Development of a conjugate vaccines for ... - Take on Typhoid · 8th International Conference on...

15
8th International Conference on Typhoid Fever and Other Invasive Salmonelloses Laura B. Martin, Head of Development Program Dhaka, Bangladesh 1 March 2013 Development of a conjugate vaccines for enteric fever

Transcript of Development of a conjugate vaccines for ... - Take on Typhoid · 8th International Conference on...

Page 1: Development of a conjugate vaccines for ... - Take on Typhoid · 8th International Conference on Typhoid Fever and Other Invasive Salmonelloses Laura B. Martin, Head of Development

8th International Conference on Typhoid Fever and Other Invasive SalmonellosesLaura B. Martin, Head of Development ProgramDhaka, Bangladesh 1 March 2013

Development of a conjugate vaccines for enteric fever

Page 2: Development of a conjugate vaccines for ... - Take on Typhoid · 8th International Conference on Typhoid Fever and Other Invasive Salmonelloses Laura B. Martin, Head of Development

NVGH enteric fever vaccine strategyBivalent vaccine against S. Typhi and S. Paratyphi A

Glycoconjugate combination vaccine• Building on NVGH development of Vi-CRM197

• Employing Novartis know-how and expertise

S. Paratyphi A component• Serovear specific O-antigen, O:2• Covalently linked to CRM197

Bivalent vaccine• VI-CRM197 + O:2-CRM197

2 | NVGH 5th Anniv | LB Martin | 22 Feb 2013 | NVGH's model in action | Business Use Only

Lipid A

Core region

n

Man Rha Gal

Glc

Par

KDO

O:2 of S. Paratyphi AExternal portion of Lipopolysaccharide (LPS)

Page 3: Development of a conjugate vaccines for ... - Take on Typhoid · 8th International Conference on Typhoid Fever and Other Invasive Salmonelloses Laura B. Martin, Head of Development

Feb Inauguration

2008 2009 2010 2011 2012 2013

NVGH milestones

Tomorrow1st out-licensing

March

Bivalent vaccine development since Kilifi KenyaBuilding on Vi-CRM197 and other NVGH projects

In-house optimization and scale-up in preparation for technology transfer

NVGH proof-of-concept development and non-clinical studies

TG1 TG3TG2€ - Wellcome Trust

O:2-CRM197 and bivalent highlights

3 | NVGH 5th Anniv | LB Martin | 22 Feb 2013 | NVGH's model in action | Business Use Only

Jan 7th internationalconference on typhoidfever and other invasiveSalmonelloses

Page 4: Development of a conjugate vaccines for ... - Take on Typhoid · 8th International Conference on Typhoid Fever and Other Invasive Salmonelloses Laura B. Martin, Head of Development

NVGH enteric fever vaccine, bivalent conjugate Unique attributes

Uses readily available, scalable GMP materials and equipment

O:2-CRM197 designed to be compatible with Vi-CRM197

Process area O:2-CRM197

O:2 sourceMOGM S. Paratyphi A

Drug sensitive for safetyGenetically modified to produce more membrane

Grows well in defined, simple media

O:2 purificationNo column chromatography

Efficient in situ extraction methodNo hazardous or expensive reagents or intermediates

Carrier proteinCRM197

Mutant diphtheria toxin(used by Novartis Vaccines and Diagnostics)

ConjugationO:2 per CRM197 < 2 Novel method developed by NVGH

Cost of Goods Similar to Vi-CRM197

Bivalent formulation No overt interactions observed

4 | NVGH 5th Anniv | LB Martin | 22 Feb 2013 | NVGH's model in action | Business Use Only

Micoli et al, PLoS One 2012 & Micoli et al, Anal Biochem 2013

Page 5: Development of a conjugate vaccines for ... - Take on Typhoid · 8th International Conference on Typhoid Fever and Other Invasive Salmonelloses Laura B. Martin, Head of Development

5 | NVGH 5th Anniv | LB Martin | 22 Feb 2013 | NVGH's model in action | Business Use Only

