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) H2O2 & 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 (ptsH or rpsU) CbsAss

Anti-HIV proteins

Secreted  RANTES derivatives 20 µm

L. jensenii 1153

 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 kDa Lactobacillus CB staining • APVT-CV-N (P51G) 17 14 • Same anti-viral activity Full-length 6 • Low mitogenic potential 3 ─ Low activation of CD25 or CD69

Additional in vitro Safety Evaluation  Co-culture model of epithelial cells and CV-Nexpressing L. jensenii (Fichorova’s lab)

kDa

w t 16 66 26 6 36 6 6 16 6 4 M 6 al p No 2 5 ba 0 n ct M er ia

• 1666, 2666, 3666: CV-N-expressing L. jensenii strains • 106 CFU/cm2 of epithelial cells

Vaginal cells

17 14

APVT-CV-N

6 3

Cervical cells

17 14

APVT-CV-N

6 3

Anti-CV-N pAb

 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 H2O2producing 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 105-107 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)

108

Macaque 3539

Macaque 3567

3 10 17 24 40 87

3 10 17 24 40 87

107 106 105 104 103

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

-N ol Lj 1153-1666 Lj 1153-2666 V C ntr M1 M2 M3 M4 M5 M6 co

NHP

0 hr 24 hr

Week 3 Week 6

In situ expressed full-length APVTCV-N (P51G) binds to gp120

 No evident immunogenicity against CV-N 1:3000

2.5 Baseline

2

7 inoculations 4/14 - 4/28

8/11/2009 8/18/2009 9/1/2009

1.5

1:6000

6 re-inoculations 9/22 - 9/29

10/5/2009

1

1:12000

10/12/2009 10/26/2009

1:24000 1:48000 1:96000 1:192000

Anti-CV-N antibodies (ELISA Signal)

Immunogenicity

11/16/2009 11/23/2009

0.5

0

M 3730

M 3559

M 3567

M 3579

rabbit anti Non-diluted Chinese rhesus macaque CVL samples CV-N se rum

 Similarly, no antibodies against L. jensenii were detected

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 • > 1010 colony-forming units (CFU)/100 mg powders Preservation Matrix Components

Live/total ratio* upon drying

Basic matrix (skim milk, trehalose, phosphate buffer, sodium ascorbate)

16%

Basic matrix + polyol + 2nd antioxidant

32%

Skim milk; 2x trehalose; 2x phosphate buffer; 2x sodium ascorbate; 2x polyol; 2x 2nd antioxidant

>90%

* live/total ratio=CFU per ml/total cells per ml

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)

108

3 days post-inoculation

108

107

107

106

106

105

105

104

104

103

103

102

102

101

101

Reconstituted in MRS

Capsule

10 days post-inoculation

Reconstituted in MRS

Capsule

 New dosage forms/delivery are being explored Collaboration with Brigitte Sanders-Beer (BIOQUAL)

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

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 Xiaowen Liu Brigham and Women’s Hospital, Raina Fichorova Yang Liu UCSF, Craig Cohen, Anke Hemmerling Rosa Yu University of Washington, Dorothy Patton, Yvonne Cosgrove Sweeney Laurel Lagenaur NIH, Dean Hamer, E. Berger, A. Gronenborn, S. Rao, Mario Roederer Peter Lee CDC, Laura Barrientos, University of South Alabama, Lewis Pannell Thomas Parks NCI, Barry O'Keefe; CAMI & UC-Berkeley, Bethany Young Holt Kimberly Smith IPM, Joe Romano, David Fairhurst Qing Xia LBL, Broad Institute, JCVI Letong Jia Stanford University, Gary Schoolnik, Mark Holodniy Wenjun Huang Planned Parenthood Mar Monte, Jill MacAfee Aaron Diamond AIDS Research Center, David Ho Former San Raffaele Institute, Paolo Lusso, Luca Vangelista colleagues Profectus Biosciences, Timothy Fouts, Antony Dimitrov Chinese CDC, Zhiqing Zhang