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

evangulck@itg.be

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

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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