Lactobacillus Crispatus

WHAT 2nd Microbiome workshop

WHEN 17 & 18 November 2016

WHERE Masur Auditorium at NIH Campus, Bethesda MD

Modulating the Vaginal Microbiome to Prevent HIV Infection

Laurel Lagenaur

For more information: www.virology-education.com Conflict of Interest I work for Osel Inc., a microbiome company developing live biotherapeutic products to prevent diseases in women The Vaginal Microbiome and HIV Infection

What’s normal /healthy/ optimal and what’s not? • Ravel Community Groups • Lactobacillus dominant vs. dysbiosis Why are Lactobacilli important for HIV prevention? • Dysbiosis = Inflammation = Increased risk of HIV acquisition • Efficacy of Pre-Exposure Prophylaxis decreased Modulation of the vaginal microbiome • LACTIN-V, live biotherapeutic-Lactobacillus crispatus • Ongoing clinical trials How we can use Lactobacilli and go a step further • Genetically modified Lactobacillus to prevent HIV acquisition HIV is Transmitted Across Mucosal Surfaces

• HIV infection in women occurs in the mucosa of

the vagina and cervix vagina cervix • Infection of underlying target cells

All mucosal surfaces are continuously exposed to a Herrera and Shattock, community of microorganisms Curr Top Microbiol Immunol 2013 Vaginal Microbiome: Ravel Community Groups Vaginal microbiomes clustered into 5 groups: Group V L. jensenii Group II L. gasseri 4 were dominated by Group I L. crispatus Lactobacillus, Group III L. iners whereas the 5th had lower proportions of lactic acid bacteria and Group 4 Diversity higher proportions of Prevotella, strictly anaerobic Sneathia, Megasphaera, organisms Atopobium

Shannon Diversity:

Ravel et al. PNAS 2011 low species high species richness/evenness richness/evenness Vaginal Microbiome Lactobacilli-dominated Healthy Vagina Dysbiosis • Low diversity, low vaginal pH • Antagonize pathogens Low Diversity High Diversity • Non-inflammatory Low Nugent High Nugent pH Bacterial vaginosis >5.5 • Decrease H O -producing Lactobacilli 2 2 pH • Increased bacterial diversity 3.6-4.5 • High pH, odor, discharge • Increased risk of STI and HIV Bacterial communities in O + Adapted from Srinivasan 2012 Vaginal Microbiota vs. Host Inflammatory Responses Increased levels of proinflammatory cytokines-IL-1α, IL- 1β, TNF-α, IFN-γ, IL-10 and IL-8

CT 1 CT 4

/ml)

)

) )

)

\ - \

)

- - \

α ( α

IL IL

1β ( 1β

1α ( 1α

TNF

- -

) )

)

IL \ IL \

IL \

/ml)

/ml) /ml)

)

) ) )

(

)

\ \ \

- - -

8 ( 8

IL IL IL

10 ( 10

IL IL

Alpha diversity IFN

- -

)

) )

IL \

\ IL IL \ • Community types Anahtar et al. Immunity 2015 Genital Inflammation is Linked to HIV infection Abundance% Abundance % Abundance % Abundance0 >50 % Abundance 0 0 0 0 >50>50 >50 >50

