Role of the Microbiome in HIV Vaccine Response Heterogeneity Ongoing and Future Microbiome Studies in HVTN Protocols

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1 Role of the Microbiome in HIV Vaccine Response Heterogeneity Ongoing and Future Microbiome Studies in HVTN Protocols Microbiome in HIV Workshop Pathogenesis, Prevention and Treatment October 20 th, 2017 Jim Kublin 11/1/2017 1

2 Many organ systems and tissues are affected by the dynamic relationship between the gut microbiota and the immune system What impact does this immuno-ecological system have on vaccination What are the consequences of this relationship with the acquisition of HIV Martin A. Kriegel, Self or non-self? The multifaceted role of the microbiota in immune-mediated diseases. Clinical Immunology (2015) 159, /1/2017 2

3 Prospects of addressing important questions within the HIV Vaccine Trials Network What is the portfolio xploratory objectives Outline Results and pilot analyses HVTN Protocols 204 and 505 Pilot studies in HVTN 096 and 602 Gnotobiotic models xperimental designs Microbial colonization 11/1/2017 3

4 HVTN Protocols with Prospective Microbiome Sampling HVTN 106: DNA (Nat, Con, and Mos env) + MVA Stool collection container at home up to 7 days prior to the first vaccination No other mucosal samples HVTN 107: ALVAC-HIV and Bivalent C gp120 alone vs MF59 vs alum adjuvants Rectal/cervical fluids/semen samples at baseline, months 6.5, 12, 12.5, and 18 Cervical/vaginal swabs for STIs at baseline, months 6.5, 12, 12.5, and 18 HVTN 108: P5 DNA/proteins Stool swab at baseline and month 6.5 Rectal/cervical fluids/semen samples at baseline, months 6.5 and 12. Cervical/vaginal swabs at baseline, months 6.5 and 12. HVTN 119: p24 Conserved lements DNA vaccine in HIV uninfected adults in the US To be harmonized with ACTG protocol of same vaccine in HIV infected adults in the US (A5369) HVTN 120: P5 ALVAC/protein with MF59 vs AS01B Stool swab at baseline and month 6.5 Rectal/cervical fluids/semen samples at baseline, months 6.5 and 12. Cervical/vaginal swab for STIs samples at baseline, months 6.5 and 12. HVTN 124: PDPHV DNA/quadrivalent protein Stool swab at baseline and month 12 Saliva, rectal/cervical fluids/semen samples at baseline, months 12 and 14. Cervical/vaginal swab for STIs samples at baseline, months 12 and 14. HVTN 602: Immunogenicity of BCG revaccination, H4:IC31, and H56:IC31 in healthy, HIV-1 uninfected adolescents Stool collection at baseline and multiple timepoints HVTN 702: P5 ALVAC/protein efficacy trial Stool swab at baseline and month 6.5 Cervical/vaginal swabs for STIs at baseline, months 6, 12, 18, 24, 30, and 36 Rectal swab for MSM STIs at baseline, and months 6, 12, 18, 24, 30, and 36 HVTN 705: Janssen Ad26/protein efficacy trial Vaginal swab and cervical secretions at baseline and months 6, 12, 18, 24, 30, and 36 11/1/2017 4

5 xploratory Microbiome Objectives in HVTN Studies Role of the microbiome in vaccine response heterogeneity Is the constitution of the human gut microbiome a predictor of specific vaccine induced immune responses? Do vaccine induced responses share cross-reactivity with specific microbiota? What mechanisms may be responsible for these associations, and are animals models, e.g. gnotobiotic mice, a useful tool to address this question? Role of the gut microbiome in immune activation What are the distinct enterotypes representing populations in the US and Southern Africa? What is the role of diet and antibiotics in clustering individuals? Are gut microbiomes associated with activation of the immune system? Are gut microbiomes associated with HIV acquisition? What other associations exist between the microbiome within study populations? Role of the genital microbiome in HIV acquisition What are the cervicotypes represented in different geographic locations and by sexual practice? What vaginal and penile microbiome types are associated with HIV and other STI acquisition? 11/1/2017 5