O:2 polysaccharide production sourceWild-type/attenuated vs GMMA producing line

Character-ization Wild-type Attenuated GMMA producing

Genetic modificaitons none ∆guaBA ∆TolR

Morphology rods

O:2 hydrolysis biomass biomass biomass + GMMA

O:2 heterogenityHMW : MMW 70 : 30 80 : 20 10 : 90

Average repeating units ~ 45 < 55 ~ 25

% O-acetylation ~ 70 > 70 ~ 50

MMW O:2 easier to handle gives, more consistent conjugates

Page 6: Development of a conjugate vaccines for ... - Take on Typhoid · 8th International Conference on Typhoid Fever and Other Invasive Salmonelloses Laura B. Martin, Head of Development

O:2 polysaccharide production processOptimized for 30 L scale

• Shake flask• Shake flask

• High cell density fermentation (30 L)• High cell density fermentation (30 L)

• O:2 antigen hydrolysis in situ (100°C, > 4 h)• O:2 antigen hydrolysis in situ (100°C, > 4 h)

• Neutralization in situ• Neutralization in situ

• Harvest (TFF Microfiltration)• Harvest (TFF Microfiltration)

• Ultrafiltration (TFF 30 kDa cut-off)• Ultrafiltration (TFF 30 kDa cut-off)

• Precipitation 1 (pH 3)• Precipitation 1 (pH 3)

• Centrifugation• Centrifugation

• Negative chromatography (Sartobind S)• Negative chromatography (Sartobind S)

• Precipitation 2 (EtOH + CaCl2)• Precipitation 2 (EtOH + CaCl2)

• Centrifugation• Centrifugation

• Ultrafiltration (TFF 10 kDa cut-off)• Ultrafiltration (TFF 10 kDa cut-off)

• Filtration (0.2 µm)• Filtration (0.2 µm)

• Purified O:2 antigen Intermediate (Bulk)• Purified O:2 antigen Intermediate (Bulk)

Exploiting sterilize in place vessel

6 | NVGH 5th Anniv | LB Martin | 22 Feb 2013 | NVGH's model in action | Business Use Only

Lipid A

Core region

n

Man Rha Gal

Glc

Par

KDO

Site of hydrolysis

Micoli et al, Anal Biochem 2013

Page 7: Development of a conjugate vaccines for ... - Take on Typhoid · 8th International Conference on Typhoid Fever and Other Invasive Salmonelloses Laura B. Martin, Head of Development

O:2 polysaccharide production processOptimized for 30 L scale

• Shake flask• Shake flask

• High cell density fermentation (30 L)• High cell density fermentation (30 L)

• O:2 antigen hydrolysis in situ (100°C, > 4 h)• O:2 antigen hydrolysis in situ (100°C, > 4 h)

• Neutralization in situ• Neutralization in situ

• Harvest (TFF Microfiltration)• Harvest (TFF Microfiltration)

• Ultrafiltration (TFF 30 kDa cut-off)• Ultrafiltration (TFF 30 kDa cut-off)

• Precipitation 1 (pH 3)• Precipitation 1 (pH 3)

• Centrifugation• Centrifugation

• Negative chromatography (Sartobind S)• Negative chromatography (Sartobind S)

• Precipitation 2 (EtOH + CaCl2)• Precipitation 2 (EtOH + CaCl2)

• Centrifugation• Centrifugation

• Ultrafiltration (TFF 10 kDa cut-off)• Ultrafiltration (TFF 10 kDa cut-off)

• Filtration (0.2 µm)• Filtration (0.2 µm)

• Purified O:2 antigen Intermediate (Bulk)• Purified O:2 antigen Intermediate (Bulk)

Exploiting sterilize in place vessel

7 | NVGH 5th Anniv | LB Martin | 22 Feb 2013 | NVGH's model in action | Business Use Only

Good purity & well characterized

O-acetylation > 40 % Protein < 0.5 % Nucleic acid < 0.5 % Endotoxin < 0.01 UI/µg

Insert chromatogram here

Micoli et al, Anal Biochem 2013

Page 8: Development of a conjugate vaccines for ... - Take on Typhoid · 8th International Conference on Typhoid Fever and Other Invasive Salmonelloses Laura B. Martin, Head of Development

