HIV-1 treatment timing shapes the human intestinal memory B-cell repertoire to commensal bacteria

HIV-1 infection causes severe alterations of gut mucosa, microbiota and immune system, which can be curbed by early antiretroviral therapy. Here, we investigate how treatment timing affects intestinal memory B-cell and plasmablast repertoires of HIV-1-infected humans. We show that only class-switched memory B cells markedly differ between subjects treated during the acute and chronic phases of infection. Intestinal memory B-cell monoclonal antibodies show more prevalent polyreactive and commensal bacteria-reactive clones in late- compared to early-treated individuals. Mirroring this, serum IgA polyreactivity and commensal-reactivity are strongly increased in late-treated individuals and correlate with intestinal permeability and systemic inflammatory markers. Polyreactive blood IgA memory B cells, many of which egressed from the gut, are also substantially enriched in late-treated individuals. Our data establish gut and systemic B-cell polyreactivity to commensal bacteria as hallmarks of chronic HIV-1 infection and suggest that initiating treatment early may limit intestinal B-cell abnormalities compromising HIV-1 humoral response.

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All statistical values calculated from the multiparameter correlations are also provided.Raw data or information on immunoglobulin gene sequences from single B cells and Ig-HTS are available upon reasonable request to the corresponding author.
The sex/gender of all human participants is indicated in the supplemental table S1, and was not considered in the study design nor in data analysis.
No reports were performed on ethnicity, or other socially relevant groupings.
Biological samples where obtained from HIV-1-infected individuals under effective antiretroviral therapy (ART) for several years at the Centre Hospitalier Regional d'Orléans.ART was started within 4 months after the diagnosis of primary HIV-1 infection (Fiebig stage II-V) in 38 patients (eART; median age 48 [28-79] years) or during the chronic stage of HIV-1 infection (Fiebig stage VI) in 40 patients (lART; median age 50 [23-80] years).The main clinical and immuno-virological characteristics of the study participants are provided in the Supplementary Table 1.
Participants in the study were randomly selected but, based on ART timing either during primary HIV-1 infection (Fiebig stage II-V) -eART goup -or the chronic stage of infection -lART group.Median age-match checking between groups was verified after the selection.
Samples were obtained as part of the research protocol called BHUANTIVIH performed in accordance with and after ethical approval from all the French legislation and regulation authorities.All donors gave written consent to participate in this study, and data were collected under pseudo-anonymized conditions using subject coding.This is a descriptive and exploratory study on difficult-to-obtain gut samples from HIV-infected individuals.Sample size determination was therefore not applied; we included and analyzed all available tissue biopsies obtained during the study (with matched blood cell samples).
For the analyses on patients' sera, we tested all bio-banked sample available in the lab (38 from eART and 40 from lART).
No data exclusions were performed.Randomization of the study populations does not fully apply; the donors were randomly-selected but first based on defined treatment timingeither eART or lART.
Blinding was not applied because: (i) this was not an interventional study, and (ii) individuals were selected according to a defined criterium (see aforementioned).Protein microarray binding analysis -Secondary conjugated antibodies used for revelation: The polyclonal goat anti-Human IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 647 (# A-21445, ThermoFischer) was used at 1 µg/ml.

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Most of the antibodies are commercially available and the reactivity validated against the appropriate species, as reported in manufactuer´s website, technical datasheets and previous studies.
Mouse anti-human CD19 A700 antibody (clone HIB19, #557921, BD Biosciences): According to manufacturer´s website the antibody is routinely tested in flow cytometry.Flow cytometric analysis of CD19 expression on human peripheral blood lymphocytes is provided in the website and technical datasheet.Supporting references are also provided.
Mouse anti-human CD38 APC antibody (clone HIT2, #560980, BD Biosciences): According to manufacturer´s website the antibody is routinely tested in flow cytometry.Flow cytometric analysis of CD38 expression on human peripheral blood lymphocytes is provided in the website and technical datasheet.Supporting references are also provided.
Mouse anti-human CD21 BV421 antibody (clone B-ly45, #562966, BD Biosciences): According to manufacturer´s website the antibody is routinely tested in flow cytometry.Flow cytometric analysis of CD21 expression on human peripheral blood lymphocytes is provided in the website and technical datasheet.Supporting references are also provided.
FlowJo software (v10.7.1, FlowJo LLC) N/A Lymphocytes were gated based on on FSC-A/SSC-A, followed by by single cell check based on on SSC-A/SSC-H.Live cells were then gated based on on a LIVE/DEAD fixable dead cell stain kit (405 nm nm excitation, Molecular Probes, ThermoFicher Scientific).B-cell subsets were then analyzed using the specified antibody cocktails following the gating strategies shown in in Supplementary Figure 1b.
Mouse anti-human CD27 BV711 antibody (clone M-T271, #564893, BD Biosciences): According to manufacturer´s website the antibody is routinely tested in flow cytometry.Flow cytometric analysis of CD27 expression on human peripheral blood lymphocytes nature portfolio | reporting summary to to confirm that a figure exemplifying the gating strategy is is provided in in the Supplementary Information.
We require information from authors about some types of materials, experimental systems and methods used in many studies.Here, indicate whether each material, system or method listed is relevant to your study.If you are not sure if a list item applies to your research, read the appropriate section before selecting a response.experimentswereperformed in duplicate or triplicate, in at least two independent experiments.Indirect immunofluorescence assays were also performed twice in two different experiments.All experiments were reproduced successfully.Protein microarray binding analyses and flow cytometry immunophenotyping data were obtained in a single experiment.Testing of purified serum IgG antibodies in the in vitro HIV-1 neutralization assay was performed once in triplicate.