Probiotic effects on immunity and microbiome in HIV-1 discordant patients

Abstract

Background: Some HIV-1 infected patients are unable to completely recover normal CD4+ T-cell (CD4+) counts after achieving HIV-1 suppression with combined Antiretroviral Therapy (cART), hence being classified as immuno-discordant. The human microbiome plays a crucial role in maintaining immune homeostasis and is a potential target towards immune reconstitution.

Setting: RECOVER (NCT03542786) was a double-blind placebo-controlled clinical trial designed to evaluate if the novel probiotic i3.1 (AB-Biotics, Sant Cugat del Vallès, Spain) was able to improve immune reconstitution in HIV-1 infected immuno-discordant patients with stable cART and CD4+ counts <500 cells/mm3. The mixture consisted of two strains of L. plantarum and one of P. acidilactici, given with or without a fiber-based prebiotic.

Methods: 71 patients were randomized 1:2:2 to Placebo, Probiotic or probiotic + prebiotic (Synbiotic), and were followed over 6 months + 3-month washout period, in which changes on systemic immune status and gut microbiome were evaluated. Primary endpoints were safety and tolerability of the investigational product. Secondary endpoints were changes on CD4+ and CD8+ T-cell (CD8+) counts, inflammation markers and faecal microbiome structure, defined by alpha diversity (Gene Richness), beta diversity (Bray-Curtis) and functional profile. Comparisons across/within groups were performed using standard/paired 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 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: HIV; immune reconstitution; prebiotics; probiotics; synbiotics.

Figure 1

CONSORT (Consolidated Standards of Reporting Trials) Flow diagram.

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).

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.

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).

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.

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.

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).

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.