Can we eliminate important bacterial pathogens with protein-‐based vaccines?
Engineered antigens derived from bacterial transferrin receptors
Dr. Tony Schryvers (University of Calgary) Vaccine Innovation Conference May 26, 2015
Host-‐Restricted Upper Respiratory Tract Pathogens
Gram-‐Negative Species
• Neisseria meningitidis • Haemophilus influenzae • Moraxella catarrhalis
Gram-‐Positive Species
• Streptococcus pneumoniae
-‐ Responsible for 20-‐25% of deaths < 5 yrs
-‐ Cause of most common bacterial infection
Common Features • highly host-‐adapted & host
specific • URT only reservoir –
transmission by respiratory route
• are naturally transformable • ‘horizontal’ gene transfer
contributes to genetic and antigenic variation
• challenge to develop a broadly protective vaccines
Non-Invasive Invasive
Bacterial Respiratory Tract Pathogens
• Responsible for a spectrum of invasive and non-‐invasive infections
• Colonization precedes infection
• Bacteria causing invasive infection possess polysaccharide capsule
S. pneumoniaeN. meningitidisM. catarrhalisH. influenzae
nontypeable
Sinusitis
Ear Infection
nontypeable
Pneumonia
Meningitis
Pneumonia
type b
type b
type b
Sepsis
0
23
45
68
90
1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009
vaccine
Type b H. influenzae (UK) 1992
S. pneumoniae PCV7 – 7 serotypes
(Calgary) 2002
Conjugate Capsular Vaccines
Reduce Infection by Targeted Serotypes
Vaccine serotypes
Conjugate Capsular Vaccines
• Designed to prevent invasive infection • Studies demonstrate that systemic vaccination
results in: – elimination of targeted serotypes from mucosal
carriage – disease reduction in non-vaccinated populations
0%
5%
10%
15%
20%
Spring '03 Spring '04 Spring '0512 months 18 months4.5 years
Carriage % Children – vaccine types
0
15
30
45
60
1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009
All SerotypesPCV7 Serotypesnon-PCV7 Serotypes
Invasive Disease Adults > 65
Conjugate Capsular Vaccines
• Highly specific immunity • reduction in carriage by vaccine serotypes coupled
with increase in non-vaccine serotypes
0%
5%
10%
15%
20%
Spring '03 Fall '03 Spring '04 Fall '04 Spring '05 Fall '0512 months 18 months 4.5 years
0%
5%
10%
15%
20%
Spring '03 Spring '04 Spring '05
12 months 18 months4.5 years
% Children – vaccine types % Children – non- vaccine types
Calgary – CASPER
Conjugate Capsular Vaccines
• Highly specific immunity - reduction in disease by vaccine serotypes coupled with increase in non-vaccine serotypes
0
75
150
225
300
2000 2001 2002 2003 2004 2005 2006 2007 2008 2009
PCV719AOther Vaccine PreventableNon-Vaccine PreventableUnknown
vaccine
Canada IMPACT
Conjugate Capsular Vaccines
PROS • Effective reduction in disease by strains expressing targeted
polysaccharide capsules • Reduction in colonization led to decreased transmission and reduction in disease in non-‐immunized (herd effect)
CONS • Not effective against other capsular types – may result in disease by other types (capsule switching)
• Not effective for non-‐immunogenic capsular types (group B meningococci)
Development of broadly cross-‐protective vaccines may need to target protein antigens
Iron Homeostasis: Role of Transferrin (Tf)
• Tf shuttles iron within body to cells requiring iron (Fe) • Tf is normally only partially saturated • Tf keeps the level of free Fe below that needed for
growth of pathogens
• Tf is a critical source of iron during invasive infection
