TYPE Original Research PUBLISHED 08 December 2022 DOI 10.3389/fimmu.2022.1066036 Probiotic effects on immunity OPEN ACCESS and microbiome in HIV-1 EDITED BY Marius Trøseid, Oslo University Hospital, Norway discordant patients REVIEWED BY Piotr Nowak, Carlos Blázquez-Bondia 1, Mariona Parera 1, Karolinska Institutet (KI), Sweden Yi-Qun Kuang, Francesc Català-Moll 1, Maria Casadellà 1, Kunming Medical University, China Dag Reikvam, Aleix Elizalde-Torrent 1, Meritxell Aguiló 2, Oslo University Hospital, Norway Jordi Espadaler-Mazo 2, José Ramon Santos 3, *CORRESPONDENCE Roger Paredes 1,3,4,5,6 and Marc Noguera-Julian 1,4,6* Marc Noguera-Julian mnoguera@irsicaixa.es 1 IrsiCaixa AIDS Research Institute, Badalona, Spain, 2AB-BIOTICS SA (Kaneka Group), Barcelona, Spain, 3 Infectious Diseases Department and Fundació Lluita contra les Infeccions, Hospital Universitari Germans SPECIALTY SECTION Trias i Pujol, Badalona, Catalonia, Spain, 4Centre for Health and Social Care Research (CESS), Faculty of This article was submitted to Medicine, University of Vic – Central University of Catalonia (UVic – UCC), Vic, Barcelona, Spain, Inflammation, 5 Universitat Autònoma de Barcelona, Cerdanyola del Vallès, Spain, 6Infectious Disease Networking a section of the journal Biomedical Research Center, Centro de Investigación Biomédica en Red de Enfermedades Infecciosas Frontiers in Immunology (CIBERINFEC), Carlos III Health Institute, Madrid, Spain RECEIVED 10 October 2022 ACCEPTED 17 November 2022 PUBLISHED 08 December 2022 CITATION Background: Some HIV-1 infected patients are unable to completely recover Blázquez-Bondia C, Parera M, Català-Moll F, Casadellà M, normal CD4+ T-cell (CD4+) counts after achieving HIV-1 suppression with Elizalde-Torrent A, Aguiló M, combined Antiretroviral Therapy (cART), hence being classified as immuno- Espadaler-Mazo J, Santos JR, discordant. The human microbiome plays a crucial role in maintaining immune Paredes R and Noguera-Julian M (2022) Probiotic effects on immunity homeostasis and is a potential target towards immune reconstitution. and microbiome in HIV-1 discordant patients. Setting: RECOVER (NCT03542786) was a double-blind placebo-controlled Front. Immunol. 13:1066036. doi: 10.3389/fimmu.2022.1066036 clinical trial designed to evaluate if the novel probiotic i3.1 (AB-Biotics, Sant COPYRIGHT Cugat del Vallès, Spain) was able to improve immune reconstitution in HIV-1 © 2022 Blázquez-Bondia, Parera, infected immuno-discordant patients with stable cART and CD4+ counts Català-Moll, Casadellà, Elizalde-Torrent, <500 cells/mm3. The mixture consisted of two strains of L. plantarum and Aguiló, Espadaler-Mazo, Santos, Paredes and Noguera-Julian. This is an open- one of P. acidilactici, given with or without a fiber-based prebiotic. access article distributed under the terms of the Creative Commons Methods: 71 patients were randomized 1:2:2 to Placebo, Probiotic or probiotic + Attribution License (CC BY). The use, distribution or reproduction in other prebiotic (Synbiotic), and were followed over 6 months + 3-month washout forums is permitted, provided the period, in which changes on systemic immune status and gut microbiome were original author(s) and the copyright evaluated. Primary endpoints were safety and tolerability of the investigational owner(s) are credited and that the original publication in this journal is product. Secondary endpoints were changes on CD4+ and CD8+ T-cell (CD8+) cited, in accordance with accepted counts, inflammation markers and faecal microbiome structure, defined by alpha academic practice. No use, distribution or reproduction is diversity (Gene Richness), beta diversity (Bray-Curtis) and functional profile. permitted which does not comply with Comparisons across/within groups were performed using standard/paired these terms. Wilcoxon test, respectively. Results: Adverse event (AE) incidence was similar among groups (53%, 33%, and 55% in the Placebo, Probiotic and Synbiotic groups, respectively, the most common being grade 1 digestive AEs: flatulence, bloating and diarrhoea. Two grade 3 AEs were reported, all in the Synbiotic group: abdominal distension (possibly related) and malignant lung neoplasm (unrelated), and 1 grade 4 AE in Frontiers in Immunology 01 frontiersin.org Blázquez-Bondia et al. 10.3389/fimmu.2022.1066036 the Placebo: hepatocarcinoma (unrelated). Synbiotic exposure was associated with a higher increase in CD4+/CD8+ T-cell (CD4/CD8) ratio at 6 months vs baseline (median=0.76(IQR=0.51) vs 0.72(0. 45), median change= 0.04 (IQR=0.19), p = 0.03). At month 9, the Synbiotic group had a significant increase in CD4/CD8 ratio (0.827(0.55) vs 0.825(0.53), median change = 0.04(IQR=0.15), p= 0.02) relative to baseline, and higher CD4+ counts (447 (157) vs. 342(73) counts/ml, p = 0.03), and lower sCD14 values (2.16(0.67) vs 3.18(0.8), p = 0.008) than Placebo. No effect in immune parameters was observed in the Probiotic arm. None of the two interventions modified microbial gene richness (alpha diversity). However, intervention as categorical variable was associated with slight but significant effect on Bray- Curtis distance variance (Adonis R2 = 0.02, p = 0.005). Additionally, at month 6, Synbiotic intervention was associated with lower pathway abundances vs Placebo of Assimilatory Sulphate Reduction (8.79·10-6 (1.25·10-5) vs. 1.61·10-5 (2.77·10-5), p = 0.03) and biosynthesis of methionine (2.3·10-5 (3.17·10-5) vs. 4·10-5 (5.66·10-5), p = 0.03) and cysteine (1.83·10-5 (2.56·10-5) vs. 3.3·10-5 (4.62·10-5) , p = 0.03). At month 6, probiotic detection in faeces was associated with significant decreases in C Reactive Protein (CRP) vs baseline (11.1(22) vs. 19.2(66), median change= -2.7 (13.2) ug/ml, p = 0.04) and lower IL- 6 values (0.58(1.13) vs. 1.17(1.59) ug/ml, p = 0.02) when compared with samples with no detectable probiotic. No detection of the probiotic was associated with higher CD4/CD8 ratio at month 6 vs baseline (0.718(0.57) vs. 0.58(0.4), median change = 0.4(0.2), p = 0.02). After washout, probiotic non-detection was also associated with a significant increase in CD4+ counts (457(153) vs. 416(142), median change = 45(75), counts/ml, p = 0.005) and CD4/CD8 ratio (0.67(0.5) vs 0.59(0.49), median change = 0.04 (0.18), p = 0.02). Conclusion: A synbiotic intervention with L. plantarum and P. acidilactici was safe and led to small increases in CD4/CD8 ratio and minor reductions in sCD14 of uncertain clinical significance. A probiotic with the same composition was also safe but did not achieve any impact on immune parameters or faecal microbiome composition. KEYWORDS probiotics, prebiotics, synbiotics, HIV, immune reconstitution Introduction important fraction of HIV-infected people will become immunodiscordant, as they fail to fully recover CD4+ counts One of the key aspects of HIV infection is a fast and and immune function (7–9) especially those who failed to widespread destruction of CD4+ T-lymphocytes (1), which receive cART on the early stages of infection. becomes more exacerbated in the latest stages of infection (2, This lack of recovery stems from the virus early replication 3). Additionally, the virus presents the ability to establish site and its reservoir sanctuary: the Gut-Associated Lymphoid reservoirs in which it can remain dormant mostly in high- Tissue (GALT) (10), where the biggest population of high CCR5 CCR5 memory