the microbiome in · alterations in the enteric microbiome are associated with disease •metabolic...

THE MICROBIOME IN HIV

Cindy Monaco, MD, PhDUniversity of Rochester School of

Medicine and Dentistry

No Disclosures

Outline

• What is the microbiome?

• Why does it matter?

• What is the relationship between the microbiome and HIV?

What is human?

• We are comprised of more than just human cells

http://scalar.usc.edu/works/lope/diagram-of-human-body

What is human?

• We are an ecosystem of bacteria, viruses, fungi, and human cells

• Human cells are outnumbered at least 3:1, not including viruses

• Genes from our microbes impact our health

Mpkb.org

What do these microbes do?

• Commensal microbes (microbes that live within us) can protect against diseases:

– Parasitic worm infection prevents allergies

– Skin/vaginal yeast infections after antibiotics

– C diff colitis after antibiotic use

– Bacterial vaginosis (BV) associated with increased risk of STIs including HSV, HIV, etc.

What do these other organisms do?

• The majority of organisms that colonize us are not able to be cultured

• How can we study those?

• Advent of NGS (next generation sequencing) aka deep sequencing, second generation sequencing– allows us to discover the unculturable microbes

at various body sites

• Dawn of “microbiome” research

Why has it taken so long to study the microbiome?

Price per base of DNA sequencing• 1869: DNA discovered• 1953: DNA revealed to be

genetic blueprint• 1970s: RNA sequencing

developed• 1977: first full DNA

sequence (bacteriophage phiX174) by Sanger sequencing (chain termination)

• 2001: human genome published (Sanger sequencing) after > 10 year effort

• 2005: first next generation sequencing (NGS) machine introduced

Rob Carlson, 3/2016, www.synthesis.cc

Sanger sequencing NGS sequencing

Microbiome components - Definitions

• Bacteria: – Microscopic (i.e. can see using microscope)

– single celled organisms

– DNA genome

– Come in various shapes

– Free-living (i.e. do not require another host cell machinery to live)

– Some require oxygen, others do not

– Differentiated by type of cell wall into gram positive and gram negative

– Can be treated with antibiotics


• Viruses– Submicroscopic (i.e. cannot see by microscope)

– Infectious agent

– Replicates only inside living cells of other organisms

– DNA or RNA genomes

– Eukaryotic viruses: viruses that infect animals or plants• Few viruses have effective treatments (HIV, HCV, Flu, CMV)

• Usually rely on vaccination (HPV, Flu, chickenpox/zoster, rotavirus, yellow fever, measles/mumps/rubella)

– Bacteriophage: viruses that infect bacteria• can be either lysogenic (replicate harmlessly in the bacteria)

• Or lytic (break open and destroy bacteria after replication)

• Can carry antibiotic resistance or toxin genes to other bacteria


• Fungus: multicellular organisms like yeast or molds

• Other prokaryotes: i.e amoeba, helminth, tapeworms, etc

What is the microbiome?

• Microbiome: all bacteria, archaea, viruses, fungi, and other microbial Eukarya

• Bacteriome: bacterial components of the microbiome

• Virome: viral microbiome, including eukaryotic viruses, bacteriophage, endogenous retroviruses– Estimated as little as 1%

sequenced

Virgin HW. Cell. 157, 2014; Clemente JC et al, Cell. 148, 2012; Cox MJ et al, Hum Mol Genet, 22, 2013

What is the microbiome?

Blum HE. Adv in Med Sci. (2017), 62

• Defined by body site

• Unique between individuals

• Diversity greater between sites than between individuals

Clemente JC et al, Cell. 148, 2012; Muszr M et al, Arch Immunol Ther. Exp (2015), 63.

Bacterial microbiome composition by body site

The bacteriome varies by age

Ottman, N et al. Aug 2012 Frontiers in Cellular and Infection Microbiology

Virome

• Much less known about the viral components of the microbiome

– lack of adequate culture methods

– highly divergent sequences between viral species

– prevents development of broad PCR-based assays

• Culture-independent, high-throughput sequencing methods

– unbiased look at the composition of the microbiome

– discovering potentially novel, unculturable viruses

Virgin HW. Cell. 157, 2014;

What is a typical gut bacteriome?