6 HVTN Pipeline 11/1/ Protocol # Short Title N HVTN 104 VRC01 Phase 1 88 HVTN 106 CHAVI DNA/MVA-CMDR 105 HVTN 100 ALVAC+gp120/MF59 RSA Phase HVTN 110 PaxVax replicating Ad 20 HVTN 098 Inovio PNNVAX-G + cytokine DNA via P 94 HVTN 112 VSV env 15 HVTN 704/HPTN085 AMP Study in MSM+TG 2,700 HVTN 703/HPTN 081 AMP Study in SSA women 1,500 HVTN 111 Correlates Study P5 (66 ppts NIAID) 132 HVTN 117/HPX2004 Janssen: trivalent/bivalent Ad26 Mosaic wgp HVTN 702 RSA 5,400 HVTN 108 Correlates Study 334 HVTN boost 100 HVTN 107 Correlates Study P5 ( ALVAC /protein alum v. MF59) - test 132 HVTN 116 VRC01 Mucosal 101 HVTN 121 AVAIL 16 HVTN 118/HPX2003 Janssen:Tetravalent Ad26 Mosaic, Mosaic+gp140 boost 142 HVTN 115, part A B cell lineage 42 HVTN 119 p24c DNA 56 HVTN 120 DNA/ALVAC (113 redesign) 320 HVTN 705 Janssen POC 2,600 HVTN 122 gp145 phase 1 45 HVTN 124 PDPHV 60 HVTN 127/HPTN087 VRC07-523LS 100 HVTN 125 DNA-FLSC 24 HVTN Boost 60 HVTN 123 Transient vs Stable Transfection Q4 Q3 Q2 Q1 Q4 Q3 Q2 Q1 Q1 Q2 Q3 Q4 Q4 Q3 Q2 Q1 Q Q1 Q2 Q3 Q RFRSH DATA

7 P5 Partners and Collaborators P5 Trial Partners Funders Manufacturers

8 Study Schema HVTN 108 HVTN 120 Group N Dose of each protein mcg mcg mcg mcg mcg mcg mcg 8 24 Total Deltoid 334 (310 vaccinees; 24 placebo) Month 0 (Day 0) Month 1 (Day 28) Month 3 (Day 84) Month 6 (Day 168) Left DNA DNA DNA DNA Right Placebo Placebo Protein + MF59 Protein + MF59 Left DNA DNA DNA DNA Right Placebo Placebo Protein + AS01 B Protein + AS01 B Left DNA DNA DNA DNA Right Placebo Placebo Protein + AS01 B Protein + AS01 B Left DNA DNA Placebo DNA Right Protein + MF59 Protein + MF59 Placebo Protein + MF59 Left DNA DNA Placebo DNA Right Protein + AS01 B Protein + AS01 B Placebo Protein + AS01 B Left DNA DNA Placebo DNA Right Protein + AS01 B Protein + AS01 B Placebo Protein + AS01 B Left Placebo Placebo Placebo Placebo Right Protein + AS01 B Protein + AS01 B Placebo Protein + AS01 B Left Placebo Placebo Placebo Placebo Right Placebo Placebo Placebo Placebo DNA-prime DNA+protein boost DNA+protein coadmin Protein primeboost Group N Dose of each protein Month 0 (Day 0) Month 1 (Day 28) Month 3 (Day 84) Month 6 (Day 168) mcg ALVAC ALVAC ALVAC/Protein/MF59 ALVAC/Protein/MF mcg ALVAC ALVAC ALVAC/Protein/AS01 B ALVAC/Protein/AS01 B mcg ALVAC ALVAC ALVAC/Protein/AS01 B ALVAC/Protein/AS01 B 4 20 N/A Placebo Total 320 (300 vaccinees; 20 placebo) 8

9 HVTN 702: Study Schema N (total 5400) Primary Vaccine Regimen Booster Month 0 Month 1 Month 3 Month 6 Month ALVAC-HIV (vcp2438) ALVAC-HIV (vcp2438) ALVAC-HIV+ Bivalent Subtype C gp120/mf59 ALVAC-HIV+ Bivalent Subtype C gp120/mf59 ALVAC-HIV+ Bivalent Subtype C gp120/mf Placebo Placebo Placebo + Placebo Placebo + Placebo Placebo + Placebo