Lactobacillus crispatus Lactobacillus crispatus Lactobacillus crispatus Lactobacillus iners Lactobacillus iners South African womenLactobacillus iners in the Prevotella bivia Prevotella bivia Prevotella bivia LactobacilluSneathias crispatus sp 99 Sneathia sp 99 Sneathia sp 99 Shuttleworthia sp 9 [BVAB-1] Shuttleworthia sp 9 [BVAB-1] Shuttleworthia sp 9 [BVAB-1] LactobacilluAerococcuss iners christensenii Aerococcus CAPRISAchristensenii study withAerococcus christensenii P. bivia were Veillonella montpellierensis Veillonella montpellierensis Veillonella montpellierensis PrevotellaPrevotella bivia amnii Prevotella amnii Prevotella amnii Atopobium vaginae Atopobium vaginae Atopobium vaginae SneathiaMegasphaera sp 99 sp 9 Megasphaera sp 9 19 times moreMegasphaera likely sp 9 to have Lactobacillus gasseri Lactobacillus gasseri Lactobacillus gasseri ShuttleworthiaMycoplasma-like sp 9 [BVAB-1] sp 9 Mycoplasma-like sp 9 Mycoplasma-like sp 9 AerococcusMycoplasma christensenii hominis Mycoplasma hominis genital inflammationMycoplasma hominis and 13 Atopobium sp 6 Atopobium sp 6 Atopobium sp 6 Shuttleworthia sp 58 [BVAB-1] Shuttleworthia sp 58 [BVAB-1] Shuttleworthia sp 58 [BVAB-1] VeilloStreptococcusnella montpellierensis agalactiae Streptococcus agalactiae Streptococcus agalactiae Lactobacillus sp 91 Lactobacillus sptimes 91 more likelyLactobacillu tos sp 91acquire HIV PrevotellaStaphylococcus amnii epidermidis Staphylococcus epidermidis Staphylococcus epidermidis AtopobiumSneathia vaginae sanguinegens Sneathia sanguinegens Sneathia sanguinegens Prevotella timonensis Prevotella timonensis Prevotella timonensis MegasphaeraCoriobacteriaceae sp 9 sp 9 Coriobacteriaceae sp 9 Coriobacteriaceae sp 9 Prevotella sp 101 Prevotella sp 101 Prevotella sp 101 LactobacilluUreaplasmas gasseri sp 1 Ureaplasma sp 1 Ureaplasma sp 1 Granulicatella elegans Granulicatella elegans Granulicatella elegans Mycoplasma-likeWillia sp 9 msia muralis Williamsia muralis Williamsia muralis MycoplasmaHIV hominis status HIV status HIV status Cytokine group Cytokine group Cytokine group

150 150 150 Trichomonas vaginalis Trichomonas vaginalis Trichomonas vaginalis

d d

d Atopobium sp 6

r r

100 100 r 100 Chlamydia trachomatis Chlamydia trachomatis Chlamydia trachomatis

b b

b Neisseria gonorrhoeae Neisseria gonorrhoeae Neisseria gonorrhoeae

s s

50 50 s 50 Shuttleworthia sp 58 [BVAB-1]

O O 0 0 0 HSV-2 HSV-2 HSV-2 StreptococcusHSV agalactiae -1 HSV-1 HSV-1 Lactobacillus sp 91 Staphylococcus epidermidis Sneathia sanguinegens Prevotella timonensis Coriobacteriaceae sp 9 Prevotella sp 101 Ureaplasma sp 1 Granulicatella elegans Williamsia muralis

HIV status Cytokine group

150 Trichomonas vaginalis

v i

r 100 Chlamydia trachomatis

s p

b Neisseria gonorrhoeae

s 50 O 0 HSV-2 HSV-1 Vaginal Microbiota May Influence Tenofovir Efficacy

• Tenofovir disoproxil - Nucleotide analog reverse-transcriptase inhibitor used in Pre-Exposure Prophylaxis

• Delivered as a 1% intravaginal gel (before and after sex) Tenofovir gel effective against HIV with Lactobacillus- dominance

Lactobacilli-dominated Non-Lactobacillus dominant 0.40 0.40 A. LaA.ct obaLacctobaillusc idllousm indoamntinant 0.40 0.40 B. NoB.n No-Lanc-Latobactobacilluscil ldusom dinomanintant

