Clinical trial NCT02706717
Conditions: HIV-1 Infection
Sponsor: Advancing Clinical Therapeutics Globally for HIV/AIDS and Other Infections
Start date: 2016-04-01; Completion date: 2017-08-28
Enrollment: 93 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

Brief summary:
The purpose of the study was to evaluate whether the probiotic Visbiome Extra Strength reduces inflammation in HIV-infected men and women when compared to a placebo (inactive medication like a dummy pill). The study evaluated whether taking Visbiome Extra Strength by mouth for 24 weeks was safe and well-tolerated for HIV-infected persons on antiretroviral therapy (ART). Probiotics are germs such as yeast or bacteria that are found in food and supplements that are used to improve the health of the digestive system. Many people refer to probiotics as "helpful bacteria." These bacteria live in the body and help the body work normally. In some medical conditions, including HIV infection, helpful bacteria are replaced with bacteria that can change the normal intestinal function and increase inflammation. The investigators tested whether giving a probiotic restored normal intestinal function and decreased inflammation.

Detailed description:
This was a phase II, randomized, double-blind, two-arm study to evaluate whether there is a significant change in sCD14 after 24 weeks of probiotic Visbiome Extra Strength (ES) therapy, and to determine the safety and tolerability of this agent in HIV-infected participants on stable antiretroviral therapy (ART). Participants were randomized 1:1 to Visbiome ES and placebo arms. Both arms initiated study treatment at Week 2 and took 1 sachet per day for the first 2 weeks and then 1 sachet twice daily for the next 22 weeks. All participants were followed for an additional 12 weeks off study product.

The study clinic visits included Entry (Week 0), and Weeks 2, 6, 14, 25, 26, and 38. Plasma for the primary outcome was collected at Weeks 0, 2, 25, and 26. The evaluations of safety (clinical assessment for signs and symptoms, diagnoses, and laboratory tests) were done at Weeks 2, 6, 14, 26, and 38.

Currently, the results are entered for the primary outcome measure and select secondary outcomes only. The results on the remaining secondary outcomes will be posted when they become available.

Arms: arm 1: None arm 2: None
Interventions: intervention 1: Visbiome Extra Strength intervention 2: Placebo
Intervention descriptions: intervention 1: From week 2 to 4, participant will receive one sachet orally daily. From week 4 to 26, participant will receive one sachet orally twice daily. intervention 2: From week 2 to 4, participant will receive one sachet orally daily. From week 4 to 26, participant will receive one sachet orally twice daily.

Protocol documents:
Prot_000.pdf: 









































































































































































































































SAP_001.pdf: 
A5350 Statistical Analysis Plan 
 
Safety, Tolerability, and Effects of the Probiotic Visbiome Extra 
Strength on Gut Microbiome and Immune Activation Markers in HIV-
Infected Participants on Suppressive Antiretroviral Therapy 
 
ClinicalTrials.gov Identifier: NCT02706717 
 
Final Version 2.0 
 
April 6, 2018 
 
This is ACTG A5350 SAP Version 2.0 with names of authors, names of publication 
writing team members and analysis timeline redacted 


A5350 Statistical Analysis Plan Final Version 2.0 
 
April 6, 2018 
 
Page 2 of 8 
Table of Contents 
Table of Contents ......................................................................................................................................................... 2 
1 
Introduction ........................................................................................................................................................... 3 
2 
Study Overview ..................................................................................................................................................... 3 
2.1 
Design .......................................................................................................................................................... 3 
2.2 
Duration ....................................................................................................................................................... 3 
2.3 
Sample Size ................................................................................................................................................. 3 
2.4 
Population ................................................................................................................................................... 3 
2.5 
Stratification ................................................................................................................................................ 3 
2.6 
Regimen ....................................................................................................................................................... 3 
2.7 
Primary Objective ....................................................................................................................................... 3 
2.7.1 
Hypothesis ....................................................................................................................................... 3 
2.8 
Secondary Objectives ................................................................................................................................ 3 
2.8.1 
Hypotheses ...................................................................................................................................... 4 
2.9 
Exploratory Objectives ............................................................................................................................... 4 
2.9.1 
Hypotheses ...................................................................................................................................... 4 
2.10 Protocol History .......................................................................................................................................... 5 
2.11 Monitoring ................................................................................................................................................... 5 
2.11.1 
Team Monitoring .............................................................................................................................. 5 
2.11.2 
Planned SMC Reviews .................................................................................................................... 5 
2.11.3 
SMC Review History ........................................................................................................................ 6 
3 
Outcome Measures ............................................................................................................................................... 6 
3.1 
Primary Outcome Measure ......................................................................................................................... 6 
3.2 
Secondary Outcome Measures .................................................................................................................. 6 
3.3 
Exploratory Outcome Measures ................................................................................................................ 6 
4 
Statistical Methods ............................................................................................................................................... 6 
4.1 
Analysis Populations .................................................................................................................................. 6 
4.2 
Participant Exclusion ................................................................................................................................. 7 
4.3 
Analysis Visit Windows .............................................................................................................................. 7 
4.4 
Statistical Summaries and Testing ............................................................................................................ 7 
 