CD4+ T-cells (4, 5), where it remains + CD4+ T-cells resides. Depletion of such cells in the gut is integrated in the host genome (3–5). The ability to maintain a coupled with a decrease in Treg numbers (11), but more slowly latency state has made it impossible to achieve a complete than its CD4+ counterpart, lowering the ratio between Treg and remission, although it can be life-long supressed in most effector CD4+ cells, especially the Th17 subtypes (11, 12). A patients with combined Antiretroviral Therapy (cART) (6). relative decrease of the Th17/Treg ratio has been correlated to a Nevertheless, even when the virus remains supressed, an higher chance of disease progression (13). Th17 have also been Frontiers in Immunology 02 frontiersin.org Blázquez-Bondia et al. 10.3389/fimmu.2022.1066036 shown to maintain gut barrier integrity, stimulating tight Materials and methods junction expression in epithelial cells (14), as well as modulating bacterial populations in the mucosa by secreting Ethics statement antimicrobial peptides (15). Hence, the skew in the Treg/Th17 ratio compromises the gut barrier integrity, and creates a The study was reviewed and approved by the Institutional feedback loop where dysbiosis and gut inflammation cause a Review Board of the Hospital Universitari Germans Trias i Pujol leakage of bacterial compounds into the bloodstream (16), which (reference PI-13-046). All participants provided written increases residual systemic inflammation (17) and leads to informed consent in accordance with the World Medical further T-cell exhaustion and senescence (18), which further Association Declaration of Helsinki, Fortaleza and Brazil, promotes dysbiosis and gut barrier. This vicious circle ends up October 2013 and personal data was managed according to causing immune exhaustion and may hinder any attempt of Spanish data protection law (LOPD 15/1999). The study immune reconstitution. concept, design, patient information and results were discussed While many approaches have been considered to recover with the FLSida Community Advisory Committee, in immunity and gut integrity, modulation of the gut microbiome accordance with AB-Biotics internal QC auditing. All available awakened great interest recently. It is now known that some information can be found in the protocol (Supplementary bacterial species directly affect the immunologic makeup of the Methods), and the study is registered in clinicaltrials.gov, gut barrier (19) by modulating tryptophan to kynurenine accession: NCT03542786. catabolism (17). This pathway is thought to promote Treg differentiation (17) and has been shown to increase with presence of some Proteobacteria (20) and decrease with Cohort description Lactobacillus species (20, 21). For this, probiotics have been widely tested with promising results for wide variety of This study took place in a two-year span between 2017 and ailments, both from the gut itself such as Inflammatory Bowel 2019 and was designed as a masked randomized, placebo- Disease (IBD) (22), diarrhoea, both HIV-induced (23) and by controlled, double-blinded three-arm study in a cohort of 100 other pathogens (24) and even outside of the gut, such as HIV+ patients with the following inclusion criteria: 18 years of age allergies and upper respiratory infections (25). or older, chronic HIV infection with stable Anti-Retroviral Recently, the gut microbiome has awakened a great interest in Treatment (cART) ongoing for longer than a year prior to the the scope of HIV clinical management, as immune recovery has start, peripheral CD4+ counts lower than 500 cells/ml in plasma, been shown to be affected by the state of the gut mucosa (26). <50 HIV copies/ml in plasma for at least 6 months before the start, Cross-sectional studies have found microbiome signatures no antibiotic treatments at least 1 month before start, lack of correlated to immune reconstitution such as higher Prevotella/ severe AIDS-defining diseases and no pregnancy. An additional Bacteroides ratio and enrichment of Faecalibacterium prausnitzii filter was later implemented, in which only those patients with at and Coprococcus comes (27) or increased abundance of least 2 samples along the trial would be selected for further Fusobacterium negatively linked to immune recovery (28). analysis, to preserve the longitudinal approach of the study. Consequently, probiotic interventions have have become an After inclusion, patients were randomly assigned to one of interesting therapeutic target, as they promote tolerogenicity the three following groups: Placebo, Probiotic or Synbiotic in a (29), displace pathogenic strains, and reduce inflammation (30), 1:2:2 ratio and matched by 3rd cART drug class: Integrase Strand which could reduce T cell depletion and senescence, opening a Transfer Inhibitors (INSTI), Non-nucleoside reverse way to improve immune reconstitution after viral suppression. transcriptase inhibitors (NNRTI) or Protease Inhibitors (PI), However, many different combinations of probiotic strains and and CD4+ nadir higher or lower than 200 cells/ml at the time prebiotic substrates have been tested with mixed results (30, 31). of screening. In this study, we performed a randomized double-blinded All recruited patients followed a 6-month treatment trial to test the safety, tolerability and effectiveness of a probiotic followed by a 3-month washout periods, with check-ups at consisting of two strains of Lactobacillus plantarum and one of months 0,1,3,6 and 9. Every follow-up visit consisted of a Pediococcus acidilactici, combined with prebiotic fibers over the sample collection of both blood and faeces (except at the 1st course of 6 months, followed by a 3-month long washout period month checkout, where only stool was collected), a physical in immunodiscordant (<500 counts/ml) HIV patients with examination and a questionnaire about quality of life, and self- stable cART. The primary endpoints consisted of safety and reported treatment adherence since last check-up. During the tolerability. The secondary endpoints were changes in CD4+, treatment period, all participants received different formulation CD8+ counts, CD4/CD8 ratio, inflammation, and gut depending on whether they received a prebiotic + probiotic permeability markers, as well as changes in the gut (Synbiotic), probiotic alone (Probiotic) or a placebo (Placebo), microbiome taxonomical and functional composition after 6 which was administered orally as dissolved powder months of intervention + 3 of washout. sachets, daily. Frontiers in Immunology 03 frontiersin.org Blázquez-Bondia et al. 10.3389/fimmu.2022.1066036 Treatment formulations Sequence filtering and quality control The probiotic used in this study consisted of a mix of 3 Raw.fastq files were first processed for quality control. Read Lactobacillales strains: L. plantarum (strains CECT7484 and Quality filtering and trimming was performed with CECT7485) and P. acidilactici (strain CECT7483). In the trimmomatic (32), with a 30-nt sliding window approach, Synbiotic group, the probiotic was co-administered with two trimming when the average phred score dropped below 20. different mixes of vegetal fibers consisting of pectin, inulin, oat, Trimmed reads were then aligned against the human Hg19 acacia, maltodextrin polydextrose and Partially Hidrolyzed Guar genomic database using bowtie2 to remove any human Gum (PHGG) that were alternatively combined with the DNA contamination. probiotic every other week (the exact formulation can be found in the protocol). The Probiotic and Placebo groups received excipient-containing envelopes that were identical to Taxonomy annotation those of the Synbiotic group to preserve the double-blind. The exact composition and manufacturing process can be found in Taxonomy assessment was performed with Metaphlan3 Supplementary Methods. (33), performing a marker gene-based quantification, using the CHOCOPhlan 201901.1 database. The obtained data was packaged into an R phyloseq structure. T-cell and inflammation marker quantification Gene function and pathway Blood samples were collected in fasting conditions at the diversity analysis same time as stool samples, when possible. A fraction of these samples was used as whole blood to perform peripheral CD4+ Parallel to taxonomic analysis, gene function and metabolic and CD8+ counts by flow cytometry at the Germans Trias i Pujol pathway quantification was performed from the raw, quality- Hospital. Soluble markers of microbial translocation in plasma filtered sequencing data using HUMAnN3.0 (33). The software (sCD14 and LBP) and inflammation markers (IL-6, D-Dimer was run with its standard configuration and built-in databases. and CRP) were quantified using sCD14 and LBP DuoSet ELISA Results from all samples were combined into a unique table development system (pg/mL), R&D systems (Minneapolis, using HUMAnN3 inner script merge_tables.sh and clustered MN), Human IL6- High Sensitivity ELISA, Invitrogen into Metacyc pathway abundances. (Waltham, Massachusetts, USA) and RayBio Human D-Dimer or CRP Elisa Kit, RayBiotech (Peachtree Corners, GA, USA), respectively. Alpha and beta diversity metrics In this study, ecological alpha diversity was studies as gene richness. To obtain it, the post-QC sequencing data was aligned Faecal DNA extraction, library against the Integrated Gene Catalog (IGC) database (34) using preparation and sequencing Bowtie2 (35). The output was sampled at different numbers of reads to obtain rarefaction curves, from which the minimum Stool samples were collected by the patient or nursing staff sampling threshold was defined as the 95th percentile of according to the GUT (DNA Stabilized-frozen Inc., Ottawa, maximum per-sample coverages, which equalled 2·107 reads. Ontario, Canada) extraction kit. Samples were stored at -80°C Samples with max coverages below this threshold were till processing. Faecal DNA was extracted using the PowerSoil discarded, and those above were subsampled to said value to DNA Extraction Kit (MO BIO Laboratories, Carlsbad, CA, remove coverage biases. USA), which was then fragmented into 300 bp clone-sized For beta diversity, the taxonomy tables were used to libraries using Nextera-XT Illumina kit (Illumina, Inc. San construct pairwise Bray-Curtis distance matrixes between Diego, CA, USA) and sequenced in an Illumina HiSeq samples. The obtain matrices were then projected into NMDS sequencer (Illumina, Inc. San Diego, CA, USA) with a coordinates using the function MetaMDS from the R vegan (36) sequencing depth target of 20 million reads. package using their default configuration. Frontiers in Immunology 04 frontiersin.org Blázquez-Bondia et al. 10.3389/fimmu.2022.1066036 Statistical testing Safety and adverse events All statistical testing was performed using R 4.0.2. Since most The proportion of patients who suffered at least one Adverse of the quantitative variables tested departed from normality, Event (AE) during the study remained comparable between all assessed by Shapiro-wilk test, comparisons across group were arms, with no significant differences found: 9 patients with AE performed using Wilcoxon Rank-Sum test, while within-group, (50%) in the Placebo, 7 (33%) in the Probiotic and 17 (53%) in longitudinal comparisons were performed using Wilcoxon rank- the Synbiotic groups (Supplementary Table 2). Most AEs sum matched-pairs test, with Benjamini-Hochberg correction. consisted of severity grade 1 and 2, being the gastrointestinal In the case of gene pathways, Kruskal-Wallis tests between conditions the most frequent, especially flatulence, dyspepsia, groups were performed to filter out those pathways which and diarrhoea, although other conditions were reported, but changed in any timepoint other than basal, before proceeding could not be associated with treatment group assignation. Two to the pairwise group/timepoint comparisons using a p < 0.05 instances of severe grade 3 AEs were reported: abdominal cut-off. Statistical results are reported as median (IQR), and distension (possibly related) and malignant lung neoplasm longitudinal tests included median change (IQR). The number (unrelated), cancer (unrelated), while one grade 4 event of patients per group for each statistical comparison performed (hepatocarcinoma, unrelat ed) was reported in the in this study can be found in Supplementary Tables 3, 4, for Placebo group. categorical and longitudinal comparisons, respectively. In addition, we studied the changes in time for numerical variables (diversity, inflammation, T-cells, and pathways) with Synbiotic formulation does not affect Linear Mixed Models (LMM) using the Lme4 package. Models CD4+ and CD8+ counts but correlates were built in two different approaches: one where the dataset was with increased CD4/CD8 ratio and split by group, in which the LMM would be built for each using reduced inflammation time as the only fixed effect, in order to study the magnitude of the change for each group and its significance, and another CD4/CD8 ratio showed a slight albeit significant increase in where an LMM was used with the entire dataset with both group the Synbiotic group at month 6 respect to baseline (median=0.76 and time as fixed effects, to assess the difference in change in (IQR=0.51) vs 0.72 (0.45), median change= 0.04 (0.19), p = slope between groups and its significance via two-way ANOVA. 0.03). At month 9, the Synbiotic group still had increased CD4/ In all cases, the patients individual IDs were inputted as the CD8 vs baseline (0.827 (0.55) vs 0.825 (0.53), median change = random variable. 0.04 (0.15), p= 0.02), CD4+ was higher (447 (157) vs 342 (73) Bray Curtis distances were compared between groups using counts/ml, p = 0.03), and sCD14 was lower (2.16 (0.67) vs 3.18 PERMANOVA. T-cell counts, inflammation and gut (0.8) p = 0.008) than Placebo (Figure 2). permeability markers were correlated with the NMDS Analysis with LMMs mirrored most of these previous coordinates using Spearman correlation. findings, a significant increasing trend was found for both CRP (ANOVA p = 0.049, slope = 4.5) and CD4/CD8 ratio (p = 0.002, slope = 0.012) in the Synbiotic group Results (Supplementary Figure 2). Finally, CD8+ counts appeared to be impacted by overall Cohort description and follow-up microbiome structure, as it was positively correlated with the 2nd coordinate of the NMDS (Supplementary Figure 1) and, as a result, From the original goal of 100 patients recruited of which 92 CD4/CD8 ratio was negatively correlated (Supplementary Table 5) passed all the inclusion criteria, 89 patients were successfully randomised for the study. At study ending, 53 patients had complete follow-up and 36 had incomplete follow-up, of which Intervention doesn’t correlate with faecal 18 provided