10 HVTN 705 Study Schema Number Month 0 Month 3 Month 6 Month Ad26.MOS4.HIV Ad26.MOS4.HIV Ad26.MOS4.HIV + Clade C gp140 Ad26.MOS4.HIV + Clade C gp Placebo Placebo Placebo Placebo 11/1/

11 Diversion of HIV-1 vaccine induced immunity by gp41 microbiota cross-reactive antibodies The DNA prime rad5 boost HIV-1 vaccine in HVTN 204 and 505 induced a gp41-reactive antibody response that was mainly non-neutralizing and derived from an intestinal microbiota gp41 cross-reactive B cell pool. This vaccine was the first vaccine containing the ectodomain of the nv gp41 component, covalently linked to gp120, to be tested in an efficacy trial and was designed to generate primarily CD8 T cell responses, although this vaccine generated nv Ab responses as well Wilton B. Williams et al., Science 349, aab1253 (2015). 11/1/

12 Gut microbiota as Potential Baseline Immunogenicity Predictors A Correlate of Protection is invaluable in vaccine development Vaccine-specific responses can only be measured in vaccine recipients: at best we identify Correlates of Risk of infection Baseline immunogenicity predictors (BIPs) are biomarkers of vaccine-induced responses that are measureable in all treatment groups at baseline Unlike host-genetics (e.g. HLA) or other vaccine responses (e.g. Tetavax in 097) as BIPs, the gut microbiota is modifiable Many probiotics have been tested in trials of infant vaccines with mixed success Björkstén, B., Diverse microbial exposure - consequences for vaccine development. Vaccine 30, New products are emerging for modifying the gut microbiota (one form of personalized medicine for IBD, metabolic disorders, cancer, periodontal disease, obesity, eczema) 5

13 Initial model predicting V1V2 responses in /1/

14 HVTN096 Pilot study The microbiome modulates baseline response and vaccine persistence

15 HVTN 096 Pilot Microbiome Study Lausanne only ICS response rates are between 50-80% BAMA response rates >90% (peak and durability timepoints) Luminex cyokines at multiple timepoints gp41-specific responses were assessed Male/female split Multiple time points: M0 (baseline), M6.5 (primary), M12 (durability) Rectal secretions: for Ab response and bacterial DNA extraction 11/1/

16 HVTN 096 BAMA Responses 16

17 xample: Vaccine Induced Response GP120

18 GP41 Has baseline variability.

19 Individual Species Abundance

20 Plot intra- vs. inter-participant distances among samples Phylum Genus Species At genus and species level there is greater inter- vs. intra- participant divergence among samples Suggests there is "stability" in the gut flora

21 Baseline IgG gp41 association Only a handful of data permutations have an association as high as the observed effect Tested each genus for an association with baseline IgG gp41 Compared negative binomial regression model vs. ranksum test, both using read counts (i.e. not relative abundance) P-values are adjusted using Holm-Bonferroni

22 Baseline IgG gp41: log-ratio, relative abundance analysis Log-ratios associated with baseline IgG gp41 include Clostridiales Family XI, Finegoldia and Peptoniphilus versus many other genuses Plot shows dark areas for pairs of genuses whose log-ratio of relative abundance is associated with baseline IgG gp41 Findings are consistent with other analyses

23 We investigated whether community structure appeared to relate to: Several immunological parameters IgGs IgAs CD4 and CD8 T Cells Using several complementary tests Log-ratio PRMANOVA/Kernal Regression Median split variables Continuous log transformed At three time-points Baseline Primary (peak) Final

24 Responses among antibodies: Immunologic Timepoint Antibody Log-ratio Permanova/ Kernel Regression (median split) Permanova/ Kernel Regression (continuous) Baseline IgG_gp IgA_gp Primary IgG_gp IgA_gp IgG_ZM96_gp IgG_Con_6_gp120/B IgG_gp70_B.CaseA_V1_V Final IgG_gp IgA_gp IgG_ZM96_gp IgG_Con_6_gp120/B IgG_gp70_B.CaseA_V1_V

25 GP41 at Baseline and GP120 persistence do not appear to associate

26 Hits: Community ~ Gp41 at Baseline (p =.0087) Whether greater or less than median Community ~ Gp120 on Day 12. (p < 10^-3) Continuous trend PRMANOVA: Community ~ gp41 + gp120 (P = , P = ) Together they explain ~27.6 % of variability in community structure.