 


A5350 Statistical Analysis Plan Final Version 2.0 
 
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1 
Introduction 
This document presents the proposed primary, secondary, and exploratory statistical analyses of ACTG A5350. It 
also presents analyses which will be included in reports for the Study Monitoring Committee (SMC) interim review(s). 
This analysis plan may be modified by the study team as necessary; major modifications will be clearly indicated. 
 
2 
Study Overview 
2.1 
Design 
A5350 is a phase II, randomized, double-blind, two-arm study to evaluate whether there is a significant change in 
sCD14 after 24 weeks of probiotic Visbiome Extra Strength therapy, and to determine the safety and tolerability of 
this agent in HIV-infected participants on stable antiretroviral therapy (ART). Participants will be followed for an 
additional 12 weeks off study product. 
A substudy, A5352s will be conducted in a subset of participants to collect colonic biopsies via flexible 
sigmoidoscopy, and to evaluate intestinal permeability and inflammation at two time points.  
2.2 
Duration 
Thirty-eight (38) weeks for each participant. 
2.3 
Sample Size 
Ninety (90) participants (45 in Arm A and 45 in Arm B) 
2.4 
Population 
HIV-infected participants (≥18 years of age) who have been on stable ART for at least 24 weeks prior to study entry, 
and have a CD4+ T-cell count >200 cells/mm3 prior to study entry, and plasma HIV-1 RNA <50 copies/mL for 48 
weeks prior to study entry. 
2.5 
Stratification 
By intent and eligibility to enroll in the substudy A5352s. 
2.6 
Regimen 
Participants will be randomized in a 1:1 ratio to either Arm A (Visbiome Extra Strength + stable ART; treatment arm) 
or Arm B (placebo for Visbiome Extra Strength + stable ART; control arm).  
Arm A: Visbiome Extra Strength 
2-week lead-in period: 1 Visbiome Extra Strength sachet per day. 
Then increase to 1 sachet twice daily for 22 weeks. 
Followed by 12 weeks of additional follow-up. 
Arm B: Placebo for Visbiome Extra Strength 
2-week lead-in period: 1 placebo sachet per day. 
Then increase to 1 sachet twice daily for 22 weeks. 
Followed by 12 weeks of additional follow-up. 
2.7 
Primary Objective 
I. 
To assess changes in sCD14 in participants treated with Visbiome Extra Strength compared to placebo.  
2.7.1 
Hypothesis 
I. 
After administration of Visbiome Extra Strength, systemic levels of sCD14 will decrease. 
2.8 
Secondary Objectives 
I. 
To assess changes in additional markers of systemic inflammation (IL-6, IP-10, sCD163, sTNF-RI, 
oxidized LDL), KT ratio, and coagulation (D-dimer) after Visbiome Extra Strength administration. 
II. 
To assess changes in markers of microbial translocation (LPS, LBP) after Visbiome Extra Strength 
administration. 
III. 
To assess changes in peripheral CD4+ lymphocyte counts and CD4+/CD8+ ratio after Visbiome Extra 
Strength administration. 