samples for at least two timepoints. Thus, 71 microbiome changes patients were finally selected for analysis, resulting in a proportion of 18 patients in the Placebo, 21 in the Probiotic No significant differences in gene richness were detected and 32 in the Synbiotic groups (Figure 1). The self-reported either between groups or longitudinally within groups mean adherences to the treatment showed no significant (Figure 3A). Beta diversity did not increase either, as no differences between groups (96% for the Placebo, 89.7% for changes in Bray Curtis distance vs each patient’s respective Probiotic and 96.6% for the Synbiotic group). Demographic and baseline could be observed (Figure 3B). NMDS showed no clinical variables (Table 1, Supplementary Table 1) were tested significant clustering based on Bray-Curtis distance and the with the corresponding statistical method depending on their intervention variable only explained 1.8% of the variance normality according to a previous Shapiro test. (Adonis R2 = 0.02, P=0.0051; Figure 3C). Frontiers in Immunology 05 frontiersin.org Blázquez-Bondia et al. 10.3389/fimmu.2022.1066036 FIGURE 1 CONSORT (Consolidated Standards of Reporting Trials) Flow diagram. Synbiotic supplementation affects change = 1.4·10-5 (3.12·10-5), p = 0.03; ASR: 1.5·10-5 (1.7·10-5) vs sulphate assimilation on the gut 8.79·10-6 (1.3·10-5) median change = 5.6·10-6 (1.3·10-5); p = 0.03; Cys: 3.1·10-5 (3.2·10-5) vs 1.8·10-5 (2.6·10-5), median change= 1.13·10-5 An exploratory analysis between groups identified 3 biochemical (2.42·10-5), p = 0.03), but no significant changes were found in the pathways with significant changes in relative abundance at month 6 other groups (Figure 4). (Kruskal-Wallis p < 0.01). Those pathways were consistently lower in A more in-depth approach with LMMs suggested a significant the Synbiotic group respective to the Placebo: L-methionine decreasing trend in the relative abundances of all three pathways biosynthesis (Met) (2.3·10-5 (3.17·10-5) vs 4·10-5 (5.66·10-5), p = over time in the Synbiotic group (Met: p= 0.003, slope = -3.12·10-6; 0.03), Assimilatory Sulphate Reduction (ASR) (median = 8.79·10-6 ASR: p = 0.003, slope = -1.59·10-6; Cys: p = 0.003, slope = -2.53·10-6) (IQR=1.25·10-5) vs 1.61·10-5 (2.77·10-5), p = 0.03), and cysteine (Supplementary Figure 4). Additionally, a test of fixed effect biosynthesis (Cys) (1.83·10-5 (2.56·10-5) vs 3.3·10-5 (4.62·10-5), p = interaction found a significant effect of intervention over the 0.03). At month 9, all three pathways increased in the Synbiotic group magnitude of change over time of pathway relative abundance vs month 6 (Met: 3.8·10-5 (4.2·10-5) vs 2.3·10-5 (3.17·10-5), median (ANOVA = 0.008, Supplementary Figure 6A). Frontiers in Immunology 06 frontiersin.org Blázquez-Bondia et al. 10.3389/fimmu.2022.1066036 TABLE 1 Study population description of demographic variables and clinical markers at baseline: Age, height, LBP, and weight represented as mean-(sd), as they followed a continuous normal distribution (Shapiro test pval < 0.05). [ALL] Placebo Probiotic Synbiotic p.overall N N = 71 N = 18 N = 21 N = 32 Age 49.9 (9.36) 52.8 (10.4) 49.1 (10.4) 48.8 (7.88) 0.316 71 CD4+ nadir 124 [74.5;236] 140 [81.8;212] 109 [74.0;186] 157 [70.0;261] 0.395 71 Gender: F 10 (14.1%) 1 (5.56%) 5 (23.8%) 4 (12.5%) 0.310 71 M 61 (85.9%) 17 (94.4%) 16 (76.2%) 28 (87.5%) BMI 24.4 [22.1;25.6] 24.8 [22.6;26.3] 24.4 [22.7;25.3] 24.1 [22.2;25.4] 0.778 69 Weight (Kg) 72.8 (11.0) 75.2 (12.6) 72.8 (12.3) 71.3 (9.08) 0.491 69 Height (cm) 172 (7.13) 174 (9.26) 171 (6.98) 171 (5.71) 0.336 71 Third drug class INSTI 47 (66.2%) 9 (50.0%) 16 (76.2%) 22 (68.8%) 0.282 71 NNRTI 18 (25.4%) 6 (33.3%) 5 (23.8%) 7 (21.9%) PI 6 (8.45%) 3 (16.7%) 0 (0.00%) 3 (9.38%) LBP (mg/mL) 5.99 (2.17) 5.71 (2.31) 6.12 (2.54) 6.06 (1.86) 0.815 70 sCD14 (mg/mL) 2.48 [2.01;2.92] 2.62 [2.03;2.95] 2.62 [2.03;3.25] 2.36 [1.90;2.61] 0.087 70 IL6 (pg/ml) 0.95 [0.65;1.72] 0.87 [0.59;2.41] 0.74 [0.64;1.67] 1.02 [0.68;1.42] 0.934 70 CRP (mg/mL) 18.1 [10.0;64.5] 17.5 [9.14;48.0] 15.3 [13.4;56.9] 20.3 [10.4;65.6] 0.901 70 D-Dimer (mg/mL) 2.76 [2.14;3.69] 2.67 [2.02;3.96] 2.56 [1.97;3.48] 2.76 [2.39;4.02] 0.290 70 CD4+ (counts/mL) 397 [337;466] 358 [344;427] 403 [318;466] 422 [338;476] 0.259 68 CD8+ (counts/mL) 696 [507;870] 734 [603;819] 690 [507;877] 657 [500;865] 0.583 68 CD4/CD8 0.55 [0.42;0.83] 0.49 [0.42;0.61] 0.56 [0.40;0.67] 0.57 [0.45;0.88] 0.333 68 Age, weight, height, and LBP and were tested by ANOVA, categorical variables were compared using Fisher’s exact test, while non-normal continuous variables (BMI, CD4 + nadir, years with cART, diagnosis, and other clinical variables) were tested by Kruskal-Wallis. Third drug row refers to the class of the 3rd ARV drug they received INSTI, Integrase Strand Transfer Inhibitor; NNRTI, Non-nucleoside reverse transcriptase inhibitors. All other variables represented as either count (percentage), or in the case of non-normally distributed as median-[Q1;Q3]. Presence of probiotic strains in faeces Microbiome analysis under this new stratification found no does not relate to changes in significant changes in alpha diversity as gene richness (Figure 6A) faecal microbiome or beta diversity as Bray-Curtis distance from baseline (Figure 6B) over time between groups with (Present) and without (Absent) To better understand the lack of microbiome changes and probiotic detection. This new variable only explained a 3.6% of the the uncertain clinical changes, we tested how well the probiotic variance of Bray-Curtis distances between samples (Adonis R2 = strains could maintain their presence in the gut. Only 6 (28.6%) 0.0362, p = 0.001). No clear group clustering could be observed patients in the Probiotic and 10 (31.3%) in the Synbiotic groups under this new definition (Figure 6C) had detectable levels of either L. plantarum or P. acidilactici in at least two stool samples at months 1, 3 or 6, although in these cases, probiotic species median relative abundance started Detection of probiotic in faeces declining after the 3rd month of treatment, long before the associates with decreased inflammation wash-out phase. In the remaining patients, probiotic species (CRP, IL-6) while non-detection were detected in only one of the timepoints in 5(23.8%) and 13 associates with increased CD4/CD8 ratio (40%) of the Probiotic and Synbiotic, respectively. None of the probiotic strains was detected in any sample obtained at baseline, At month 6, the Present group was associated with a month 9 or in the Placebo group at any time point (Figure 5A). significant decrease of CRP (median=11.1 (IQR=22) vs 19.2 To better filter any potential probiotic strain-specific effect, as (66), median change= -2.7 (13.2) ug/ml, p = 0.04), while the well as uncover potential factors that may affect probiotic Absent group related to an increase in CD4/CD8 ratio (0.72 (0.57) engraftment, a new variable was defined. Two groups were vs 0.56 (0.4), median change = 0.4 (0.2) p=0.015) vs baseline. IL-6 created that separated patients with or without detectable levels of differed between both groups at month 6, being lower in Present P. acidilactici in at least 2 stool samples belonging to different time than Absent group (0.58 (1.13) vs 1.17 (1.59) ug/ml, p = 0.02). At points, excluding those patients in the Placebo group. P. acidilactici month 9, an increase vs baseline