0.35 0.35 0.35 0.35

n 61% efficacy

n 18% efficacy n

EfficaEfcyf,i c a6c1y%, 61% n EfficEfacfyic,a 1c8y,% 18%

o o

0.30o 0.30 0.30o

i 0.30

t c

95% CI, 11 to 84% c 95% CI, -77 to 63% c

95% CI, 11 to 84% c 95% CI, -77 to 63%

n n

0.25n

0.25n i

0.25 i 0.25

P=0.644 V

V P=0.644

0.20 f 0.20

f 0.20

l i

0.15 0.15l i

0.15 i 0.15

o o

r 0.10 P=0.013 o r 0.10

r 0.10 P=0.013

r 0.10

P P 0.05 0.05 0.05 0.05 HR = 0.39 (95% CI: 0.20; 0.83) HR = 0.82 (95% CI: 0.40; 1.65) 0.00 HR = 0.39 (95% CI: 0.20; 0.83) HR = 0.82 (95% CI: 0.40; 1.65) 0.00 0.00 0.00 0.0 0.5 1.0 1.5 2.0 2.5 0.0 0.5 1.0 1.5 2.0 2.5 0.0 0.0 0.5 0.5 1.0 1.0 1.5 1.5 2.0 2.0 2.5 2.5 Years in Study Years in Study Years in Study Years in Study Tenofovir Placebo Tenofovir Placebo Tenofovir Placebo Tenofovir Placebo

Lactobacillus dominant women at risk (Cumulative number of infections) Non-Lactobacillus dominant women at risk (Cumulative number of infections) Lactobacillus dominant women at risk (Cumulative number of infections) Non-Lactobacillus dominant women at risk (Cumulative number of infections) Tenofovir 205 (0) 204 (1) 183 (3) 129 (7) 46 (9) 0 (9) Tenofovir 140 (0) 137 (2) 123 (6) 87 (10) 32 (13) 0 (14) Tenofovir 205 (0) 204 (1) 183 (3) 129 (7) 46 (9) 0 (9) Tenofovir 140 (0) 137 (2) 123 (6) 87 (10) 32 (13) 0 (14) Placebo 202 (0) 196 (4) 173 (12) 123 (19) 51 (22) 0 (22) Placebo 141 (0) 137 (4) 116 (12) 84 (17) 28 (17) 0 (17) Placebo 202 (0) 196 (4) 173 (12) 123 (19) 51 (22) 0 (22) Placebo 141 (0) 137 (4) 116 (12) 84 (17) 28 (17) 0 (17) Tenofovir was rapidly depleted by Gardnerella but not Lactobacillus

Tenofovir (supernatant) Tenofovir (cell)

1.5 Abiotic e

g 0.003

L. iners L. iners

a )

h G. vaginalis G. vaginalis L

C 1.0

d l

g 0.002

(

i v

0.5 v

f o

o 0.001

T T 0.0 0 10 20 30 0.000 Time (hours) 0 10 20 30 Time (hours) 4 hours: G. vag vs. L. iners: P=0.002 4 hours: G. vag vs Abiotic: P=0.005 G. vag vs. L. iners: P<0.001

24 hours: 24 hours: G. vag vs. L. iners: P<0.001 G. vag vs. L. iners: P<0.001 G. vag vs Abiotic: P<0.001 What Can We Do to Decrease HIV Infection in Women?

• Treat their Bacterial vaginosis- Metronidazole

Lactobacillus dominant Dysbiosis Abx Post-Abx Restoration

↓ H O + Lactobacillus 2 2 Live Biotherapeutic Product (LBP) Growth of diverse commensal and pathogenic bacteria LACTIN-V: Lactobacillus crispatus CTV-05

• Give women exogenous Lactobacillus Osel Product LACTIN-V (Lactobacillus crispatus) for Treatment of Bacterial Vaginosis LACTIN-V Clinical Studies Phase 1 • Safe and well-tolerated at all dose levels* * Hemmerling et al., S Trans Dis 2009 Phase 2a • Following Metronidazole treatment • Colonization up to 78% of women treated for BV** ** Hemmerling et al., S Trans Dis 2010 • Colonization correlated with reduced BV recurrence

Phase 2b (sponsor NIAID/DMID ) • Prevention of recurrent BV • 1st patient enrolled Q2 2016 • Locations: University of California, San Diego, San Francisco General Hospital, Cook County Health and Hospitals System, Chicago, Washington University School of Medicine in St. Louis • Double Blind, Placebo Controlled, Randomized Clinical Trial Microbiome Changes During Successful LACTIN-V Colonization

Lactobacillus crispatus Gardnerella vaginalis Resolution of infection Megasphaera spp. Atopobium vaginae Snethia BVAB2 Lactobacillus iners Lactobacillus iners

Lactobacillus crispatus

ABX LACTIN-V

Enrollment (Ngugi et al., 2011)

L. crispatus administered following ABX replenishes the vaginal microbiome Increase in Lactobacillus and L. crispatus at follow-up

Ngugi, et al., STD, 2011 Enrollment Day 10 Day 28 Follow up visits

Two potential mechanism: 1) colonizes directly (evidence) and 2) fosters growth of endogenous vaginal lactobacilli

Modulating the vaginal microbiome: Can we go further?