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IV. 
To assess markers of monocyte and lymphocyte activation and senescence after Visbiome Extra 
Strength administration.  
V. 
To assess change in gastrointestinal microbial diversity from fecal samples after Visbiome Extra 
Strength administration. 
VI. 
To assess measures of enterocyte death (I-FABP) after administration of Visbiome Extra Strength.  
VII. 
To assess safety and tolerability of Visbiome Extra Strength.   
VIII. 
To assess diversity of the gut microbiome 12 weeks after completion of 24 weeks of Visbiome Extra 
Strength. 
2.8.1 
Hypotheses 
I. 
After administration of Visbiome Extra Strength, other markers of systemic inflammation interleukin (IL)-
6, interferon gamma-induced protein (IP)-10, soluble tumor necrosis factor receptor I (sTNF-RI), soluble 
CD163 (sCD163), oxidized low-density lipoprotein (LDL), kynurenine to tryptophan (KT) ratio, and 
coagulopathy (D-dimer) will decrease. 
II. 
After administration of Visbiome Extra Strength, markers of microbial translocation lipopolysaccharide 
(LPS) and lipopolysaccharide-binding protein (LBP) will decrease. 
III. 
After administration of Visbiome Extra Strength, peripheral CD4+ lymphocyte counts and CD4+/CD8+ 
ratio will increase. 
IV. 
After administration of Visbiome Extra Strength, markers of lymphocyte and monocyte activation and 
senescence will decrease. 
V. 
Visbiome Extra Strength administration will increase gastrointestinal microbial diversity in fecal samples. 
VI. 
After administration of Visbiome Extra Strength, markers of enterocyte death as measured by I-FABP 
(intestinal fatty acid binding protein) will decrease. 
VII. 
Visbiome Extra Strength will be safe and well tolerated. 
VIII. 
The gut microbiome changes will persist after completion of Visbiome Extra Strength administration. 
2.9 
Exploratory Objectives 
I. 
To assess changes in a marker of insulin sensitivity (HOMA-IR) after administration of Visbiome Extra 
Strength. 
II. 
To assess changes in fasting lipid parameters (low-density lipoprotein [LDL], high-density lipoprotein 
[HDL], non-HDL cholesterol, and triglycerides) after administration of Visbiome Extra Strength.  
III. 
To explore the correlations between the gut microbiome and soluble and cellular markers performed in 
the study. 
IV. 
To explore the correlations between the microbial translocation markers and other soluble and cellular 
markers performed in the study. 
V. 
To assess durability of the probiotic effect on inflammatory and microbial translocation markers 12 
weeks after discontinuation of Visbiome Extra Strength therapy. 
VI. 
To assess the differences in composition of the baseline microbiome based on ART. 
VII. 
To assess changes in chronic gastrointestinal symptom score after administration of Visbiome Extra 
Strength. 
VIII. 
To assess the relationship between reported dietary intake and composition of the microbiome. 
2.9.1 
Hypotheses 
I. 
After administration of Visbiome Extra Strength, insulin sensitivity, as measured by homeostatic model 
assessment (HOMA-IR), will improve. 
II. 
After administration of Visbiome Extra Strength, fasting lipid parameters will improve. 
III. 
After administration of Visbiome Extra Strength, changes in gut microbiome will be associated with 
changes in markers of systemic inflammation and coagulation as well as changes in cellular immune 
activation markers.  


