group Patient HIV-1 subtype sex origin age Years since HIV diagnosis
Days on HAART
Viral load (copies/ml)
CD4 T cell count (cells/µl)
SC
P1 B M Belgium 65 20 6290 <50 250
P2 D F Belgium 55 11 2092 <50 1316
P3 B M Venezuela 36 4 Unknown <50 244
P4 A1 M Belgium 37 11 2420 <50 1486
EC
P5 B M Belgium 36 7 0 <50 633
P6 B M Belgium 62 13 0 <50 614
P7 B M Belgium 61 9 0 <50 437
P8 B M Belgium 59 9 0 <50 675
SNC
P9 D F Congo 51 17 5259 216000 726
P10 B M Belgium 40 14 2964 233000 361
P11 B M Belgium 47 11 3451 13800 345
P12 B F Belgium 39 15 4616 50000 732
TN
P13 B M Belgium 38 10 0 4280 361
P14 B M Belgium 27 1 0 69600 582
P15 B M Brazil 34 1 0 26000 425
P16 B M Belgium 41 9 0 10100 310
HAART
P17 CRF02_AG F Belgium 52 12 4294 <50 707
P18 B M Belgium 51 12 4259 <50 973
P19 B M Belgium 58 16 5987 <50 806
P20 B M Belgium 67 9 3409 <50 438Results: 1) Magnitude and breadth 2) Virus inhibition assay 3) Autologous neutralization 4) Real time PCR
prDNA
TNSNC
HAART EC SC
0
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Materials and methods
Blood HIV-1Donor
plasma
PBMC
Isolation IgG
Isolation total RNA
Isolation total RNA
Stimulation peptides
Stimulation bispecific monoclonal AB
Neutralization assay against autologous gp160 (result 3)
Real time PCR for ribosomal, unspliced and multiple spliced RNA (Result 4)
Real time PCR for proviral DNA and beta actin (Result 4)
Virus inhibition assay (Result 2)
ELISPOT (Result 1)
PBMC from different patient groups were stimulated with peptide pools encoding the whole HIV genome. A)Represented the mean number of peptide pools inducing pos ELISPOT responsesB)The mean number of IFN- spot forming cells per million PBMC
Purified CD4+ T-cells from SC (A) and from EC (B) were infected with different virus isolates and cultured in the absence or presence of CD8+ T-cells (1:1 ratio). On day 13 the ratio of p24 production in the supernatant of cultures with CD8+ T-cells and without CD8+ T-cells was calculated and the % inhibition of viral production is represented.
Pseudoviral constructs were generated from the plasma samples. The same plasma was also used to purify IgG that was used in the PV-TZMbl neutralization assay to look for autologous neutralization. This assay was performed for SC (A), SNC (B), TN (C) and HAART patients (D). Percent neutralization is shown over a concentration range of individual patient IgG. Each patient is represented by one symbol.
Boston, 27.02 – 02.03.2011 CROI – 18th Conf on Retrovir & Opport Infect