was observed for CD4/CD8 ratio was chosen because it had presence in all samples where L. (0.67 (0.5) vs 0.59 ± (0.49), median change = 0.04 (0.18), p = 0.02), plantarum was detectable, but not the other way around (Figure 5B). and CD4+ counts (457 (153) vs 416 (142), median change = 45 Frontiers in Immunology 07 frontiersin.org Blázquez-Bondia et al. 10.3389/fimmu.2022.1066036 FIGURE 2 Evolution of T-cell counts, ratio, inflammation, and bacterial translocation markers between months 0 and 6, among treatment group. Comparisons within group and between time points were performed by Wilcoxon test, in its paired form for longitudinal differences and unpaired for cross-group comparisons. Significance was coded as follows: *(p < 0.05), **(p < 0.01). (75), counts/ml, p = 0.005) in Absent but not in Present However, these trends changed when stratifying by treatment group (Figure 7). group, especially for CRP, which increased in the Synbiotic Analysis with LMM found little overall effect on immune arm (p = 0.013, slope = 7.4) (Supplementary Figure 3C). Also, status and inflammation, with a slight increase of CD4/CD8 CD4/CD8 ratio increase was specific to the subjects in Absent ratio (p = 0.008, slope = 0.012) and D-Dimer (p = 0.047, slope group who were in the Synbiotic arm (p = 0.005, slope = 0.236) in the Absent group (Supplementary Figure 3A). = 0.015). Frontiers in Immunology 08 frontiersin.org Blázquez-Bondia et al. 10.3389/fimmu.2022.1066036 A C B FIGURE 3 Effect of probiotics on the gut microbiome. (A) Gene richness counts for each intervention arm and month. Comparisons between group at each timepoint were performed using Wilcoxon test, while the paired version was used for within-group comparisons between months. (B) Distribution of Bray Curtis-Distances from baseline for each month and intervention arm. (C) NMDS showing sample ordination according to Bray-Curtis distance. Group distance dissimilarity was tested using ADONIS. Probiotic presence in faeces is not patients with supressed HIV after long, stable cART in a double- coupled with lower abundances of ASR blind, randomized trial. We found that Synbiotic intervention pathways with strains of L. plantarum and P. acidilactici along with prebiotic fibers is safe and associated with slight increases of Sulphate assimilation pathways seemed slightly reduced in CD4+ counts, CD4/CD8 ratio, and a decrease of the gut the probiotic Present vs Absent group, but the differences were leakiness, as measured with sCD14 which is a proxy for statistically non-significant (Figure 8). Analysis with LMMs bacterial endotoxin entering the bloodstream. However, the found no significant trends for any of the three previously clinical significance of such improvements is uncertain, studied pathways neither in the Present nor in the Absent especially as they manifested 3 months after the intervention group. Interestingly, after stratifying by intervention, pathway stopped. A more in-depth analysis suggests that the presence of relative abundances did not change over time in the Present the probiotic strains in stool was associated with lower levels of patients within the Probiotic group but increased in the Absent proin flammatory cytokines (IL-6 an d CRP), while group (Met: p = 0.026, slope = 2.13·10-6; ASR: p=0.024, slope = improvements in CD4/CD8 ratio appeared to be linked to the 1.11·10-6; Cys: p = 0.021, slope = 1.256·10-6), while the Synbiotic prebiotic fibers. The lowering in proinflammatory cytokines group showed a relative abundance decline in both Present and seems in agreement with previous reports using the same Absent groups (Supplementary Figure 5). Fixed effect interaction probiotic composition in animal models of IBD (37). This tests on unified models found no significant effect of presence/ could point to a trade-off between immune activation and absence in any of the pathways, with and without stratification modulation by the prebiotic and probiotic, respectively. Of (Supplementary Figure 6). note, the reduction in inflammation markers happened during the intervention period, when no changes in gut permeability markers could be observed, suggesting that the Discussion immunomodulatory activity of the probiotics may be independent of the gut barrier status. Some gut barrier- We assessed the efficacy and safety of long-term probiotic unrelated anti-inflammatory mechanisms have been described, supplementation on immunodiscordant (CD4+ counts < 500) as Kawashima, Tadaomi et al. (38) found, lactic acid bacteria can Frontiers in Immunology 09 frontiersin.org Blázquez-Bondia et al. 10.3389/fimmu.2022.1066036 FIGURE 4 Changes in gene function at both start and end of the treatment period, among treatment group. Across group comparisons were performed with regular Wilcoxon test and longitudinal comparisons were performed using matched-samples Wilcoxon test. Significance was coded as follows: *(p < 0.05). induce IgA secretion to the gut lumen, which is a known the gastrointestinal (GI) tract, some lactobacilli show a nomadic immunomodulatory agent (39, 40). behaviour (44) and tend to stay most commonly on the upper GI The health-promoting properties of lactic acid bacteria have hence they tend to be underrepresented and transient in stool been widely reported in vivo and in vitro (41, 42). Nevertheless, samples (45). In turn, implantation itself is not a strict the actual implantation and ecological viability of such strains is requirement to generate change in the gut ecosystem or the a multidimensional problem where host related factors and host’s physiology (46). In this study, P. acidilactici was more inherent ecological features of the host microbiome (such as consistently found than Lactobacillus plantarum in faeces but, normal ecological succession across the intestine) (43). Within unlike the later, the former has been reported as having good Frontiers in Immunology 10 frontiersin.org Blázquez-Bondia et al. 10.3389/fimmu.2022.1066036 A B FIGURE 5 Detection of the probiotic strains in stool samples. (A) Relative abundances (in -log10) of P. acidilactici and L. plantarum in faeces for each of the intervention arms. (B) Heatmap representing presence/absence of each arm in each sample. Rows represent single patients and columns represent the month each sample belongs to. White cells represent lack of sample. adhesion to the lower GI and higher presence in stool (47). especially of short chain fatty acids (51), but little is known Noteworthy, such differences may be strain-specific and not about their relationship with sulphate metabolism. The fact that generalizable to other formulations (43). Hence, assessing the the Synbiotic group showed a consistent decline in such effectiveness of the probiotic strains in this study has been pathways could point to metabolic modulation and a problematic, due to the low proportion of patients having a population shift by the prebiotic fibers in the small intestine, detectable presence of those, but also to the spatial and temporal although such changes may have been partially represented in complexity that characterizes the gut microbiome. the faecal samples. Additionally, we found that synbiotic intake was linked to Previous studies also found a lack of change in neither decreases in the Assimilatory Sulphate Reduction (ASR) and peripheral blood T-cells and/or gut permeability markers with