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IV. 
After administration of Visbiome Extra Strength, changes in markers of microbial translocation will be 
associated with changes in systemic markers of inflammation and coagulation as well as changes in 
cellular immune activation markers.  
V. 
The improvements in markers of systemic inflammation and microbial translocation will persist for 12 
weeks after completion of 24 weeks of Visbiome Extra Strength.    
VI. 
At baseline, the composition of the microbiome will differ by antiretroviral class including protease 
inhibitors (PIs), nonnucleoside reverse transcriptase inhibitors (NNRTIs), and integrase inhibitors 
(InSTIs). 
VII. 
After administration of Visbiome Extra Strength, gastrointestinal symptom scores will improve. 
VIII. 
The composition of the microbiome will be influenced by dietary intake as reported through the dietary 
assessments. 
2.10 
Protocol History 
I. 
Protocol Version 1.0 (finalized January 5, 2016) 
II. 
Protocol Version 1.0 Clarification Memo #1 (February 22, 2016) clarifying that all A5350 on-treatment 
visits must be fasting, adding to items that were inadvertently left out of the protocol, and confirming that 
fasting is required for the A5352S lactulose/mannitol visits. 
III. 
Protocol Version 1.0 Letter of Amendment #1 (April 15, 2016) specifying the pregnancy test sensitivity 
and defining reproductive potential in A5350 and removed Week 24 hematology evaluations in A5352S. 
2.11 
Monitoring 
2.11.1 Team Monitoring 
Accrual, a summary of AEs, and sample/data availability will be reviewed regularly by the protocol core team once 
initial programming is complete. This summary will be pooled over the study arms. In addition, baseline 
characteristics as well as premature study treatment and study discontinuations (and reasons) will be reviewed 
regularly by the protocol core team.  
2.11.2 Planned SMC Reviews 
Per the protocol, an ACTG-appointed Study Monitoring Committee (SMC) will review accrual, adverse event 
summaries, off-treatment and off-study rates and reasons, and sample/data availability (if available from the DMC), 
broken down by study arm. The first SMC review will occur 6 months after the first participant is enrolled and then 
every 6 months as long as participants remain in follow-up. In addition, longitudinal changes in CD4+ T-cell count and 
HIV-1 RNA levels will be reviewed. Note that stored specimens will be assayed in batches after follow-up is 
concluded, and, therefore these data will not to be available at interim reviews. The SMC may also be convened if a 
reason is identified by the DAIDS clinical representative, study chairs, or study statistician in consultation with the 
team. 
Open reports (pooled over treatment arms) will be distributed to the SMC and team members participating in the 
SMC review.  In addition a closed report (by treatment arm) will be distributed to the SMC and study statisticians.  
The following summarizes the components of these reports: 
Open Report: 
 
Screening and Entry Analyses 
 
Study Status and Extent of Follow-up Analyses 
 
Study Treatment Status 
 
Data Completeness (if available from the DMC) 
 
Immunologic and Virologic Studies 
 
Safety 
Closed Report:  
 
Screening and Entry Analyses 
 
Study Status and Extent of Follow-up Analyses 
 
Study Treatment Status 
 
Data Completeness (if available from the DMC) 
 
Immunologic and Virologic Studies 
 
Safety 


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2.11.3 SMC Review History 
From SMC Review Letter: 
“The SMC met via teleconference on Wednesday, October 26, 2016, to review the interim safety reports. After 
carefully reviewing the accrual and safety data for both A5350 and A5352s, the SMC concluded that no significant 
safety concerns were identified and the studies should proceed as planned.” 
 
3 
Outcome Measures 
3.1 
Primary Outcome Measure 
I. 
Change in sCD14 from baseline to week 26. 
3.2 
Secondary Outcome Measures 
I. 
Change in the following markers from baseline to week 26. 
a. 
Systemic inflammation markers (IL-6, IP10, sCD163, TNF-RI, oxidized LDL), kynurenine to 
tryptophan (KT) ratio, and coagulation marker (D-dimer). 
b. 
Microbial translocation markers (LPS, LBP). 
c. 
CD4+ lymphocyte counts and CD4+/CD8+ ratio. 
d. 
Monocyte (%CD14+CD16-, %CD14+CD16+, %CD14lowCD16hi) and lymphocyte (%CD38+, 
%HLA-DR+, %CD38+HLA-DR+ in CD4+ and CD8+) activation and senescence (%CD28-
CD57+ in CD4+ and CD8+). 
e. 
Gastrointestinal microbial diversity (Shannon index, richness) from fecal samples. [Not 
analyzed at SDAC] 
f. 
Enterocyte death (I-FABP). 
II. 
Occurrence of adverse events.  
III. 
Diversity of gut microbiome (Shannon index, richness) at week 38. [Not analyzed at SDAC] 
3.3 
Exploratory Outcome Measures 
I. 
Change from baseline to week 26 in HOMA-IR. 
II. 
Change from baseline to week 26 in LDL, HDL, non-HDL cholesterol, and triglycerides. 
III. 
Change from week 26 to week 38 in systemic inflammation markers (IL-6, IP10, sCD163, sTNF-RI, 
oxidized LDL, and KT ratio). 
IV. 
Gastrointestinal microbial diversity from fecal samples at entry and post-entry visits. [Not analyzed at 
SDAC] 
V. 
Change in gastrointestinal symptoms from baseline to 26 weeks. 
VI. 
Diversity of gut microbiome and dietary intake composition. [Not analyzed at SDAC] 
 
4 
Statistical Methods 
4.1 
Analysis Populations 
Intent-To-Treat (ITT): All randomized participants. 
Modified Intent-To-Treat (mITT): All randomized participants who started study product. 
Per-Protocol (PP): All eligible, randomized participants who 1) have baseline and week 25/26 sCD14 
measurements, 2) remain on study product through week 26 (participants with more than 50% on average of missed 
study product may be excluded), 3) have not used prohibited medications, 4) do not have a confirmed virologic failure 
(as defined per section 6.2.3 at or prior to week 26, and 5) do not experience inflammatory conditions, receive 
vaccines, or have concurrent illness (as defined per section 6.2.3). 