O:2-CRM197 conjugation processAlso used in the iNTS conjugate vaccines

O-Antigen derivatization with dihydrazide

Further O-Antigen derivatization with SIDEA

Conjugation with CRM197 N

O

O

O

O O

O N

O

O

SIDEA

O

O

ON

O

O

HC NHNHC(CH2)4CONHNHOAg

O O

O

NH-CRM197HC NHNHC(CH2)4CONHNHOAg

OCRM197-NH2

+ H2NHN

O

NaBH3CN

C OOAgKDO

HC NHNHC(CH2)4CONHNH2OAg

O

C NNHC(CH2)4CNHNH2OAg

O

ADH O

NHNH2

O

8 | NVGH 5th Anniv | LB Martin | 22 Feb 2013 | NVGH's model in action | Business Use Only

Micoli et al, PLoS One 2012

Page 9: Development of a conjugate vaccines for ... - Take on Typhoid · 8th International Conference on Typhoid Fever and Other Invasive Salmonelloses Laura B. Martin, Head of Development

O.2-CRM197 production processOptimized for 200 mg scale

• O:2 activation• O:2 activation

• Dried O:2 derivatized with ADH• Dried O:2 derivatized with ADH

• Purification of O:2-ADH• Purification of O:2-ADH

• Dry O:2-ADH• Dry O:2-ADH

• O:2-ADH derivatized with SIDEA• O:2-ADH derivatized with SIDEA

• Purification of O:2-ADH-SIDEA• Purification of O:2-ADH-SIDEA

• Dry O:2-ADH-SIDEA• Dry O:2-ADH-SIDEA

• Addition of CRM197 to activated O:2-ADH-SIDEA• Addition of CRM197 to activated O:2-ADH-SIDEA

• Purification of O:2-CRM197• Purification of O:2-CRM197

• Filtration (0.2 μm)• Filtration (0.2 μm)

• O:2-CRM197 Bulk Drug Substance• O:2-CRM197 Bulk Drug Substance

9 | NVGH 5th Anniv | LB Martin | 22 Feb 2013 | NVGH's model in action | Business Use Only

Good purity & well characterized

Selective chemistry through KDO, terminal sugar, of core

PS : Protein 1.3 Yield CRM197 80 % O-acetylation > 40 %

Page 10: Development of a conjugate vaccines for ... - Take on Typhoid · 8th International Conference on Typhoid Fever and Other Invasive Salmonelloses Laura B. Martin, Head of Development

O:2-CRM197 immunogenicitySimilar responses from wild-type, attenuated and ∆TolR strains

Historical study comparisonsStrain: CD-1 miceGroup size: 8Dose: 1 µg Route: SC, 200 µL

10 | NVGH 5th Anniv | LB Martin | 22 Feb 2013 | NVGH's model in action | Business Use Only

O:2 antigen source

HMW : MMW

Anti-O:2 serum IgG responses are not highly dependent on Source of O:2 antigen Ratio of the MW populations in the O:2 Level of O-acetylation

Page 11: Development of a conjugate vaccines for ... - Take on Typhoid · 8th International Conference on Typhoid Fever and Other Invasive Salmonelloses Laura B. Martin, Head of Development

O:2-CRM197 induces functional antibodiesIncreasing antibody = increased killing

O:2-CRM197 produces antibodies that can kill S. Paratyphi A

Impact of combining Vi-CRM197 with O:2-CRM197 . . . 11 | NVGH 5th Anniv | LB Martin | 22 Feb 2013 | NVGH's model in action | Business Use Only

Anti-O:2 ELISA Units

0.01 0.1 1 10

Per

cent

S. P

arat

yphi

A k

illed

0

20

40

60

80

100

AttenuatedWild type

Serum Bactericidal AssayCFU: 103 bacteria (in exponential phase)Sera: serial diluted Day 56 BRC’: 10%Incubation time: 2 hControls: ∆BRC’, ∆sera no SBA (0% killing)

Page 12: Development of a conjugate vaccines for ... - Take on Typhoid · 8th International Conference on Typhoid Fever and Other Invasive Salmonelloses Laura B. Martin, Head of Development

Bivalent vaccine antibodies are bactericidalAgainst both Vi+ bacteria (Citrobacter) and S. Paratyphi A

Serum Bactericidal AssayCFU: 103 bacteria (in exponential phase)Sera: serial diluted Day 56 BRC’: 10%Incubation time: 2 hControls: ∆BRC’, ∆sera no SBA (100% survival)

