Bioengineered Lactobacillus as Next Generation Probiotics

Bioengineered Lactobacillus
as Next Generation Probiotics
Qiang Xu, PhD
MucoCept Team
M2010 Satellite Symposium on Bacterial Therapeutics

Strategy
Commensal Lactobacillus species are
important in host defense
BV & STI’s are known cofactors for HIV
transmission
Our Strategy:
• Select optimal human vaginal strains
• Express potent HIV inhibitors at high levels
• Develop technologies to preserve
Lactobacillus as dry powder for ambient
temperature storage
• Promote high level and persistent vaginal
colonization

Key Issues
Colonization & competition with native flora
In situ protein expression level & bioactivity
Immunological responses
Preservation of Lactobacillus
Bio-containment
• Environmental survival
• Strain transmission
Regulatory hurdle for GMO

Strain Selection
Primary screening in vitro
• Bacterial identification (by 16S rDNA gene sequence)
• H 2O 2 & lactate production, doubling time, aerotolerance
• Endogenous plasmid
• Self-aggregation (a surrogate marker for in situ colonization)
• Antibiotic resistance
Secondary screening in vitro
• Competition with commensal bacteria and mucosal pathogens
─ Gardnerella vaginalis (& Candida albicans)
• Susceptibility to possible Lactobacillus phages
• Bacterial adherence in a human epithelial model
Properties related to bacterial preservation
• Carbohydrate fermentation profile
• % of viable cells during fermentation and after drying
• Vaginal colonization in a NHP model
Suitability for genetic modification
Cell banks made

Genetic Modification
L. jensenii 1153 was selected from >300
human vaginal isolates of lactobacilli
Expression cassette [employing strong native
promoters* and signal sequence (CbsAss)]
integrated in the bacterial chromosome
• *Verified in situ
20 µm
L. jensenii 1153
(ptsH or rpsU)
CbsAss Anti-HIV proteins
Secreted
RANTES derivatives
Antibody fragments
Virus entry inhibitors
• Cyanovirin-N (P51G)

Cyanovirin-N (CV-N)
& its Derivative
A small acid-stable, monomeric protein (NCI)
• A cyanobacterial protein – easy to express
• No post-translational modification site
A potent HIV envelope-targeting inhibitor
Retains activity in seminal plasma
We expressed a modified version in
Lactobacillus
• APVT-CV-N (P51G)
• Same anti-viral activity
• Low mitogenic potential
kDa
17
14
─ Low activation of CD25 or CD69
6
3
CB staining
Full-length

Additional
in vitro Safety Evaluation
Co-culture model of epithelial cells and CV-Nexpressing
L. jensenii (Fichorova’s lab)
• 1666, 2666, 3666: CV-N-expressing L. jensenii strains
• 10 6 CFU/cm 2 of epithelial cells
kDa
17
14
6
3
17
14
6
3
wt
Anti-CV-N pAb
1666
2666
3666
1646
Malp2 50 nM
No bacteria
Vaginal cells
APVT-CV-N
Cervical cells
APVT-CV-N
Expressed CV-N
binds to gp120
No evident
upregulation of
proinflammatory
markers by the
cervicovaginal
epithelium
Raina Fichorova, Oral presentation for Abstract # 355

Vaginal Colonization
in NHP
A key issue for safety and efficacy, prior to
human dosing
Establishment of Chinese rhesus macaque
model (Yu et al., 2009)
• Harbor endogenous vaginal Lactobacillus, mostly H 2O 2producing
L. johnsonii
• Allow clearance of vaginal Lactobacillus with
azithromycin, and restoration of endogenous L.
johnsonii colonization
• Support persistent colonization of a human vaginal
isolate of L. jensenii at 10 5 -10 7 CFU per vaginal swab
collected
Collaboration with Dean Hamer (NCI), ABL, SRI,
Brigitte Sanders-Beer (BIOQUAL)

Vaginal Colonization
in NHP
CV-N-Lactobacillus* dosed vaginally using hydroxyethyl cellulose (HEC)
Lactobacillus recovered
(CFU/vaginal swab)
10 8
10 7
10 6
10 5
10 4
10 3
Macaque 3539 Macaque 3567
3 10 17 24 40 87 3 10 17 24 40 87
Number of Days After Bacterial Inoculation
*Tested in >20 Chinese rhesus macaques

In Situ CV-N Expression
Assayed by
Collection of CVL by 2× 2-ml washes
CV-N detected by Western blots
Time post inoculation
0 hr
24 hr
Week 3
Week 6
CV-N
control
Lj 1153-1666 Lj 1153-2666
M1 M2 M3 M4 M5 M6
NHP
In situ expressed
full-length APVT-
CV-N (P51G)
binds to gp120

Immunogenicity
Anti-CV-N antibodies (ELISA Signal)
2.5
2
1.5
1
0.5
0
No evident immunogenicity against CV-N
Baseline
8/11/2009
8/18/2009
9/1/2009
10/5/2009
10/12/2009
10/26/2009
11/16/2009
11/23/2009
7 inoculations
4/14 - 4/28
6 re-inoculations
9/22 - 9/29
M3730 M3559 M3567 M3579 rabbit anti
CV-N
serum
Non-diluted Chinese rhesus macaque CVL samples
Similarly, no antibodies against L. jensenii were detected
1:3000
1:6000
1:12000
1:24000
1:48000
1:96000
1:192000