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4.2 
Participant Exclusion 
Participants found to be ineligible for A5350 by the site, study statistician(s), or data manager will be reviewed by the 
study co-chairs for confirmation of their ineligibility.  
4.3 
Analysis Visit Windows 
For all analyses, the following visit windows will be used: 
 
Screening: evaluations completed within 45 days of randomization. 
 
Entry (week 0): entry evaluations must occur on/after randomization and at least 24 hours after screening. 
 
Week 2: pre-treatment evaluation scheduled within a week [1, 3] window but prior to treatment initiation, 
treatment must begin within the next 72 hours after evaluation. 
 
On-treatment evaluations: 
o 
Week 4: weeks (3, 5] 
o 
Week 6: weeks (5, 8] 
o 
Week 14: weeks [12, 16] 
o 
Week 25 & 26: weeks [23, 28] 
 
Week 38: post-treatment evaluation scheduled within a week [36, 40] window. 
If multiple results are available within the Week 6 or 14 visit windows the result closest to the scheduled visit will be 
used. If multiple results are available within the Week 38 window the result closest to 12 weeks after the last dose of 
study treatment (corresponding to protocol-defined 12 week washout) will be used. 
Results within the weeks [23, 28] visit window will use the following logic to determine Week 25 and Week 26 results: 
 
If two results are collected on treatment the earlier will be Week 25 and the latter Week 26. 
 
If two results are collected off treatment the earlier will be Week 25 and the latter Week 26. 
 
If one result is collected on treatment and one result is collected off treatment the on treatment will be Week 
25 and the off treatment Week 26. 
 
If only one result is collected it will be Week 25. 
 
If three or more results are available priority will be given to those collected on treatment and assigned 
Weeks based on the above rules.   
For markers run in duplicate at entry and Week 2 and also at Weeks 25 and 26, “baseline” will refer to the average of 
entry and Week 2 and “Week 25/26” will refer to the average of Week 25 and Week 26.  If only one result is available 
at either of these time points the single result will be used.  For per-protocol analyses the “Week 25/26” calculation 
will only use results collected while on treatment. 
4.4 
Statistical Summaries and Testing 
All statistical tests will be two-sided with a nominal alpha level of 0.05 and no adjustment for multiple testing. 
Specimens collected 1 day after the last dose of study treatment will be considered “on treatment”. 
Specimens collected within 6 days of an influenza vaccination will not be used for analysis unless the vaccination and 
specimen collection occurred on the same date and the site has documented that the vaccination was given after 
specimen collection. 
Outcome measures will be transformed for analyses and summaries on the log10 scale, as appropriate, if determined 
to not be approximately normally distributed. 
Because the stratification is to ensure balance between the treatment arms within A5352s and is not felt to affect 
A5350 outcomes, the stratification will not be taken into account in analyses. 
Safety analyses will summarize the highest grade AE by participant using the mITT population and will compare 
treatment arms using the Wilcoxon rank-sum test. All AEs and AEs related to study treatment will be analyzed.  
For all continuous outcome measures, tabular and graphical data summaries will be provided for both cross-sectional 
values across study weeks and change from baseline to post-baseline study weeks and will use the PP population. 
Mean changes in outcome measures will be compared between arms with either a two-sample t-test with equal 
variances or an adjusted linear regression model, as appropriate. The mean difference and 95% confidence interval 
(CI) will be provided along with the associated p-value.  
For the primary analysis, two supplemental linear regression analyses will be performed. The first will additionally 
adjust for baseline sCD14 values (continuous) while the second will assess differential Visbiome Extra Strength 


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effects by baseline sCD14 tertile by additionally adjusting for the sCD14 tertile main effect and the study arm by 
sCD14 tertile interaction. 
Correlations will be assessed with scatter plots as well as the Spearman correlation coefficient and associated p-
value using the PP population.