Trucount

Nine parameter flow cytometric analysis was performed on 200uL of whole blood. Lymphocytes were assessed by forward and side scatter, and stained for the following: anti-CD19-PECy5 (clone HIB19),anti- CD20- APC-H7 (clone L27), anti-CD10- APC (clone HI10a), anti-CD21- PE (clone B-ly4), anti- CD27- PECy7 (clone M-T271) (all from BD Biosciences, San Jose, CA) and anti-CD38- Alexa Fluor 700 (clone HIT2, Biolegend, San Diego, CA). At least 20,000 CD19+ B cells events were acquired on an LSRII cytometer driven by FACSDiVa version 6.2 software. Analysis was performed using FlowJo Version 10 software. BD TruCOUNT (BD Biosciences) beads were used for absolute counts of CD19+ B cells. For CD86 FITC (clone 2331, BD Biosciences, San Jose, CA) whole blood was stained for 10 minutes in the dark at room temperature, then lysed for 2 minutes with 2mL of BD Lyse (BD Pharmigen, San Diego, CA) and washed with 2mL of 0.1% BSA+ PBS, then fixed with 200uL of BD Stabilizing Fixative (BD Biosciences). For Ki-67(clone B56, BD Biosciences),whole blood was stained as above, but permeabilized with BD Cytofix/Permeabilization (BD Biosciences) on ice for 20 minutes, washed with 2mL of Perm Wash (BD Biosciences) and stained with 20uL of anti-Ki-67 FITC for 30 minutes on ice, washed twice with 2 mL of Perm Wash and fixed.

The ANRS-EP56 study comprised three groups of patients. The first group included ART-naïve patients diagnosed with PHI (n = 19), symptomatic or not, defined by a negative or weakly positive ELISA, and at least one of the following criteria: incomplete HIV Western Blot, a positive p24 antigenemia and/or detectable plasma HIV-1-RNA. The second and the third groups comprised chronic HIV-infected patients (CHI) either UT (UT-CHI, n = 17) or ART-treated (ART-CHI, n = 23) with HIV-RNA levels <20 copies/mL since at least 6 months. Non-inclusion criteria included active HCV or HBV infection and ongoing bacterial or opportunistic infection. Patients were prospectively enrolled in the study conducted in three clinical sites in Paris, France between April 2015 and July 2016. Written informed consent was provided by study participants according to French ethical laws. The ethical committee of Ile de France IV approved the study. In addition, a statement of ethical clearance was also received from Institut Pasteur, Paris, which was the sponsor of the study, in charge of administrative and ethical issues. Seventeen healthy donors (HD) were also included in the study. Lymphocyte, CD4, and CD8 counts were measured in all patients routinely using the BD Multitest™ CD3/CD8/CD45/CD4 and BD Trucount™ tubes.

Blood for full blood counts, CD4+ T cell count measurement and viral load quantification was collected in EDTA anticoagulant vacutainer tubes [Becton Dickinson (BD), Franklin Lakes, New Jersey, USA]. ANCs were enumerated by full blood count using the automated XN 1000 Hematology Analyzer (Sysmex, Kobe, Hyogo, Japan). CD4 counts were measured using BD Trucount and analyzed on a four-parameter FACS Calibur flow cytometer (BD). Viral loads were determined using the NucliSENS EasyQ HIV-1 v2.0 kit with a detection limit of 20 copies/ml (BioMérieux, Marcy-l'Étoile, France).

Blood for full blood counts, CD4+ T cell count measurement and viral load quantification was collected in Ethylenediaminetetraacetic acid (EDTA) anticoagulated vacutainer tubes (Becton Dickinson (BD), Franklin Lakes, New Jersey, USA). ANCs were enumerated by full blood count using the automated XN 1000 Haematology Analyser (Sysmex, Kobe, Hyōgo, Japan). CD4 counts were measured using BD Trucount and analysed on a four-parameter FACS Calibur flow cytometer (BD). Viral loads were determined using the NucliSENS EasyQ HIV1 v2.0 kit with a detection limit of 20 copies/ml (BioMérieux, Marcy-l'Étoile, France).

Blood samples were collected in ethylenediaminetetraacetic acid (EDTA) anticoagulated vacutainer tubes (Becton Dickinson (BD), Franklin Lakes, NJ, USA) for CD4+ T cell count measurement and viral load quantification. CD4 counts were measured using BD Trucount and analysed on a four-parameter FACSCalibur flow cytometer (BD). Viral loads were determined using the NucliSENS EasyQ HIV-1 v2.0 kit with a detection limit of 20 copies/ml (BioMérieux, Marcy-l’Étoile, France). Individuals initiating ART were prescribed a fixed-dose three drug oral combination pill of efavirenz, emtricitabine and tenofovir. For participants who started antiretroviral therapy in acute HIV infection, raltegravir was prescribed as a fourth drug until 90 days after viral suppression.