• Human gastrointestinal tract is colonized by trillions of microorganisms

• Differs between the developed world and agrarian cultures

• Prevotella-rich and Bacteroides-poor community associated with agrarian cultures

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Prevotella

Bacteroides

De Filippo et al., PNAS, 2010

What can alter the microbiome?

• Diet– Plant versus animal based– Even artificial sweetener

• Medications, especially antibiotics or antivirals, heartburn medications

• Sexual practices – Anal intercourse associated with increased Prevotella in fecal samples – Differences in vaginal microbiome

• Hormones and hormonal contraceptives• Immunosuppression (steroids, AIDS, cancers)• Some environmental factors (pets, family members)• Hygiene practices (skin microbiome with antimicrobial soaps,

vaginal microbiome and douching)

Why Does the microbiome matter?

Effect of intestinal bacterial microbiome in health

• For a long time we have known that diet can affect many diseases in including aging, cardiovascular disease, dementia– May be due to the gut bacterial microbiome

• Intestinal bacterial communities and their metabolites– shape and maintain gut immunity– influence systemic immune response– alter behavior– produce metabolites used by us as energy source. – promote gut epithelial barrier integrity

Dillon SM et al, AIDS, 2017 31:511-521

Alterations in the enteric microbiomeare associated with disease

• Metabolic disorders such as obesity and diabetes mellitus– Fecal transplant from obese mice to lean mice led to

weight increase despite no change in diet.

• Neuropsychiatric disorders such as schizophrenia, autistic disorders, anxiety disorders and major depressive disorders.

• Gut-brain axis– signals originating in the gut can also influence brain

function through interface with the enteric and central nervous systems

– gut microorganisms are capable of producing and delivering neurotransmitters such as serotonin and GABA

Evrensel A and Ceylan ME. Clin Psychopharmacol Neurosci. 2015 Dec; 13(3): 239–244; Perry RJ et al, Nature. 2016 June; 534: 213–217

The gut microbiome and stroke

• Ischemic stroke – highly prevalent disease – limited therapeutic options– Inflammation is key component

• Treated mice with augmentin (a PCN antibiotic) for 2 weeks – decreased diversity of intestinal

bacteriome

• Then induced transient middle cerebral artery occlusion (MCAO). – 60% reduction in stroke volume in

Augmentin-treated mice.

• Similar effects with oral vancomycin• Ciprofloxacin and Flagyl treatment

reduced survival following MCAO• Suggesting that particular microbial

species cause differential effects on brain injury recovery

Benakis C et al, Nature Medicine volume22 , pages516–523 (2016)

Control Augmentin

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Control Augmentin

The gut microbiome and Austism

• Autism spectrum disorders (ASDs), a complex neurobiological disorder– impaired social interactions and communication – leads to restricted, repetitive, and stereotyped patterns of behavior, interests,

and activities

• Causes of poorly understood – Complex interplay of genetic and environmental factors

• Many also experience significant GI symptoms, which correlate with ASD severity– Many ASD kids have gotten more Abx therapy in first 3 years of life

• Several studies showed altered gut bacterial microbiomes in ASD kids compared with neurotypcial children– lower abundances of fermentative bacteria (e.g., Prevotella copri)– lower overall bacterial diversity

• ASD mouse model demonstrated that Bacteroides fragilis treatment could alter gut microbiota and blood metabolite profiles, correct increased gut permeability, and improve ASD symptoms

Kang DW et al, Microbiome20175:10; Fung TC et al, Nature Neuroscience volume20, pages145–155 (2017)

The gut microbiome and Austism

• 2 open-label studies in children with ASD, – 8 weeks of oral vancomycin transient

improvements in both GI symptoms and ASD symptoms

– Fecal Transplant: 2-week antibiotic treatment, a bowel cleanse, and then an extended fecal microbiota transplant (FMT) using a high initial dose followed by daily lower maintenance doses for 7–8 weeks. • 80% reduction of GI symptoms at the end of treatment,

persisted 8 weeks after treatment.

