UTILIZATION OF MODERN SCIENTIFIC TECHNOLOGIES TO PRODUCE COMPATIBLE ANTIGENS FOR SWINE VACCINES

Jeffrey E. Galvin, PhD

Zoetis

December 2015

1

A LONG HISTORY WITH PCV2 AND M. HYOPNEUMONIAE (M.HYO)

• RespiSure®

• RespiSure One®

• Suvaxyn® MH

• Suvaxyn® MH-One

• Respifend®

• Suvaxyn® PCV

• Fostera® PCV

• Relsure® PCV

2

3

M. hyo medium components

Medium/Broth Base

Porcine serum

Yeast extract

Amino acid supplement

Carbohydrate

Sterile H2O

Antibodies: Specific for

any antigens that

source pigs were

exposed.

PCV2 antigen

Potency Assay Detection Issues Combination of Antigen Fluids

(antibody binding to PCV2)

TECHNICAL PROBLEM: ANTI-PCV2 ANTIBODIES IN M. HYOPNEUMONIAE ANTIGEN FLUIDS

M. HYO ANTIGEN AND PCV ANTIBODIES: WHERE ARE THEY AND HOW TO REMOVE THEM?

Fermentation

Centrifugation

Mhyo

100%

90%

10%

sup

Pellet (cells)

Anti-PCV

100%

99%

<1%

10%

90%

PCV2 COMPATIBLE ANTIGEN: M.HYO CELLS ONLYA SPECTACULAR FAILURE

Study -32929 LS Mean M. hyo Lung Lesion Results

Treatment Description Percent Lung

Lesions LS Mean

Range Percent

Lung with Lesions

T01 Placebo 11.1% 1.4 – 30.0%

T02 M. hyo (low) + cPCV 14.2% 1.2 – 67.5%

T03 M. hyo (med) + cPCV 11.3% 0.5 – 41.0%

T04 M. hyo (high) + cPCV 12.7% 3.8 – 54.0%

Data on file, Study Report No. 3127W-60-10-929, Zoetis Inc.

M. hyo Antigen Strategy: Collect Cells via Liquid/Solid Separation

All in vivo work was conducted after ethical review, and in compliance with local, state, and national

regulations per CP 109 on animal care and use

ALTERNATIVE STRATEGIES TO ADDRESS ANTI-PCVANTIBODIES

Approach Comments & Challenges

Remove antibodies from swine serum

•Treating swine serum may have an effect on antigen profile. •Moving away from the conventional Mhyo antigen production model.

Grow Mhyo without serum Absolute requirement of serum for Mhyo growth.

Alternative source of serum to grow Mhyo

•Safety concern of using heterologous serum.•Unknown effect of changing serum on efficacy of antigen.•Moving away from the conventional production model.

Remove antibodies from antigen fluid

•Protein A (expensive and need to remove cells)•Filtration

Other

•CDCD swine serum•Act-O-Vial (2 mL only) •Two bottle format (inconvenience of use)•Alternative potency assay

WESTERN BLOT ANALYSISM. HYO P65 M. HYO P46 M. HYO P97

3

188

98

49

38

28

1714

62

6

Std. 1 2 3

3

188

98

49

38

28

17

14

62

63

188

98

49

38

28

17

14

62

6

Std. 1 2 3 Std. 1 2 3W

hole

Flu

id

Cell

Fre

e S

up

ern

ata

nt

Cell

Pelle

t (1

0x)

Wh

ole

Flu

id

Cell

Fre

e S

upern

ata

nt

Whole

Flu

id

Cell

Fre

e S

upern

ata

nt

Cell

Pelle

t (1

0x)

Cell

Pelle

t (1

0x)

10%

90%

PROTEIN A

• Virulence factor of Staph aureus (interferes with opsonization and

subsequent phagocytosis)

• Immunoglobulin binding activity described in 1940

• Protein described and named in 1964 (found in “fraction A”)

• Large scale purification from “excretor” strains in 1970’s

• Gene cloned and expressed in E. coli in 1983

• rProtein A coupled to an immobile phase (i.e., sepharose) became

available in the 1990s1

Images from:

1. Graille, M. et al. Proc. Natl. Acad. Sci. USA, 5399–5404 (2000).

2. Vila, J. et al. Proc. Natl. Acad. Sci. USA, 14812-14816 (2003).

2

PROTEIN A – BINDING CHARACTERISTICS

SpeciesAntibody

Subtype

Binding

Affinity

Human IgG1 +++

IgG2 +++

IgG3 -

IgG4 +++

IgA +/-

IgD +/-

IgE +/-

IgM +/-

Porcine ++

Bovine ++

Canine ++

Caprine -

Equine ++

Ovine +/-

Mouse IgG1 +

IgG2a +++

IgG2b +++

IgG3 ++

IgM +/-

Rat -

Relative Binding Affinity of Protein A for Various

Antibody Species and Subclasses1 2

References:

1. Richman et al., The binding of Staphlococci protein A by the sera of different animal species. J.

Immunol. 128:2300-2305. 1982.

2. Harlow, E. and D. Lane (Eds). Antibodies: A Laboratory Manual. First Edition. Published by

Cold Spring Harbor Laboratory Press. 1988.