Regulatory Considerations
Environmental Persistence
• No persistence in the environment
Genetic Stability
• The engineered strain is genetically stable
Bacterial Transmission
• No sexual transmission of L. jensenii from female to
male macaques, n=2
Availability of Rescue Therapy
• Engineered Lactobacillus contain no antibiotic
resistance marker
• Engineered Lactobacillus cleared from colonized
macaques using azithromycin as vaginal suppository

Lactobacillus Manufacture
Issues:
Cell viability
Cell recovery upon rehydration
Appropriate bacterial dosage form
Shelf life at room temperature storage
Focus:
Fermentation
Formulation & preservation
Bacterial dosage
Poster Presentation, Abstract # 241

Formulation & Preservation
Fermentation optimization
Basic preservation matrix
• Trehalose, skim milk, and an antioxidant (sodium ascorbate)
• Transient buffering
Production of highly viable Lactobacillus
• By freeze (or spray) drying
• >10 10 colony-forming units (CFU)/100 mg powders
Preservation Matrix Components
Basic matrix
(skim milk, trehalose, phosphate buffer, sodium ascorbate)
Live/total ratio*
upon drying
16%
Basic matrix + polyol + 2nd antioxidant 32%
Skim milk; 2x trehalose; 2x phosphate buffer; 2x sodium
ascorbate; 2x polyol; 2x 2nd antioxidant
* live/total ratio=CFU per ml/total cells per ml
>90%

Lactobacillus Powder Delivery
Vaginal administration to 6 macaques
• Dried powder in capsule vs. reconstituted in 1.0 ml MRS
Vaginal swabs collected 3 and 10 days after final
bacterial inoculation
Lactobacillus recovered
(CFU/vaginal swab)
10 8
10 7
10 6
10 5
10 4
10 3
10 2
10 1
3 days post-inoculation 10 days post-inoculation
Reconstituted
in MRS
Capsule
New dosage forms/delivery are being explored
Collaboration with Brigitte Sanders-Beer (BIOQUAL)
10 8
10 7
10 6
10 5
10 4
10 3
10 2
10 1
Reconstituted
in MRS
Capsule

Current Focus
Conduct pre-IND consultation with FDA on the use
of bioengineered Lactobacillus
• Evaluate vaginal colonization, clearance, and biocontainment
in human volunteers (Phase 0)
─Use of bioengineered Lactobacillus with a
surrogate marker (β-glucuronidase)
• Working with the MIP R33 Team
Expand the platform, along with our lead program
• Co-express HIV inhibitors (single or multiple strains)
• Develop multipurpose microbicides against other STIs
• Mucosal delivery of antigens
Confirm bioactivity of in situ-expressed proteins
Refine dosage forms/delivery to promote more
efficient vaginal colonization

Studies in Progress
by Other Investigators
CV-N-expressing Lactobacillus strains in yogurt for
feeding and gut CV-N expression
• Bharat Ramratnam
Alpaca antibodies to inhibit HSV by targeting
integrins
• Richard Markham
Mannose binding Lactobacillus as anti-HIV strategy
• Lin Tao
Antiviral factors from Lactobacillus
• Ruth Ingrid Connor
Lactobacillus on genital HIV shedding
• Jane Hitti
And more…

Acknowledgements
NIH U19 AI60615 (PO: Drs. Jim Turpin/Roberta Black)
NIH U01 AI066708 (PO: Dr. Carolyn Deal/Jonathan Glock); MIP II, MIP IV (PO: Dr. Turpin)
USAID funding through IPM; CONRAD Foundation/GMP; GATES Foundation GCE
Xiaowen Liu
Yang Liu
Rosa Yu
Laurel Lagenaur
Peter Lee
Thomas Parks
Kimberly Smith
Qing Xia
Letong Jia
Wenjun Huang
Former
colleagues
External Collaborators:
Bioqual, Inc., Brigitte Sanders-Beer
Southern Research Institute, Carol Lackman-Smith, James Cummins
Advanced Bioscience Laboratories, Inc., Deborah Weiss, Jim Treece
University of Pittsburgh, Sharon Hillier, Lorna Rabe
Brigham and Women’s Hospital, Raina Fichorova
UCSF, Craig Cohen, Anke Hemmerling
University of Washington, Dorothy Patton, Yvonne Cosgrove Sweeney
NIH, Dean Hamer, E. Berger, A. Gronenborn, S. Rao, Mario Roederer
CDC, Laura Barrientos, University of South Alabama, Lewis Pannell
NCI, Barry O'Keefe; CAMI & UC-Berkeley, Bethany Young Holt
IPM, Joe Romano, David Fairhurst
LBL, Broad Institute, JCVI
Stanford University, Gary Schoolnik, Mark Holodniy
Planned Parenthood Mar Monte, Jill MacAfee
Aaron Diamond AIDS Research Center, David Ho
San Raffaele Institute, Paolo Lusso, Luca Vangelista
Profectus Biosciences, Timothy Fouts, Antony Dimitrov
Chinese CDC, Zhiqing Zhang