• Significantly improved behavioral ASD symptoms, persisted 8 weeks after treatment ended.

Kang DW et al, Microbiome20175:10; Fung TC et al, Nature Neuroscience volume20, pages145–155 (2017)

Inflammatory Bowel Disease linked to alterations in enteric bacteriome and virome

Norman JM,…, Monaco CL, et al. Cell, 2015

Gut microbiome may predict response to chemotherapy medications

• Melanoma patients undergoing anti-PD-1 immunotherapy (n=112)

• Significantly higher gut bacterial diversity in patients who responded to therapy (R)

• Significant difference in mortality depending on gut bacterial diversity

Gopalkrishnan V et al, Gut microbiome modulates response to anti-PD-1 immunotherapy in melanoma patients, Science. 2018 Jan 5;359(6371):97-103.

Div

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Diversity

Compositional differences in the gut

microbiome are associated with responses to anti-PD-1 immunotherapy

• Higher relative abundance of Ruminococcaceae bacteria (p<0.01) in responding patients.

• Metagenomic studies revealed functional differences in gut bacteria in responders (R) including enrichment of anabolic pathways.

• Immune profiling suggested enhanced systemic and anti-tumor immunity in responders

• Verified results in gnotobioticmice (germ-free mice) with transplanted fecal microbiomes

• This has profound implications for all chemotherapy

Gopalkrishnan V et al, Gut microbiome modulates response to anti-PD-1 immunotherapy in melanoma patients, Science. 2018 Jan 5;359(6371):97-103.

Cardiovascular disease and the gut microbiome

Blum HE. Adv in Med Sci. 62 (2), 2017.

THE MICROBIOME AND HIV

HIV• Major global public health issue

– > 39 million dead

– ~ 36 million people living with HIV

• Sub-Saharan Africa disproportionally affected– 66% of new HIV infections globally

– 24.7 million people with HIV in 2013

• ART has transformed HIV from a fatal disease to a chronic condition

WHO 2014.; Maartens G et al, Lancet, 2014; Brenchley JM. Mucosal Immunology. 2013; SMART study et al, NEJM, 355 (2006);

Prevalence of HIV/AIDs in Africa (% population in 2011) (World Bank)

Over 15%5-15%2-5%1-2%0.5-1%0.1-0.5%Insufficient data

Uganda

HIV

• ~ 50% of deaths in people on ART in developed world are not due to AIDS– non-AIDS- defining cancers (23·5%),

cardiovascular disease (15·7%), and liver disease (14·1%).

– 50% increased risk of MI after adjustment for vascular risk factors

– ART does not fully correct this risk

• May be a consequence of chronic immune activation– Linked to CVD, renal, bone disease and

increased mortality

– Uncertain mechanism, microbial products likely involved

Maartens G, Celum C, and Lewin SR, HIV infection: epidemiology, pathogenesis, treatment and prevention, Lancet, 2014; Brenchley JM. Mucosal Immunology. 2013, April 1-9; Marchetti G, et al. Clin Micro Rev. 2013, 26(1): 2-18;

Why look at the microbiome and HIV?