27 dbrda Visualize association between community and immune response Participant

28 xample: Bacteroides ~ Con 6 GP120 rr 2 = 0.34; p < 0.01

29 This pattern is present in other GP120s -and significant in 2/3 cases Final Immunologic Timepoint Antibody (IgG) Log-ratio Permanova/ Kernel Regression (median split) Permanova/ Kernel Regression (continuous) A244_gp_120_gDneg/29 3F/mon Con_6_gp120/B MN_gp120_gDneg/293T

30 This pattern persists, for GP120 at the final microbiome timepoint Immunologic Timepoint Antibody (IgG) Log-ratio Permanova/ Kernel Regression (median split) Permanova/ Kernel Regression (continuous) Baseline gp Primary gp Con_6_gp120/B Final gp Con_6_gp120/B MN_gp120_gDneg/293T A244_gp120_gDneg/293F/mon

31 HVTN 096 Pilot Summary Sample collection and processing: Rectal swab samples that are preprocessed for eluting antibodies yield little/no DNA for amplification/sequencing DNA yield was much improved if after the procedure for eluting antibodies the weck and pellet are included for DNA extraction Fecal samples contained a diversity of organisms Relative abundance at the phylum level included bacteroidetes, proteobacteria, firmicutes, actinobacteria and bacteria. Relative abundance at the genus level included Prevotella, bacteroides, enteroacteriaceae, blautia, and clostridiales family. The abundance of organisms within individuals who had multiple samples remained relatively stable at the phylum and genus levels, with a few exceptions. Inter- vs. intra-participant divergence was greater with species > genus > phylum Suggesting relative stability within individuals in their gut microbiota Instability within individuals may be attributed to the use of antibiotics preliminary analysis revealed no trends. HVTN 096 elicited robust CD4+ T-cell and antibody responses to the HIV antigens in the vaccine, and some of these responses were associated with certain microbiota predominance Presence of Clostridiales Family XI, Finegoldia and Peptoniphilus were associated with gp41-specific IgG responses at baseline Baseline gp41-specific IgG was not predictive of vaccine-induced IgG to any antigens The microbiome may contribute to high baseline gp41 responses that may also reduce gp120 response to the HIV vaccine. 11/1/

32 HVTN 602 Collaborative Phase 1 trial in RSA adolescents evaluating BCG revaccination, H4:IC31, and H56:IC13 11/1/

33 HVTN 602 Rationale To prepare for correlates analysis for 3 vaccines undergoing efficacy evaluations in BCG vaccinated adolescents H4:IC31 H56:IC31 BCG revaccination (attenuated M. bovis) To obtain a comprehensive view of immune responses occurring after boost vaccination in a high TB burden population 11/1/

34 Study Objectives Identify good potential biomarkers for planned efficacy trials: Validate assays* (e.g. mycobacterial growth inhibition) Identify immune responses with good BM characteristics Identify peak response timepoint for each assay Characterize immune responses: Response to H4:IC31, H56:IC31, BCG* Innate gene expression and responses to vaccines and IC31* Adaptive Ag-specific gene expression and responses (BCG*) Baseline/existing immunity* (e.g. inflammation profile, gene expression) Innate predictors of adaptive responses (*as this relates to IC31 and BCG) (*valuable even if no efficacy is observed in sister studies) 11/1/

35 HVTN 602 Study Schema Arm Number Dose Day 0 Day 56 Group mcg H4:IC31 H4:IC31 Group 2 18 TBD # H56:IC31 H56:IC31 Group 3* x 10 5 CFU BCG - Group 4 9 Saline Saline Total 63 # Dose for H56 will be decided in July based on C-035 study data. *Since BCG causes a recognizable local injection site reaction, the BCG revaccination arm will be unblinded. 11/1/

36 Immunogenicity time points: HVTN 602 H4:IC31 (G1) H56:IC31 (G2) BCG (G3) d0 H4:IC31 (G1) H56:IC31 (G2) d56 Baseline d0 d1 d3d7 Innate d14 2 wks-post 1 st vacc d28 (BCG only) 4 wks-post 1 st vacc d70 2 wks-post 2 nd vacc m6 4 mos-post 2 vacc 6 mos post 1 st vacc m8 6 mos-post 2 nd vacc (safety) BCG peak response? Peak response 11/1/