L I - \ )

)

) )

\ \

\ I L - )

IL -

IL - - IL • Colonize the mucosa with live recombinant Lactobacillus

• Natural defense, lactic acid, • “Anti-inflammatory”

• Genetically modified to produce an HIV entry inhibitor

L - \ ) • Protection at the siteI of HIV infection

HIV HIV HIV inhibitor HIV inhibitor Lactobacillus Lactobacillus Strain selection L. jensenii 1153

• Screened >20 isolates of L. crispatus, L. jensenii and L. gasseri from healthy women • Selected L. jensenii 1153 - Growth rate-doubling time - D-Lactic acid production - Adherence to tissue - Activity against Gardnerella and Staphylococcus - Transform (no endogenous plasmids)

• Broad Institute/J. Craig Venter Institute -sequenced genome 1153 Inhibitor Selection Cyanovirin-N (NCI) - Identified by National Cancer Institute drug screen (Cyanobacterium) - Activity against all subtypes - Inhibited >85% HIV viruses in a representative panel of major subtypes, including Tier 2, CRF, T/F - CV-N active pH 4-8 (vaginal pH range) (vaginal pH range) Constructing a recombinant L. jensenii 1153-1666

• Stably integrated mCV-N Integration plasmid gene into the Lactobacillus chromosome by orfA` mCVN orfA` homologous recombination

orfA L. jensenii 1153 chromosome • Biologically active level of inhibitor expression using a Integrate into chromosome constitutive ribosomal promoter

mCV-N L. jensenii 1153-1666 secreting mCVN Liu et al. 2006, AAC mCV-N Protein Seen on Coomassie Stain Proteins in 300 µl

• Parental L. jensenii 1153 is Strains Lj1153 Lj1153-1666 compared to recombinant L. pH 6.6 4.9 6.6 5.7 4.9 OD 600 0.5 2.0 0.5 1.0 2.0 jensenii 1153-1666 on kDa protein gel 188 98 62 49 • Global protein expression 38 28 unchanged 17 14 • Lactic acid production and 6 growth unchanged 3 Coomassie Blue Staining Intravaginal Inoculation of L. jensenii 1153-1666 in Macaques

In initial studies….. Pellet + 3% hydroxyethyl cellulose Lactobacilli

Vaginal Epithelial Cell Freshly prepared Rhesus macaque overnight culture Brown and Hopps Modified Gram stain Recovery of L. jensenii 1153 -1666 from Rhesus Macaques following 5-day dose

Followed 10macaques10 for 6 weeks

109 d

e Vaginal colonization

r 8

e 10 v

o 7

c 10

i i 6

n 10

e s

n 105

s 4 u

l 10

i c

a 103

o t

c 2

a 10 L

101 Lactobacillus CFU recovered CFU Lactobacillus 100 7 14 21 28 35 42 49 Days PI L. jensenii 1153 expressing mCV-N Proof of Principle

• Colonized rhesus macaques

• Cervical vaginal lavage Lj 1153-1666 Lj 1153-2666 taken weekly M1 M2 M3 M4 M5 M6

Week 0 • mCV-N protein (before inoculation) Day 1 detected by (24 hrs after inoculation) immunoblot for at least Week 3 6 weeks Week 6 Proof of Principle

• Colonized rhesus macaques

• Immunohistochemistry on Pre-immune Anti-CV-N vaginal biopsies for mCV-N

protein vaginal lumen

• mCV-N protein localized on mucosal surface at the site of mucosal surface virus entry 1:4000 1:4000 We selected a strain, and an HIV inhibitor. We made a recombinant L. jensenii

