C.L. Ellis 1,2 , C.-S. Li 2 , S. Mann 3 , Z.-M. Ma 4 , H. Overman 2 , A. Maniar 1 ,

1. Effect of HIV-infection & highly-active antiretroviral therapy on the human gut microbiome investigated by phylogenetic-targeted pyrosequencing: A meta-genomic profile of duodenal biopsies, aspirates, and stool over time C.L. Ellis1,2, C.-S. Li2, S. Mann3, Z.-M. Ma4, H. Overman2, A. Maniar1, T. Yotter1, E. Tsuchida5, T.H. Knight1, J.C. Rutledge1, C.J. Miller1,4, R.B. Pollard1, D.M. Asmuth1 1University of California Davis Medical School , Sacramento, CA, USA 2University of California Davis, Davis, CA, USA 3Mather Veteran's Administration Hospital, Internal Medicine, Mather, CA, USA 4California National Primate Research Center, Davis, CA, USA 5CARES Clinic, Sacramento, CA, USA IAS 2011, July 18, Rome, Italy Abs. MOAA0104

2. Conflict of Interest Disclosures • None to declare

3. We house >trillion gut microbes (~99% Bacteria) outnumbering our own cells & genes Majority of the microbial ‘communities’ composing this gut microbial meta-genome, called the gut microbiome (or microbiota), cannot be studied by classical culturing Characterize based on evolutionary- or phylo- genetic signatures within specific 16S ribosomal RNA (rRNA) gene sequences (rDNA) • 16S rDNA Quantitative PCR • 16S rDNA Sequencing • environmental or meta- genomics Hamaday M & Knight R. Gen Res (2009)

4. HIV Disease & The Gut Microbiome:Overall Research Questions • Is the gut microbiome altered during acute/chronic HIV disease (i.e. any microbial communities depleted or that actually thrive in an infected/inflammed gut)? • What about after therapy and immune reconstitution? • Any relationships to gut inflammation, barrier destruction, microbial translocation, and systemic immunopathy/inflammation Marchettia G et al. Ellis CL et al. Merlini E. et al. Gori A et al. Estes et al. McKenna P et al. Hunt P et al. Wolf BW et al. Anukam KC et al. BrenchleyJM Jiang W et al. Gautraux MD et al.

5. Higher-Taxonomic qPCR Assessment of Stool Microbiota in HIV Patients We observed an unexpected increase in the proportion of ‘yet-unidentified’ microbiota not covered by common primers which accounted for majority of seronegative microbiota Ellis CL et al. CROI, 317 (2011)

7. % Enterobacteriales (Gamma-Proteobacteria)

8. What Taxa Compose That ‘Yet-Unidentified’ Proportion? • Next-generation sequencing superior to traditional sanger sequencing for this ecological question • Example: 454 pyro-sequencing • Pros: • Better coverage (orders of magnitude), accuracy, sensitivity (parallel sample analysis) • Cons: • Computationally challenging • $$$$$$$$ Benson A et al. PNAS (2010)

9. Hypothesis-Generating Study Objectives • Our objective with HIV/AIDS patients was to apply pyrosequencing, during a HAART trial, in order to: • Develop a panel of potential taxa which may compose the yet-unidentified stool microbiota and thereby guide qPCR-based characterization • Explore, using different intestinal specimen-types, the creation of a spatial meta-genomic 16S pilot profile before and after HAART at the stable higher-taxonomic community level

10. Pilot Clinical Trial - Cohort Design • 14 chronically-infected HIV patients with race/ethnic/sex variation • Distal duodenal biopsy and aspirate specimen, and a stool specimen, at 0 mo and 9 mo > HAART • 2 paired biopsies, 2 aspirates, and • 16 stools (1/2 are paired/longitudinal) • 3 seronegative control subjects volunteered (family/friends of patients) • 1 biopsy, 2 stools • Total: 1/4 of a 96-well plate split for submission to UN-L CAGE (Core for Applied Genomics & Ecology), USA

11. Wet Lab: • Microbial gDNA extracted by beadbeating* and phenol/chloroform-based methods • The chosen primer set for pyro-seq covered V1, V2 and V3 16S rRNA gene regions •  long read lengths, spikes *actinobacterial taxa Dry Lab (Bioinformatics): CLASSIFIER algorithm: Ribosomal Database Project Website (Free) Full method incl. taxa binning/classification, scoring, sequence matching/alignment described: Benson A et al. PNAS (2010)