• SIV-infected vs uninfected macaque monkeys – same diet, separately housed – 24 wks post intra-rectal SIV infection

• Pathogenic SIV is associated with an expanded enteric virome– >10-fold increase in viral sequences – New viruses found– Expansion of adenoviruses – Adenovirus-positive enteric

epithelial cells assoc with enteropathy (damage to gut)

• Bacterial microbiome was unchanged

• No difference in microbiome in non-pathogenic infection of African green monkeys

• Alterations in the enteric virome may be important in the pathogenesis of AIDS

Handley S et al., Cell. 2012

The gut during HIV infection

Gut Lumen

Gut epithelium

Blood vesselCD4Tcell

CD4Tcell

CD4Tcell

GALTLamina Propria

CD4Tcell HIV

HIV

Adenovirus

Bacteria

Bacteriophage


Gut Lumen

Gut epithelium


CD4Tcell

GALTLamina Propria

CD4Tcell HIV

HIV

Adenovirus

Bacteria

Bacteriophage


Gut Lumen

Gut epithelium

Blood vessel

CD4Tcell

GALTLamina Propria

CD4Tcell HIV

HIV

Adenovirus

Veazey et al, Science 1998

Pe

rip

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al L

N

G

ALT

Bacteria

Bacteriophage

CD4Tcell


Gut Lumen

Gut epithelium

Blood vessel

CD4Tcell

GALTLamina Propria

CD4Tcell HIV

HIV

Adenovirus

Bacteria

Bacteriophage


Gut Lumen

Gut epithelium

Blood vessel

CD4Tcell

GALTLamina Propria

CD4Tcell HIV

HIV

Adenovirus

sCD14:- LPS binding protein- increased levels in HIV- marker of microbial translocation- Linked to increased mortality in HIVSandler et al, JID, 2011

Bacteria

Bacteriophage

HIV gut microbiome studies

Gootenberg DB et al, Current Opinion in Infectious Diseases, 2017

Effect of intestinal microbiome in HIV

• Fecal microbiome in most studies shows increases in potentially “pathogenic” bacterial families with decreases in “beneficial” bacterial

– In western world see increase Prevotella species, Firmicutes and Bacteroides reduced

– In developing world, Prevotella species already high (likely due to higher fiber/plant diet)

Dillon SM et al, AIDS, 2017 31:511-521

Purpose of this study

• What is the effect of HIV, ART, and immune status on the enteric virome and bacteriome?

Monaco CL et al, Cell Host and Microbe, 2016

ISS clinic in Mbarara, Uganda• Founded November 1998

• Initially compassionate care to HIV-positive patients

• UARTO cohort: Uganda AIDS Rural Treatment Outcomes

– 40 HIV negative subjects

– 40 HIV-infected on ART ≥ 5 yrs

– 42 HIV-infected, ART naïve

– HIV neg and HIV untreated enrolled on initial test for HIV

– Matched stool and plasma


CD4 T cell count correlates with sCD14


Adenoviridae and Anelloviridae are expanded in AIDS


Bacterial richness and diversity differ by CD4 T cell count

Richness Diversity


Discriminant Bacterial Families


HIV Negative versus CD4 T cell count < 200

Conclusions• CD4 < 200 was associated with changes in the

enteric DNA virome • Adenoviridae sequences were increased in

patient with advanced HIV disease– Similar to SIV

• CD4 <200 associated with decreased bacterial richness and diversity

• Enterobacteriaceae associated with low CD4 T cell count– Similar to SIV

• Decreased Ruminococcaceae associated with CD4 <200 in multivariate model

Model of gut in HIV

Gut Lumen


CD4Tcell

CD4Tcell

GALTLamina Propria

CD4Tcell HIV

HIV

Adenovirus

• Severe immunodeficiency destabilizes enteric commensals

• Intestinal dysfunction and disease

• Treatment of enteric pathogens, in conjunction with ART, may limit HIV-associated enteropathy

• May minimize systemic immune activation

Shigella

Ruminococcus

Gut epithelium

Model of gut in HIV

Gut Lumen


CD4Tcell

GALTLamina Propria

CD4Tcell HIV

HIV

Adenovirus





Shigella

Ruminococcus

Gut epithelium

Model of gut in HIV

Gut Lumen

Blood vessel

CD4Tcell

GALTLamina Propria

CD4Tcell HIV

HIV

Adenovirus





Shigella

Ruminococcus

Gut epithelium

What about the vaginal microbiome?

