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
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
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