sulphur-containing amino acids biosynthesis pathways. ASR, probiotics only. Serrano-Villar et al. (52) found a significant unlike the Dissimilatory Sulphate Reduction (DSR), takes decrease in inflammation markers (IL-6 and CRP) but no sulphate without producing hydrogen sulphide as a final differential improvement in either circulatory T-cells nor product, which has been shown to impair butyrate oxidation, sCD14 after 48 weeks, using a synbiotic formulation of the primary source of energy of enterocytes, and has been linked Saccharomyces boulardii with various additives. However, this to gut inflammation and Ulcerative Colitis (48, 49). DSR is study was performed on late presenter, cART-naïve individuals, exclusive of anaerobic bacteria that undergo sulphate while our study was conducted on immunodiscordant patients respiration, while ASR is more ubiquitous (50). Intake of with stable cART. Fructose and Glucose Oligosaccharides (FOS and GOS Previous studies using HIV infected immune discordant respectively) has been extensively used to modulate the cohort reported diverse results. Presti et al. (53) tested a microbiome, and has been linked to metabolic changes, probiotic treatment consisting of different strains of Frontiers in Immunology 11 frontiersin.org Blázquez-Bondia et al. 10.3389/fimmu.2022.1066036 A C B FIGURE 6 Differences by presence or absence of the probiotic strains on the gut microbiome. (A) Gene richness counts for each group arm and month. Comparisons between group at each timepoint were used simple Wilcoxon test, while the paired version was used for within-group comparisons between months. (B) Distribution of Bray Curtis-Distances from baseline for each month and group. (C) NMDS showing sample ordination according to Bray-Curtis distance. Group distance dissimilarity was tested using ADONIS. streptococcus, bifidobacteria, and lactobacilli, without prebiotics, Several limitations in this study should be considered. over 12 weeks, finding no differences in gut integrity (sCD14) First, since patients were randomized by class of third and inflammation markers (D-Dimer, IP-10) but a significant antiretroviral drug, same-class ARV drgus might have decrease in Proteobacteria. Stiksrud et al. (54) found a decrease different effects of the gut microbiome. Additionally, the in inflammation (IL-6 and D-Dimer) but no differences in cohort selected for this study was composed by bacterial translocation markers and CD4+ counts after 8 weeks immunodiscordant individuals, whose low CD4+ counts of intervention with formulation of skimmed milk, enriched after viral suppression made them prone to infections that with various species of Lactobacillus and Bifidobacterium vs a required antibiotic treatment, causing many dropouts. placebo of skimmed-milk only and a control groups. Geng et al. However, no significant biases were created by dropouts (55) reported an improvement in gut integrity (D-Dimer, DAO) among the groups, although final sample size (n=71) was and an enhanced CD4+ recovery in immune discordant patients clearly smaller than original target (n=100) and may have using pre-digested protein supplementation. Despite the been underpowered to detect some effects. Importantly, faecal differences from the previously described studies (whether samples hold an inherent bias and may not be representative from study design or probiotic formulation), our study found of the actual gut microbiome composition, especially from the improvement in inflammation and translocation markers but upper GI. Also, diet and concomitant treatments may affect not an overall improvement in immune reconstitution. In faecal microbiome composition. While we found low rates of addition, we could assess probiotic-related gut microbiome at concomitant medications and these were balanced among species-level resolution of the microbiome, coupled with groups, dietary information was not available, although functional evidence and a washout period which adds extreme diets were excluded. This may affect our capability robustness to any finding of potential signal of treatment effect. to detect any correlation between clinical outcomes and Frontiers in Immunology 12 frontiersin.org Blázquez-Bondia et al. 10.3389/fimmu.2022.1066036 FIGURE 7 Evolution of T-cell counts, ratio, inflammation, and bacterial translocation markers between months 0 and 6, separated presence/absence of probiotic strains. Comparisons within group and between time points were performed by Wilcoxon test, in its paired form for longitudinal differences and unpaired for cross-group comparisons. Significance was coded as follows: *(p < 0.05), **(p < 0.01). microbiome features. Finally, the fact that treatment intake Conclusions was self-reported, could have led to many patients not taking the treatment but reporting otherwise during the 6 months A synbiotic intervention with L. plantarum and P. acidilactici follow-up, thus overestimating the actual intake of the was safe and well tolerated. Synbiotic intervention led to small probiotics. All these shortcomings add up to the limited increases in CD4/CD8 ratio and minor reductions in sCD14 after effect of probiotics in immune reconstitution and affect the 6 months and continued 3 months after discontinuing the capability to translate research results from into clinical intervention, but such changes are of uncertain clinical practice and warrant further research. significance. A probiotic with the same composition but without Frontiers in Immunology 13 frontiersin.org Blázquez-Bondia et al. 10.3389/fimmu.2022.1066036 FIGURE 8 Longitudinal and across-group comparisons of Assimilatory Sulphate Reduction and sulphur amino acid pathways across intervention groups. Comparisons were performed across between groups by Wilcoxon test and longitudinally within-group with paired Wilcoxon test. prebiotics was also safe but did not achieve any impact on (reference PI-13-046). All participants provided written immune parameters or faecal microbiome composition. informed consent in accordance with the World Medical Association Declaration of Helsinki, Fortaleza and Brazil, Data availability statement October 2013 and personal data was managed according to Spanish data protection law (LOPD 15/1999). The study The data presented in the study are deposited in the EBI- concept, design, patient information and results were discussed ENA repository, accession number PRJEB56022. with the FLSida Community Advisory Committee, in accordance with AB-Biotics internal QC auditing. All available Ethics statement information can be found in the protocol (Supplementary Methods), and the study is registered in clinicaltrials.gov, The study was reviewed and approved by the Institutional accession: NCT03542786. The patients/participants provided Review Board of the Hospital Universitari Germans Trias i Pujol their written informed consent to participate in this study. Frontiers in Immunology 14 frontiersin.org Blázquez-Bondia et al. 10.3389/fimmu.2022.1066036 Author contributions AB-Biotics for their non-financial support and feedback, and the staff from MISTRAL. RP, JE-M, JRS, and MN-J conceived and designed the study. RP and JRS recruited the study participants and performed their clinical evaluations. MA performed study monitoring. MP, AE- Conflict of interest T, and MC performed sample processing for sequencing and inflammation markers experiments, under the supervision of JE-M and MA are full-time employees of AB-BIOTICS SA, MN-J and RP. CB and FC-M performed the bioinformatic and the company owning IP rights for the probiotic strains used in statistical analyses with the supervision of RP, MN-J, CB, RP, this study. MN-J is co-founder and shareholder of Nano1Health and MN-J wrote the paper, which was reviewed, edited, and SL, outside the scope of the submitted work. approved by all authors. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Funding This project has received funding from the European Union’s Publisher’s note Horizon 2020 Research and Innovation Programme under Grant Agreement N° 847943, and the Spanish Ministry of Economy and All claims expressed in this article are solely those of the Competitiveness (MINECO), reference RTC-2016-5223-1. This authors and do not necessarily represent those of their affiliated research was supported by CIBER -Consorcio Centro de organizations, or those of the publisher, the editors and the Investigación Biomédica en Red- (CB 2021), Instituto de Salud reviewers. Any product that may be evaluated in this article, or Carlos III, Ministerio de Ciencia e Innovación and Unión Europea claim that may be made by its manufacturer, is not guaranteed – NextGenerationEU. or endorsed by the publisher. Acknowledgments Supplementary material We thank all the enrolled participants who contributed to The Supplementary Material for this article can be found this study, the staff from Fundació Lluita contra la Sida (FLS), online at: https://www.frontiersin.org/articles/10.3389/ IrsiCaixa and Hospital Germans Trias I Pujol. We also thank fimmu.2022.1066036/full#supplementary-material References 1. Doitsh G, Galloway NLK, Geng X, Yang Z, Monroe KM, Zepeda O, et al. Cell prolonged undetectable plasma viral loads. J Infect Dis (2005) 191:1670–9. death by pyroptosis drives CD4 T-cell depletion in HIV-1 infection. Nature (2014) doi: 10.1086/429670 505:509–14. doi: 10.1038/nature12940 9. Negredo E, Massanella M, Puig J, Pé rez-Á lvarez N, Gallego-Escuredo JM, 2. Pantazis N, Papastamopoulos V, Paparizos V, Metallidis S, Adamis G, Villarroya J, et al. Nadir CD4 T cell count as predictor and high CD4 T cell intrinsic Antoniadou A, et al. Long-term evolution of CD4+ cell count in patients under apoptosis as final mechanism of poor CD4 T cell recovery in virologically combined antiretroviral therapy. AIDS (2019) 33:1645–55. doi: 10.1097/ suppressed HIV-infected patients: Clinical implications. Clin Infect Dis (2010) QAD.0000000000002248 50:1300–8. doi: 10.1086/651689 3. Deeks SG, Overbaugh J, Phillips A, Buchbinder S. HIV Infection. Nat Rev Dis 10. D’Angelo C, Reale M, Costantini E. Microbiota and probiotics in health and Prim (2015) 1:1–22. doi: 10.1038/nrdp.2015.35 HIV infection. Nutrients (2017) 9(6):615. doi: 10.3390/nu9060615 4. Chomont N, El-Far M, Ancuta P, Trautmann L, Procopio FA, Yassine-Diab 11. Kanwar B, Favre D, McCune JM. Th17 and regulatory T cells: Implications B, et al. HIV Reservoir size and persistence are driven by T cell survival and for AIDS pathogenesis. Curr Opin HIV AIDS (2010) 5:151–7. doi: 10.1097/ homeostatic proliferation. Nat Med (2009) 15:893–900. doi: 10.1038/nm.1972 COH.0b013e328335c0c1 5. Kuo HH, Lichterfeld M. Recent progress in understanding HIV reservoirs. 12. Hartigan-O’Connor DJ, Hirao LA, McCune JM, Dandekar S. Th17 cells and Curr Opin HIV AIDS (2018) 13:137–42. doi: 10.1097/COH.0000000000000441 regulatory T cells in elite control over HIV and SIV. Curr Opin HIV AIDS (2011) 6. Collaboration TATC. Life expectancy of individuals on combination 6:221–7. doi: 10.1097/COH.0b013e32834577b3 antiretroviral therapy in high-income countries: A collaborative analysis of 14 13. Nilsson J, Boasso A, Velilla PA, Zhang R, Vaccari M, Franchini G, et al. HIV-1- cohort studies. Lancet (2008) 372:293–9. doi: 10.1016/S0140-6736(08)61113-7 driven regulatory T-cell accumulation in lymphoid tissues is associated with disease 7. Gazzola L, Tincati C, Bellistrì GM, Monforte ADA, Marchetti G. The absence progression in HIV/AIDS. Blood (2006) 108:3808–17. doi: 10.1182/blood-2006-05-021576 of CD4+ T cell count recovery despite receipt of virologically suppressive highly 14. Kinugasa T, Sakaguchi T, Gu X, Reinecker HC. Claudins regulate the active antiretroviral therapy: Clinical risk, immunological gaps, and therapeutic intestinal barrier in response to immune mediators. Gastroenterology (2000) options. Clin Infect Dis (2009) 48:328–37. doi: 10.1086/595851 118:1001–11. doi: 10.1016/S0016-5085(00)70351-9 8. Benveniste O, Flahault A, Rollot F, Elbim C, Estaquier J, Pé dron B, et al. 15. Salzman NH, Hung K, Haribhai D, Chu H, Karlsson-Sjöberg J, Amir E, et al. Mechanisms involved in the low-level regeneration of CD4+ cells in HIV-1- Enteric defensins are essential regulators of intestinal microbial ecology. Nat infected patients receiving highly active antiretroviral therapy who have Immunol (2010) 11:76–83. doi: 10.1038/ni.1825 Frontiers in Immunology 15 frontiersin.org Blázquez-Bondia et al. 10.3389/fimmu.2022.1066036 16. Marchetti G, Bellistrì GM, Borghi E, Tincati C, Ferramosca S, La Francesca 37. Loré n V, Manyé J, Fuentes MC, Cabré E, Ojanguren I, Espadaler J. M, et al. Microbial translocation is associated with sustained failure in CD4+ T-cell Comparative effect of the I3.1 probiotic formula in two animal models of colitis. reconstitution in HIV-infected patients on long-term highly active antiretroviral Probiotics Antimicrob Proteins (2017) 9:71–80. doi: 10.1007/S12602-016-9239-5 therapy. AIDS (2008) 22:2035–8. doi: 10.1097/QAD.0b013e3283112d29 38. Kawashima T, Ikari N, Kouchi T, Kowatari Y, Kubota Y, Shimojo N, et al. 17. Vujkovic-Cvijin I, Swainson LA, Chu SN, Ortiz AM, Santee CA, Petriello A, The molecular mechanism for activating IgA production by pediococcus et al. Gut-resident lactobacillus abundance associates with IDO1 inhibition and acidilactici K15 and the clinical impact in a randomized trial. Sci Rep (2018) 8 Th17 dynamics in SIV-infected macaques. Cell Rep (2015) 13:1589–97. (1):5065. doi: 10.1038/S41598-018-23404-4 doi: 10.1016/j.celrep.2015.10.026 39. Guo Y, Wang B, Wang T, Gao L, Yang ZJ, Wang FF, et al. Biological 18. Vallejo AN, Weyand CM, Goronzy JJ. T-Cell senescence: A culprit of characteristics of IL-6 and related intestinal diseases. Int J Biol Sci (2021) 17:204. immune abnormalities in chronic inflammation and persistent infection. Trends doi: 10.7150/IJBS.51362 Mol Med (2004) 10:119–24. doi: 10.1016/j.molmed.2004.01.002 40. Wang Y, Liu L, Moore DJ, Shen X, Peek RM, Acra SA, et al. An LGG- 19. Blaschitz C, Raffatellu M. Th17 cytokines and the gut mucosal barrier. J Clin derived protein promotes IgA production through upregulation of APRIL Immunol (2010) 30:196–203. doi: 10.1007/s10875-010-9368-7 expression in intestinal epithelial cells. Mucosal Immunol (2017) 10:373–84. 