• Greater than 90% of HIV transmission worldwide occurs following heterosexual intercourse– Women are twice as likely to contract HIV as men

• Young African women have up to 8-fold increased HIV prevalence compared to young men

• The vaginal microbiota plays an important protective role in preventing– pre-term birth– bacterial vaginosis (BV)– yeast infections– sexually transmitted diseases including HIV

M. I. Petrova et al, FEMS microbiology reviews 37, 762-792 (2013); B. A. White et al, Trends in endocrinology and metabolism: TEM 22, 389-393 (2011);

What is the vaginal microbiome?

• There is no single core vaginal bacteriome, but instead several major community groupings distinguished by whether they are Lactobacillus dominant or deficient

• Lactobacilli inhibit invasive bacterial species through– reduction of vaginal pH

– Competing for nutrients and adherence to the vaginal epithelium

• Pathological changes such as the dysbiosis associated with bacterial vaginosis (BV)– Can occur rapidly

– Almost always accompanied by depletion of lactobacilli

M. I. Petrova et al, FEMS microbiology reviews 37, 762-792 (2013); B. A. White et al, Trends in endocrinology and metabolism: TEM 22, 389-393 (2011); J. J. Schellenberg, F. A. Plummer, International journal of inflammation 2012, 131243 (2012); H. Borgdorff et al., The ISME journal 8, 1781-1793 (2014).

Vaginal microbiome in HIV

• BV is directly associated with

– increased inflammation in the vagina

– increased risk of STIs including HPV, HSV, and HIV

• HIV-infected women with altered vaginal bacterial flora (BV)

– have higher concentrations of HIV-1 RNA in genital secretions

– associated with a 3-fold increased risk of HIV transmission to a discordant male partner

M. I. Petrova et al, FEMS microbiology reviews 37, 762-792 (2013); B. A. White et al, Trends in endocrinology and metabolism: TEM 22, 389-393 (2011); J. J. Schellenberg, F. A. Plummer, International journal of inflammation 2012, 131243 (2012); H. Borgdorff et al., The ISME journal 8, 1781-1793 (2014).

Lactobacillus-Deficient Vaginal Bacteriomes Are Associated with Increased HIV Acquisition

• FRESH cohort in South Africa

• 4 times higher rate of HIV acquisition in women with high diversity vaginal bacteriomes

• Higher levels of activated mucosal CD4 T cells are higher in women with high-risk bacteria (Prevotella and other anaerobes)

• Lower levels in women with Lactobacillus crispatus

Gosmann C et al, Immunity, 46(1): 29-37, 2017

Microbiome and PREP

• In clinical trials of women the efficacy of PREP was poor

• CAPRISA 004 trial showed 39% efficacy for coital vaginally-applied tenofovir gel

• Variable adherence = major contributing factor

• Why is higher adherence required in women?

Klatt, NR et al, Science 02 Jun 2017: Vol. 356, Issue 6341, pp. 938-945

HIV infection rate with vaginal Lactobacillus dominance and

non-Lactobacillus bacterial dominance


• Separated into Lactobacillus dominant (LD) or non-Lactobacillus dominant (non-LD) bacteriome

• LD group: HIV incidence rate was 61% lower in women assigned to tenofovir gel versus placebo gel [P = 0.013]

• Non-LD: HIV incidence rate was 18% lower with tenofovir gel versus placebo gel (P = 0.644)

• The efficacy of tenofovir gel in preventing HIV infection in the subgroup of women with >50% adherence: 78% (P = 0.003) in the LD group versus 26% (P = 0.558) in the non-LD group.

• Tenofovir concentrations significantly lower in non-LD vs LD women (P = 0.0077).

