Immune and Viral Correlates of “Secondary Viral Control” after Treatment Interruption in Chronically HIV-1 Infected Patients

Upon interruption of antiretroviral therapy, HIV-infected patients usually show viral load rebound to pre-treatment levels. Four patients, hereafter referred to as secondary controllers (SC), were identified who initiated therapy during chronic infection and, after stopping treatment, could control virus replication at undetectable levels for more than six months. In the present study we set out to unravel possible viral and immune parameters or mechanisms of this phenomenon by comparing secondary controllers with elite controllers and non-controllers, including patients under HAART. As candidate correlates of protection, virus growth kinetics, levels of intracellular viral markers, several aspects of HIV-specific CD4+ and CD8+ T cell function and HIV neutralizing antibodies were investigated. As expected all intracellular viral markers were lower in aviremic as compared to viremic subjects, but in addition both elite and secondary controllers had lower levels of viral unspliced RNA in PBMC as compared to patients on HAART. Ex vivo cultivation of the virus from CD4+ T cells of SC consistently failed in one patient and showed delayed kinetics in the three others. Formal in vitro replication studies of these three viruses showed low to absent growth in two cases and a virus with normal fitness in the third case. T cell responses toward HIV peptides, evaluated in IFN-γ ELISPOT, revealed no significant differences in breadth, magnitude or avidity between SC and all other patient groups. Neither was there a difference in polyfunctionality of CD4+ or CD8+ T cells, as evaluated with intracellular cytokine staining. However, secondary and elite controllers showed higher proliferative responses to Gag and Pol peptides. SC also showed the highest level of autologous neutralizing antibodies. These data suggest that higher T cell proliferative responses and lower replication kinetics might be instrumental in secondary viral control in the absence of treatment

Breadth and magnitude of T cell responses against HIV peptides.

<p>(A) To evaluate T cell response breadth, PBMC from different patient groups were stimulated with peptide pools spanning the whole HIV genome. In the Y-axis the number of pools tested for each gene or gene group is indicated. In the X-axis the mean number (+SD) of peptide pools inducing positive ELISPOT responses in the HAART, TN, SNC, EC and SC patient groups is represented. (B) Based on the same dataset, the magnitude of the response was calculated as the mean number of IFN-γ spot forming cells (SFC) per million PBMC for each group of patients. Only peptide pools against which a positive response was measured, were included in this calculation. For each patient the SFC of positive peptide pools within each protein were cumulated and the mean (+SD) within each patient group is represented. No statistical differences were found.</p

Levels of intracellular HIV-1 molecular markers in PBMC.

<p>Levels of proviral DNA (prDNA), unspliced RNA (usRNA), and multiply spliced RNA (msRNA) are shown. Patient groups with detectable plasma viremia (TN and SNC) are shown in red, and those with undetectable plasma viremia (HAART-treated, EC, and SC) are shown in blue. Horizontal bars represent median values. Undetectable values, left-censored at the corresponding detection limits (which were different for every sample, as they depended on total cellular inputs in the real-time PCR reactions), are depicted by open circles. Individual prDNA and usRNA values are means of two independent measurements. The statistical significance of the comparison between usRNA levels of SC and HAART-treated patients was calculated by use of unpaired <i>T</i>-test.</p

Autologous neutralizing antibodies.

<p>IgG was purified and Env was cloned from the same plasma sample in each patient. The neutralizing capacity of IgG was tested in the TZMbl assay, using single cycle chimeric pseudoviruses, containing the respective autologous Env. This assay could be performed for 2 SC (A), 2 SNC (B), 3 TN (C) and 2 HAART patients (D). Percent neutralization in Y-axis is shown over a concentration range of individual patient IgG, given in X-axis. Each patient is represented by one symbol and in many patients several autologous Env clones were tested.</p

Replication characteristics of virus from secundary controllers.

<p>Ten million purified CD4+ T-cells from 4SC and 4SNC are cultivated with PHA and IL-2 stimulated PBMC. Panel A shows the virus replication in the primary culture (y-axis shows OD of p24 ELISA). These viruses were used in a secondary culture to evaluate fitness, by infecting new PHA and IL-2 stimulated PBMC at an equal MOI, with Ba-L virus as a reference. In panel B, the mean (and SEM) virus production in three donor PBMC is expressed as p24 in the supernatant. Green symbols are the SC; blue symbols are SNC and red symbol is BAL virus.</p

Patients characteristics at enrollment

a<p>SC = secondary controller</p>b<p>EC = elite controller</p>c<p>SNC = secondary non-controller</p>d<p>TN = therapy-naïve patients</p>e<p>HAART = patients under highly active antiretroviral treatment</p>f<p>Viral load under AZT</p>g<p>UK: unknown: this patient had already started HAART in another center.</p>h<p>in SC and SNC treatment was stopped for at least 6 months</p

T cell proliferation against Gag and Pol peptides.

<p>PBMC from different patient groups were stimulated with Gag peptide pools 1–6 and Pol pools 1–12, spanning the entire Gag-Pol region. The mean stimulation index (SI)+SD for all 4 SC, EC, TN, HAART and SNC is shown. Statistical differences were measured using Mann-Whitney and Kruskall-Wallis test (P<0.05) and represented in the upper right insert.</p

Evaluation of T cell polyfunctionality.

<p>PBMC from all study subjects were stimulated with peptide pools, selected for positive responses in the IFN-γ ELISPOT screening in each individual separately. In A the gating strategy for identification of multifunctional CD3+CD8+ and CD3+CD8− (CD4+) T cell responses is shown. After physical gating the mononuclear cells and excluding the dead cells, CD3+CD8+T-cells and CD3+CD8−(CD4+ T-cells) were identified. Within each of these populations the expression of IFN-γ, IL-2, TNF-a or CD107a was plotted against side scatter, to allow Boolean gating for the ultimate quantification of polyfunctionality. Polyfunctionality was analyzed using Flowjo by assessing the percentages of CD3+CD8− (hence CD4+) T cells (in B) and CD3+ CD8+ T cells (in C) that produce one, two, three or four cytokines. Percentages of co-expression for all selected peptide pools within one patient were summed and the mean+SD was calculated for each group. In D and E is the number of CD4+ T-cells respectively CD8+ T-cells that produce one, two, three or four cytokines presented. No statistical differences between groups were found.</p

CORIOLIS. Rapport d’activités 2016

Rapport d'activité Coriolis pour l'année 2016 dans le cadre de la convention cadre Coriolis 2014-202