37 Relative abundance: Genus level Intra- vs inter participant distances Shows that the distances between samples was greater for samples from different participants Plot shows Jensen-Shannon divergence: Unifrac and Weightedunifrac were qualitatively similar 37

38 Germ Free microbiome pilot study using the C4B gp120 heptamer/alum vaccine 38 11/1/2017

39 11/1/

40 arly B-cell development also occurs within the mouse intestinal lamina propria (LP), where the associated V(D)J recombination/ receptor editing processes modulate primary LP immunoglobulin repertoires. B-cell development occurs in the intestinal mucosa, where it is regulated by extracellular signals from commensal microbes that influence gut immunoglobulin repertoires. 11/1/

41 Microbiome, metabolites and host immunity Maayan Levy, ran Blacher, ran linav Current Opinion in Microbiology, Volume 35, 2017, 8 15

42 Mouse xperiments (i) Characterize baseline immunity of each gnotobiotic model The Milieu Intérieur of mice and men Milieu totalis (mycome, virome?) Variety with inbreeding, crosses, knock out/in. 001 first GF and SPF experiments underway 426c heptamer protein and Ova injected Antibodies, B and T cell assays 11/1/

43 11/1/

44 TM4 V1-3 Candidate Vaccine Binding affinities of the indicated germline VRC01-class antibodies to selected 426c variants. 11/1/

45 xperimental Design Preliminary DC Results 11/1/

46 Humoral Immune Results i 450 Abs monomer coated plate- averages Dilution G1-G6 average G7-8 average 1-6 average 7-9 average 10 J1-6 average J7-9 average J Abs heptamer coated plate- averages Dilution G1-G6 average G7-8 average 1-6 average 7-9 average 10 J1-6 average J7-9 average J10

47 Humoral Immune Response ii inj vs. GF inj: p = * J inj vs. GF inj: p = * 11/1/

48 B Cell Results 11/1/

49 Mouse xperiments (ii) 002 reconstitute GF mice with predefined minimal microbial community What community to select? Metabolic qualities/functions (Metabolon) What in silico computations can assist? 003 introduce new genetic lineage to B6 derived germ free Selected based on known directionality of immune responses Predominant B-cell and humoral? Strongest T-cell responses? Knock-in for 426c directed Ab response Using multiple models to derive mechanisms Th1 (e.g. IgG2b ) Th2 (IgG1) serum responses For fractional dose clinical findings For durability and isotype 11/1/

50 Mining the Human Gut Microbiota for Immunomodulatory Organisms. Cell, Volume 168, Issue 5, 2017, e11 Figure 1. xperiment Design and Bacterial Colonization(A) Four-week-old GF mice were monocolonized with human gut bacteria and analyzed after 2 weeks for colonization, impact on immune system, and genomic activity. (B) Innate and adaptive immune responses were analyzed by flow cytometry of cells extracted from SI, PPs, colons, mlns, and SLOs. (C) Cladogram of the human gut microbiota. Microbes were identified in the Human Microbiome Project (HMP) database except for SFB. Blue diamonds denote the genera included; red stars mark the species. Species where more than one strain was analyzed are in bold type. The outer ring represents a bar graph of the prevalence of each genus.

51 Maayan Levy et al. Genes Dev. 2016;30: Chart of metabolic pathways with highlighted examples of microbiome-derived or -modulated pathways involved in modulation of the immune response.

52 Mouse xperiments (iii) Considerations and new work that will need to be addressed Stability of minimal microbial communities Procedures Tissue-specific isolation and analysis of mouse cells of high purity and yield from fat tissue, lung, bronchoalveolar lavage fluid (BALF) and intestine (e.g. LPMC). Reconstitution via gavage/co-housing Procurement of additional vaccines GSK gp120s with alum, MF59 and AS01B Sanofi ALVAC CHUV DNA vectors 11/1/

53 Study Participants HVTN Leadership Andrew Fiore-Gartland Justin Taylor Jenny Lund David Fredericks Sujatha Srinivasan Georgia Tomaras Kevin Hager Jacob Cram Acknowledgments 11/1/