OH

HUMAN BIO-THERAPEUTICS THAT UTILIZE PROTEIN A

Antibody Brand name Company Approval date Type TargetIndication

(Targeted disease)

Tocilizumab ( or

Atlizumab )

Actemra and

RoActemra2010 Humanised Anti- IL-6R

Rheumatoid

arthritis

Brentuximab

vedotinAdcetris 2011 Chimeric CD30

Anaplastic large

cell lymphoma

(ALCL) and

Hodgkin lymphoma

Ofatumumab Arzerra 2009 Human CD20

Chronic

lymphocytic

leukemia

Bevacizumab Avastin Genentech/Roche 2004 humanized

Vascular

endothelial growth

factor (VEGF)

Colorectal cancer,

Age related

macular

degeneration (off-

label)

Belimumab Benlysta GlaxoSmithKline 2011 humaninihibition of B- cell

activating factor

Systemic lupus

erythematosus

Tositumomab Bexxar GlaxoSmithKline 2003 murine CD20Non-Hodgkin

lymphoma

Alemtuzumab Campath Genzyme 2001 humanized CD52

Chronic

lymphocytic

leukemia

Certolizumab

pegol[19] Cimzia UCB (company) 2008 humanizedinhibition of TNF-α

signalingCrohn's disease

Cetuximab Erbitux

Bristol-Myers

Squibb/Eli

Lilly/Merck KGaA

2004 chimericepidermal growth

factor receptor

Colorectal cancer,

Head and neck

cancer

Trastuzumab Herceptin Genentech 1998 humanized ErbB2 Breast cancer

Adalimumab Humira Abbot 2002 humaninhibition of TNF-α

signaling

Several auto-

immune disorders

Canakinumab Ilaris Novartis 2009 Human IL-1β

Cryopyrin-

associated periodic

syndrome (CAPS)

http://en.wikipedia.org/wiki/Monoclonal_antibody_therapy#FDA_approved_therapeutic_antibodies

32967: STUDY DESIGN (MHYO ANTIGEN CHARACTERIZATION)

Goal: Identify an efficacious M.hyo process that is PCV2 compatible

● Treatment: 1-dose, 3 weeks of age, M. hyo. challenge 6 weeks, 16 animals/group

T01 Negative Control

T02 M.Hyo (whole fluid)—1X

T03 M.Hyo (UF Concentration)—1X

T04 M.Hyo (UF Concentration + Centrifugation)—1X

T05 M.Hyo (UF Concentration + Centrifugation)—2X

T06 M.Hyo (Centrifugation)—1X

T07 M.Hyo (Centrifugation)—2X

T08 M.Hyo (Centrifugation + Heat)—1X

T09 M.Hyo (Supernatant)—1X

T10 M.Hyo (Supernatant followed by Protein A Treatment)—1X

T11 RespiSure One® (Positive Control)—1X

All vaccines blended with PCV2 (except T01 and T11)

T02 through T10 originated from same M.hyo fermentation

STUDY 32967: MHYO EFFICACY (MHYO ANTIGEN CHARACTERIZATION STUDY)

UF + Cell Pellet

UF + Cell Pellet

(2x Antigen)

Cell Pellet

Cell Pellet

(2x Antigen)

Cell Pellet + Heat

UF

Respisure One ®

Cell Free Supernatant

Whole Fluid

Cell Free

Supernatant

+ Protein A

PCV2 Compatible Efficacious

Antigen Characteristics

1. No mycoplasma cells

• “Non-Bacterin” Approach

• Reduced Overall Biomass

• Safety

• Targeted Immune Response

2. Patented* antigen composition with low levels of anti-PCV2 and other

antibodies

• Compatible with PCV2 antigen

• Further reduction of extraneous proteins that can affect efficacy

and safety

A NEW PARADIGM FOR M. HYOPNEUMONIAE ANTIGEN

*US Patent No. 9,120,859 B2

SUMMARY OF EFFICACY AND SAFETY STUDIESStudy Intent # Doses Age

(weeks)

Outcomes Study #

Efficacy - PCV2 1 3 • Aid in preventing viremia

• Aid in preventing lymphoid depletion

• Aid in preventing tissue colonization

• Aid in reducing virus shedding

B822R-US-12-046

Efficacy - MH 1 3 • Aid in reducing enzootic pneumonia B823R-US-13-136

Safety 1 3 • Low injection site reaction rate (1.1%)

• All ISR resolved by day 3

• No anaphylactic rxns

B921R-US-12-009

Safety 2 3 & 5 • Low injection site reaction rate (0.6 to 2.0%)

• 99.6% of animals had no ISRs at 2 days post -

immunization (remaining (n=2) resolved between

day 2 & 6)

• No anaphylactic rxns

B921R-US-12-113

Efficacy - MH 2 3 & 5 • Aid in reducing enzootic pneumonia B822R-US-12-112

Efficacy - PCV2 2 3 & 5 • Aid in preventing viremia

• Aid in preventing lymphoid depletion

• Aid in preventing tissue colonization

• Aid in reducing virus shedding

B822R-US-12-111

EU MDA Efficacy

– PCV2

1 3 • Significant reduction of viremia

• Significant reduction in PCV2 colonization of

lymphoid tissue

• Significant reduction in PCV2 fecal shedding

B828R-US-13-239

EU MDA Efficacy

– MH

1 3 • Significant reduction of enzootic pneumonia B828R-US-13-137

One-dose vaccine with flexible, 2-dose option to help protectagainst both PCV2 and M. hyo

Same protection against PCV2 and M. hyo as you expect from FOSTERA® PCV & RespiSure-ONE® but is not a mix of the two

Uniquely developed to balance antigen levels and target theanimal’s immune response

Ease of administration, no measuring or mixing required