20. Vujkovic-Cvijin I, Dunham RM, Iwai S, Maher MC, Albright RG, doi: 10.1038/MI.2016.57 Broadhurst MJ, et al. Dysbiosis of the gut microbiota is associated with HIV 41. Smits HH, Engering A, van der Kleij D, De Jong EC, Schipper K, Van Capel disease progression and tryptophan catabolism. Sci Transl Med (2013) 5:193ra91– TMM, et al. Selective probiotic bacteria induce IL-10-producing regulatory T cells 193ra91. doi: 10.1126/scitranslmed.3006438 in vitro by modulating dendritic cell function through dendritic cell-specific 21. Jenabian M-A, El-Far M, Vyboh K, Kema I, Costiniuk CT, Thomas R, et al. intercellular adhesion molecule 3-grabbing nonintegrin. J Allergy Clin Immunol Immunosuppressive tryptophan catabolism and gut mucosal dysfunction following (2005) 115:1260–7. doi: 10.1016/J.JACI.2005.03.036 early HIV infection. J Infect Dis (2015) 212:355–66. doi: 10.1093/infdis/jiv037 42. Kim HG, Lee SY, Kim NR, Lee HY, Ko MY, Jung BJ, et al. Lactobacillus 22. Sheil B, Shanahan F, O’Mahony L. Probiotic effects on inflammatory bowel plantarum lipoteichoic acid down-regulated shigella flexneri peptidoglycan- disease. J Nutr (2007) 137(3 Suppl 2):819S–24S. doi: 10.1093/jn/137.3.819S induced inflammation. Mol Immunol (2011) 48:382–91. doi: 10.1016/ J.MOLIMM.2010.07.011 23. Zhang XL, Chen MH, Geng ST, Yu J, Kuang YQ, Luo HY, et al. Effects of probiotics on diarrhea and CD4 cell count in people living with HIV: A systematic 43. Walter J. Ecological role of lactobacilli in the gastrointestinal tract: review and meta-analysis. Front Pharmacol (2021) 12:570520/BIBTEX. Implications for fundamental and biomedical research. Appl Environ Microbiol doi: 10.3389/FPHAR.2021.570520/BIBTEX (2008) 74:4985. doi: 10.1128/AEM.00753-08 24. Monachese M, Cunningham-Rundles S, Diaz MA, Guerrant R, Hummelen 44. Martino ME, Bayjanov JR, Caffrey BE, Wels M, Joncour P, Hughes S, et al. R, Kemperman R, et al. Probiotics and prebiotics to combat enteric infections and Nomadic lifestyle of lactobacillus plantarum revealed by comparative genomics of HIV in the developing world: A consensus report. Gut Microbes (2011) 2(3):198– 54 strains isolated from different habitats. Environ Microbiol (2016) 18:4974–89. 207. doi: 10.4161/GMIC.2.3.16106 doi: 10.1111/1462-2920.13455 25. Wang Y, Li X, Ge T, Xiao Y, Liao Y, Cui Y, et al. Probiotics for prevention 45. Zmora N, Zilberman-Schapira G, Suez J, Mor U, Dori-Bachash M, and treatment of respiratory tract infections in children: A systematic review and Bashiardes S, et al. Personalized gut mucosal colonization resistance to empiric meta-analysis of randomized controlled trials. Med (United States) (2016) 95(31): probiotics is associated with unique host and microbiome features. Cell (2018) e4509. doi: 10.1097/MD.0000000000004509 174:1388–1405.e21. doi: 10.1016/J.CELL.2018.08.041/ATTACHMENT/ 39F12DBE-F56A-4CD7-9089-962332F15476/MMC7.PDF 26. Meyer-Myklestad MH, Medhus AW, Lorvik KB, Seljeflot I, Hansen SH, Holm K, et al. Human immunodeficiency virus–infected immunological 46. Wieërs G, Belkhir L, Enaud R, Leclercq S, Philippart de Foy JM, Dequenne I, nonresponders have colon-restricted gut mucosal immune dysfunction. J Infect et al. How probiotics affect the microbiota. Front Cell Infect Microbiol (2020) 9:454. Dis (2022) 225:661. doi: 10.1093/INFDIS/JIAA714 doi: 10.3389/FCIMB.2019.00454 27. Lu W, Feng Y, Jing F, Han Y, Lyu N, Liu F, et al. Association between gut 47. Balgir PP, Kaur B, Kaur T, Daroch N, Kaur G. In vitro and In vivo survival microbiota and CD4 recovery in HIV-1 infected patients. Front Microbiol (2018) and colonic adhesion of pediococcus acidilactici MTCC5101 in human gut. BioMed 9:1451/FULL. doi: 10.3389/FMICB.2018.01451/FULL Res Int (2013) 2013:583850. doi: 10.1155/2013/583850 28. Lee SC, Chua LL, Yap SH, Khang TF, Leng CY, Raja Azwa RI, et al. 48. Jørgensen J, Mortensen PB. Hydrogen sulfide and colonic epithelial Enrichment of gut-derived fusobacterium is associated with suboptimal immune metabolism: implications for ulcerative colitis. Dig Dis Sci (2001) 46:1722–32. recovery in HIV-infected individuals. Sci Rep (2018) 8(1):14277. doi: 10.1038/ doi: 10.1023/A:1010661706385 s41598-018-32585-x 49. Figliuolo VR, dos Santos LM, Abalo A, Nanini H, Santos A, Brittes NM, 29. Zeuthen LH, Fink LN, Frokiaer H. Epithelial cells prime the immune et al. Sulfate-reducing bacteria stimulate gut immune responses and contribute to response to an array of gut-derived commensals towards a tolerogenic inflammation in experimental colitis. Life Sci (2017) 189:29–38. doi: 10.1016/ phenotype through distinct actions of thymic stromal lymphopoietin and J.LFS.2017.09.014 transforming growth factor-b. Immunology (2008) 123:197–208. doi: 10.1111/ 50. Kushkevych I, Cejnar J, Treml J, Dordević D, Kollar P, Vı́tězová M. Recent j.1365-2567.2007.02687.x advances in metabolic pathways of sulfate reduction in intestinal bacteria. Cells 30. Hummelen R, Changalucha J, Butamanya NL, Koyama TE, Habbema JDF, (2020) 9(3):698. doi: 10.3390/CELLS9030698 Reid G. Efect of 25 weeks probiotic supplementation on immune function of HIV 51. Serrano-Villar S, Vá zquez-Castellanos JF, Vallejo A, Latorre A, Sainz T, patients. Gut Microbes (2011) 2:80–5. doi: 10.4161/gmic.2.2.15787 Ferrando-Martı́nez S, et al. The effects of prebiotics on microbial dysbiosis, 31. Rawi MH, Zaman SA, Pa’ee KF, Leong SS, Sarbini SR. Prebiotics metabolism butyrate production and immunity in HIV-infected subjects. Mucosal Immunol by gut-isolated probiotics. J Food Sci Technol (2020) 57:2786. doi: 10.1007/S13197- (2016) 10(5):1279–93. doi: 10.1038/mi.2016.122 020-04244-5 52. Serrano-Villar S, De Lagarde M, Vá zquez-Castellanos J, Vallejo A, 32. Bolger AM, Lohse M, Usadel B. Trimmomatic: A flexible trimmer for Bernadino JI, Madrid N, et al. Effects of immunonutrition in advanced human illumina sequence data. Bioinformatics (2014) 30:2114–20. doi: 10.1093/ immunodeficiency virus disease: A randomized placebo-controlled clinical trial bioinformatics/btu170 (Promaltia study). Clin Infect Dis (2019) 68(1):120–30. doi: 10.1093/cid/ciy414 33. Beghini F, McIver LJ, Blanco-Mı́guez A, Dubois L, Asnicar F, Maharjan S, 53. Presti RM, Yeh E, Williams B, Landay A, Jacobson JM, Wilson C, et al. A et al. Integrating taxonomic, functional, and strain-level profiling of diverse randomized, placebo-controlled trial assessing the effect of VISBIOME ES microbial communities with bioBakery 3. Elife (2021) 10:e65088. doi: 10.7554/ probiotic in people with HIV on antiretroviral therapy. Open Forum Infect Dis eLife.65088 (2021) 8(12):ofab550. doi: 10.1093/OFID/OFAB550 34. Li J, Jia H, Cai X, Zhong H, Feng Q, Sunagawa S, et al. An integrated catalog 54. Stiksrud B, Nowak P, Nwosu FC, Kvale D, Thalme A, Sonnerborg A, of reference genes in the human gut microbiome. Nat Biotechnol (2014) 32:834–41. e t a l . Re du ce d l e v el s o f d-d im er a n d c ha n g es i n gu t m ic r o b io t a doi: 10.1038/NBT.2942 composition after probiotic intervention in HIV-infected individuals on stable ART. J Acquir Immune Defic Syndr (2015) 70:329–37. doi: 10.1097/ 35. Langmead B, Trapnell C, Pop M, Salzberg SL. Ultrafast and memory- QAI.0000000000000784 efficient alignment of short DNA sequences to the human genome. Genome Biol (2009) 10:R25. doi: 10.1186/gb-2009-10-3-r25 55. Geng ST, Zhang JB, Wang YX, Xu Y, Lu D, Zhang Z, et al. Pre-digested protein enteral nutritional supplementation enhances recovery of CD4+ T cells 36. Oksanen J, Simpson GL, Blanchet FG, Kindt R, Legendre P, Minchin PR, and repair of intestinal barrier in HIV-infected immunological non-responders. et al. Vegan: Community ecology package. R package version 2.6-4. In: Vegan Front Immunol (2021) 12:757935/FULL. doi: 10.3389/FIMMU.2021.757935/ community ecol (2012). Available at: https://cran.r-project.org/package=vegan. FULL Frontiers in Immunology 16 frontiersin.org