We showed that our L. jensenii recombinant could colonize the macaque and express mCV-N protein in situ on the mucosa

Now we wanted to challenge macaques with (S)HIV to test whether infection could be prevented SHIV Repeated Dose Challenge • Two arm study • 12 macaques received L. jensenii expressing mCV-N • 12 control (8 macaques received HEC placebo gel/4 no gel • Macaques were challenged each week for 6 weeks with (S)HIV • Monitored for viral infection each week

• Repeated dose challenges measure the rate of infection per exposure • Infection rate about 33% macaques/exposure (100-1000X higher than amount found in human)

• Monitor time to infection Tested recombinant L. jensenii 1153-1666 SHIV Repeated Dose Vaginal Challenge

Studies performed at BIOQUAL Inc. • 300 TCID50 SHIVSF163P3 • Controls: 35% infection per exposure • L. jensenii 1153-1666 : 13% infection per 61 challenge 8 infections=13.1% exposure L. jensenii 1153-1666 • 63% reduction in acquisition p<0.004 31 challenges/11 infected=35.4% • 6X reduction in viral load

Lagenaur et al. 2011 Mucosal Immunology

A second smaller experiment confirmed these results Based on the results • Moved forward with formulation of the product that could be

used in the real life a prototype tablet 100 mg, 5x1010 CFU -Developed a vaginal tablet (VT)

Stability-Tablets of MucoCept-mCV are potent,-N tablets stable, colonized macaques

12 12 Storage 10 10 1012 1012

Temperature 6 6

o 6 )

4 C 1

g /

o 3 5 U 11 11 25 C

10 10 11 11 1 F 10 10

o 1

37 C i (

New formulation less potent, but i

y e

c 90-day

o s n

more stable at 37 C n

e e

t 10 10 j

10 10 10 10 o

. 10 10

g o 109 109 L 0 60 120 180 240 300 360 109 109 0 60 120 Days Days Lagenaur et al. 2011 PLoSOne Next Steps in Assay and Product Development

• Developed ELISA to analyze mCV-N protein-limit of detection 1ng • Developed qPCR to detect L. jensenii 1153-1666 • Submitted documents for pre-IND meeting with FDA (initial comments received) • Formulation needs to be sufficient for first in human studies (our stability of sufficient) • Transfer strain and process to an industrial partner • IND-submission IND=Investigation New Drug Application Summary MucoCept Product Development

• Identified a need: to develop novel HIV prevention stragegies for women • Shown that formulated L. crispatus can be delivered and can colonize women • Selected a Lactobacillus jensenii (vaginal colonizer) and an HIV inhibitor • Constructed a recombinant L. jensenii that secretes mCV-N, tested fitness • Tested colonization, protein expression, efficacy, safety of the recombinant L. jensenii Vaginal Biopsy-Healthy woman • Formulated the concept into a product (Vaginal Tablet) • Tested colonization and efficacy of the tablet • Developed assays to detect mCV-N protein and recombinant L. jensenii • With submission of an IND, we are moving toward phase 1 safety trial in women Acknowledgments

Bioqual, Inc. UCSF Hanne Andersen, PhD Peter Lee, MD Brigitte Sanders, PhD Thomas Parks, PhD Craig Cohen, MD, MPH Trine Nilsen, PhD Anke Hemmerling, MD, PhD, MPH University of California, Davis KT Moortgat, PhD Christopher Miller, DVM, PhD Iwona Swedek Tim Carroll, PhD, Angela Marcobal, PhD Linda Fritts Caitlyn Dela Cruz Former Osel colleagues Chia-Hwa Chang, PhD NIH Teresa Chang, PhD Dean Hamer, PhD Andrew Cheng, PhD Kirsten Essenmacher, PhD Jay Berzofsky, MD, PhD Courtney Frasier, PhD Letong Jia Nancy Miller, PhD Wenjun Huang Carole Bewley, PhD Xiaowen Liu, PhD Yang Liu, PhD Pat Martin, PhD David Simpson, PhD Kimberly Smith Qing Xia Qiang Xu, PhD Rosa Yu, PhD Yonghong Zhu, PhD