12 | VADER course 1 | LB Martin | 5 Feb 2013 | NVGH's Enteric Fever Vaccines | Business Use Only

Study DesignStrain: CD-1 mice Route: SC, 200 µLGroup size: 8 Immunization: days 1, 14 & 42Dose: 1 µg antigen Bled: day 56Vaccines: Vi-CRM197, O:2-CRM197 or bivalentELISA results: no immunologic interference

Vi-CRM197 + O:2-CRM197 likely to provide coverage against enteric fever

S. Paratyphi A killing correlates with anti-O:2 levels

Anti-O:2 ELISA Units

0.001 0.01 0.1 1 10

Perc

ent

NVG

H308

kill

ed

0

20

40

60

80

100

Citrobacter killing correlates with anti-Vi levels

Anti-Vi ELISA Units 0.001 0.01 0.1 1 10

Perc

ent

NVG

H328

kill

ed

0

20

40

60

80

100

Vi-CRM197BivalentO:2-CRM197

Page 13: Development of a conjugate vaccines for ... - Take on Typhoid · 8th International Conference on Typhoid Fever and Other Invasive Salmonelloses Laura B. Martin, Head of Development

Proof of principle for a bivalent, Typhoid / Paratyphoid A, vaccine• Technical and animal immunogenicity

Activities for 2013 and beyond• Prepared to transfer the robust processes to a commercialization partner

- Vi-CRM197 for typhoid fever- Bivalent (Vi-CRM197 + O:2-CRM197) for enteric fever

• Support partner for further technical and clinical development

Aiming for developing country access to enteric fever vaccine • Initial registration and roll out in India• WHO prequalification and wider distribution throughout S. Asia

NVGH enteric fever vaccine, what is nextReducing the risk and meeting the mission

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Page 14: Development of a conjugate vaccines for ... - Take on Typhoid · 8th International Conference on Typhoid Fever and Other Invasive Salmonelloses Laura B. Martin, Head of Development

AcknowledgementsWorking together with collaborators and contributorsNVGH Enteric Fever Development Project TeamsFrancesca Micoli, Massimiliano GaviniSimona Rondini, Luisa LanzilaoGiulia BernardiMae Shieh, Breda Rogulj Allan Saul, Giorgia Scapecchi

Technical DevelopmentVito Di Cioccio Antonito Baccante, Emilia Cappelletti, Martina Carducci, Anna Maria Colucci, Graziella Di Salvo, Carlo Giannelli, Giulia Iannello, Filipe Marques, Federico Pippi, Ivan Pisoni, Silvia Sanzone, Luigi SollaiRegulatory Affairs and Clinical DevelopmentAudino Podda Alessandra Anemona, Jochen Auerbach, Venere Basile, Qasim Khan, Elisa Marchetti, Michela Squaglia

Novartis Vaccines and DiagnosticsP. Costantino (Vaccine Chemistry), R. RappuoliTechnical Development; Toxicology; Regulatory Affairs; Clinical Serology; Protocol Review Committee; Data Safety Management Board; Biostatics Clinical Data Management, Pharmacovigilance

cGMP ManufacturersGenIbet Biopharmaceuticals (Portugal)Areta International (Italy)

Clinical PartnersVolunteers, their families & trial site staff ofAga Khan Univ, Pakistan King Edward Memorial Hospital, India Research Institute for Tropical Medicine, PhilippinesCenter for Evaluation of Vaccines, Belgium

CollaboratorsCVD - Univ Maryland BaltimoreNIBSCNICHD/NIH Oxford UniversityUniv CapetownUniv SienaUniv TriesteWellcome Trust Sanger Institute

External FundingSclavo Vaccines Association with grants from Fondazione Monte dei Paschi Regione ToscanaEU 7th Framework - ADITECWellcome Trust Strategic Award

14 | NVGH 5th Anniv | LB Martin | 22 Feb 2013 | NVGH's model in action | Business Use Only

Page 15: Development of a conjugate vaccines for ... - Take on Typhoid · 8th International Conference on Typhoid Fever and Other Invasive Salmonelloses Laura B. Martin, Head of Development

Think what is possible

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