12. DATA

13. Higher-Taxonomic Microbial Community Snapshot

14. Stool Summary • G(+) Clostridia and G(-) Bacteroidetes dominated most samples • Also a great abundance of G(+) Erysipelotrichi and Actinobacteria (owing to beadbeating) in all samples (inconsistent pre/post therapy changes) • Actinoappear to be increased in some HIV+ specimens vs controls

15. Anti-HIV Activity of Biochemicals from Strains within the Actinobacteria class Tanaka et al. Jpn J Cancer Res (1986)

16. Higher-Taxonomic Microbial Community Snapshot

17. Distal Duodenal Biopsies Summary • Facultative anaerobic taxa, e.g. G(-) Gammaproteobacteria, were present in greater numbers in biopsies (site-specific) vs the stool (mixture) • Though obligate anaerobes were also detected in high abundance…. • Interestingly, Cyanobacteria (primitive BGA) was also detected

18. UnexpectedCyanobacteria/blue-green algae observed in a pre-HAART biopsy of a non-vegetarian patient at >26% vs. control

19. Obesity and The Gut Microbiota Cyanobacterial sequences detected in obese mouse cecum: May represent descendants of non-photosynthetic ancestral cyanobacteria that have adapted to life in lightless animal guts Ley R et al. PNAS (2005)

20. Extracts & Compounds from Algae & Cyanobacteria Possess Anti-HIV Activity Scheffer et al. Ecotox Enviro Safety (2000)

21. Higher-Taxonomic Microbial Community Snapshot

22. Distal Duodenal Luminal Aspirates Summary • A liquid-phase sample-type new to this sequencing approach: a spread of facultative anaerobes but also some obligate anaerobes • also Fusobacteria (a potent LPS-producing G[-] taxa) and, again, Cyanobacteria

23. Summary/Conclusion • 1. Pyrosequencing for patient gut microbiome profiling: orders of magnitude > information/insight than traditional methods! • much more sensitive to inter-subject differences • 2. Our prelim pyro dataset revealed taxa which likely accounted for the previously-unidentified seropositive microbiota (Erysipelotrichi and Fusobacteria as well as Actinobacteria andCyanobacteria) • Does increased anti-HIV active microbes (Actino- and Cyano- taxa) indicate a mutualistic symbiosis-based response to protect the infected host? Cause or effect? • A link to gut inflammation/translocation, immunopathy, CVD? • 3. Post-therapy immune reconstitution likely affects host-microbiota interactions, however, fewconsistent 0mo9mo changes observed • HAART may not be sufficient to restore any alterations in overall gut microbiome composition during HIV disease • Much opportunity for more research

24. LIMITATIONS • Low n, especially controls • Only 2 time points for each patient >Currently expanding our n and surveying multiple timepoints over the 9mo of HAART However, objectives were met, we now have a: 1- Panel of potential taxa to guide qPCR design 2- Spatial pilot profile of the HIV+ gut microbiome

25. Take Home Message Attention to the ‘other’ druggable genome(s): Sequencing of a patient's personal microbial (meta)genome or microbiome could help design future ancillary therapeutic-interventions Targeted pro-biotics, pre-biotics, or both (syn-biotics), and selective anti-biotics (or all?) *Some success in IBD, Atherosclerosis, Obesity

26. Acknowledgements • Project PI and Mentor: David M. Asmuth MD • Major Prof/Project Co-PI: John C. Rutledge MD • Thesis Committee: Jonathan A. Eisen PhD Charles L. Bevins MD, PhD • Other Dissertation Mentors • Mark A. Underwood MD, MAS Britt M. Burton-Freeman PhD Amber L. Hartman PhD Eugene S. Lee MD, PhD Project Team: Andrew Tritt, BS Zhong-Min Ma PhD Surinder K. Mann MD Chin-Shang Li PhD Jian Wu MD, PhD Thomas H. Knight MS Tammy Yotter BS, RN, CCRPJian Wu MD, PhD Timothy L. Hayes PhD Archana H. Maniar MD Paolo V. Troia-Cancio MD Heather A. Overman BS Natalie J. Torok MD Anthony Albanese MD Christopher J. Miller DVM, PhD Richard B. Pollard MD *Our Subjects/Patients and Their Families THANK YOU FOR YOUR ATTENTION AND ANY FEEDBACK! Correspondence:david.asmuth@ucdmc.ucdavis.edu NIH - NCRR