Metabolism of tenofovir by G. vaginalis and BV-

associated bacteria


• Genital tenofovir concentrations negatively correlated with G. vaginalis protein abundance (correlation coefficient r = –0.19, P = 0.0014) and other anaerobic bacteria (Prevotella, r = –0.14, P = 0.023)

• Suggests relationship between BV-associated bacteria and tenofovir levels.• Used an in vitro culture system to assess potential metabolism of tenofovir by bacteria• Tenofovir concentrations in culture with G. vaginalis decreased rapidly by 50.6% compared

with marginal changes in either L. iners (P = 0.0037), L. crispatus (P = 0.0019), or control (P < 0.0001) at 4 hours

• Adenine, metabolite of tenofovir, only formed in presence of G. vaginalis• Microbiome metabolism of drugs has profound implications for treatment and prevention of

HIV

Limitations

• Main way used to examine bacterial microbiome is 16S rRNAsequencing, where small part of a region of the bacterial ribosome is amplified

• Sequencing different regions of 16S rRNA used may identify different bacteria (different results)

• Difficult to get species level identification of bacteria, usually more family level– Need species level for therapy

• All microbiome studies are associative – unless do in vivo or in vitrostudies to validate findings

• With shot-gun sequencing for viruses– Dependent on database used for comparison – need similar sequence

to identify– large amount of sequence data is “dark matter” – no homology to

known organisms– Sequencing depth vs cost issue

Conclusions

• Vaginal and enteric bacterial microbiomesdiffer in HIV infection from healthy control

• May offer novel therapeutic options to

– Prevent chronic inflammation in HIV

– Help prevent transmission and acquisition of infection

• Still a lot of work to be done

QUESTIONS?

Ways to measure intestinal microbiome

• Keep in mind that gut bacteria composition varies depending on location (Stomach vs upper small intestine vs middle vs lower small intestine vs colon)

• Fecal (stool or rectal swabs)– Easiest to collect– May not represent mucosal bacterial population

• Weck-cel (or similar)– Swab to rectal mucosal surface, have to leave in a little bit– Can get mucosal bacterial communities

• Biopsy from colonoscopy– Theoretically measures mucosal adherent bacteria– But only get after colonic prep – wash out a lot of bacteria

• Swab/fecal sample during colonoscopy– Same as above

Uganda Cohort CharacteristicsPatient Characteristics

HIV Neg(n = 40)

HIV on ART(n = 40)

HIV no treatment(n = 42) P value

16S rRNA sequencing (n) (total 110) 37 39 34VLP NGS sequencing (n) (total 65) 21 21 23Demographics, median (IQR)

Age (years) 43 (38-48) 44 (38-49) 29 (24-34) < 0.0001Males, n (%) 20 (50) 20 (50) 11 (26.2) 0.0404BMI (SD) 24 (22-28) 24 (21-27) 23 (20-27) 0.4305

Laboratory measures, median (IQR)CD4 T cell count (cells/µl) NA 396 (283-490) 225 (113-382) 0.0003

>500, n (%)* NA 8 (20) 4 (9.5)200-500, n (%)* NA 29 (72.5) 16 (38.1)<200, n (%) NA 3 (7.5) 22 (52.4)

CD4 Percent NA 25 (21-31) 15 (10-24) < 0.0001HIV Viral Load (copies/ml) NA 20 (20-20) 95,571 (24455-285548) < 0.0001CD4 nadir (cells/µl) NA 116 (58-167) 225 (110-382) 0.0001

Symptoms over last 30 days

Nausea/Vomiting, n (%) 13 (32.5) 5 (12.5) 15 (35.7) 0.0389

Diarrhea, n (%) 10 (25) 4 (10) 9 (21) 0.2000

Constipation, n (%) 17 (42.5) 13 (32.5) 10 (23.8) 0.1969

Loss of Appetite, n (%) 21 (52.5) 10 (25) 20 (47.6) 0.0286

Medications last 30dNRTI, n (%) NA 40 (100) NANNRTIn, n (%) NA 35 (85) NAYears on ART, median (IQR) NA 6.7 (6.1-7.1) NABactrim last 30d, n (%) 0 (0) 38 (95) 38 (90.5) < 0.0001Other Antimicrobials 30d, n (%) 15 (37.5) 14 (35) 4 (9.5) 0.0066


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