• There is limited information regarding iron homeostasis on mucosal surfaces
• Tf known to be critical iron source on the mucosl surface for some bacteria (Neisseria gonorrhoeae, Actinobacillus pleuropneumoniae)
Human Neisseria meningitidis meningitis, sepsis
Neisseria gonorrhoeae gonorrhea
Haemophilus influenzae meningitis, sepsis
Haemophilus influenzae (NT) otitis media, COPD, pneumonia
Moraxella catarrhalis otitis media, COPD, pneumonia
Cattle Sheep Goat Mannheimia haemolytica BRD (shipping fever)
Pasteurella multocida BRD
Histophilus somni BRD, invasive disease
Moraxella bovis pinkeye
Pasteurella trehalosi sepsis / RD
Pig Actinobaccilus pleuropneumoniae pneumonia
Haemophilus parasuis Glasser’s disease (invasive disease & pneumonia)
Actinobacillus suis sepsis, hemorrhagic/embolic lesions of lung
Bacterial Transferrin Receptors
FbpA
TbpA
FeFbpB/C
TonB
ExbBExbD
TbpB
Tf
Receptor-‐mediated iron acquisition from transferrin (Tf)
• Surface lipoprotein,TbpB (transferrin binding protein B) captures Fe loaded Tf
• TbpB transfers Fe-‐Tf to TbpA, an integral outer membrane protein
• TbpA removes Fe from Tf and transports Fe across the outer membrane
• FbpA (ferric binding protein A) shuttles iron to inner membrane complex
• FbpBC inner membrane complex transports Fe into the cell
TbpB Vaccines – A Brief History
1990: patent on Tf/Lf receptors filed
Meningococcal Vaccines: • 1990 Pasteur Merieux (currently Sanofi Aventis) licensed Tbps • 1990-‐1998 native TbpB shown to be immunogenic and protective in mice/rabbits, 3 TbpB variants provide broad coverage
• 1998 human Phase I clinical trial (w/o adjuvant) -‐ disappointing results • 2001 license abandoned
Animal Vaccines: • recombinant proteins tested directly in pigs and cattle Key Relevant Research Findings: • human gonococcal and pig experiments demonstrate Tbps critical for survival and disease
2001 -‐2006: noncompete clause, insufficient funding 2008: • AHFMR Team Grant & sabbatical, structure-‐based vaccine design
TbpB Vaccines - Rationale for Structure-based Antigen Engineering Approach
Hypotheses:
• host Tf binds to TbpB during systemic immunization & interferes with development of optimal protective response • explains poor performance in Phase I trial & only moderate success for animal vaccines
• solution: design non-‐Tf binding antigens
• TbpBs bind to same region of Tf requiring conserved binding interface, antigenic variation involves immunodominant ‘decoy’ B-‐cell epitopes • solution: engineer antigens with enhanced ability to induce cross-‐protective immune response
• remove decoy epitopes and preserve conserved binding interface
barrel barrel
barrel
barrel
handle
handle
C-‐lobe
C-‐lobe
N-‐lobe
N-‐lobe
anchorpeptide
TbpB Structure (porcine pathogen -‐ A. pleuropneumoniae)
Trevor Moraes
Site-‐Directed Mutants
Single mutations designed to reduce/eliminate Tf binding.
Panel A: Targeted residues
Panels B – G: Superposed mutant and
wild-‐type structures
Mutations had no effect on overall structure
Site-‐Directed Mutants
Single mutation results in dramatic loss in Tf binding
Affinity binding constants for wild-type andmutant TbpBs
Protein Mutation Loop Kd MethodApH49 TbpB WT 55 nM ITCApH49 TbpB F171A L8 NDB ITCApH49 TbpB WT 44 nM SPRApH49 TbpB F171A L8 TBD SPRApH87 TbpB WT 60 nM SPRApH87 TbpB Y95A L3 585 nM SPRApH87 TbpB Y121A L5 203 nM SPRApH87 TbpB Y174A L8 8.9 uM SPRApH87 TbpB R179E L8 6.1 uM SPRAsH57 TbpB WT 120 nM SPRAsH57 TbpB F63A L1 326 nM SPRAsH57 TbpB F152A L5 495 nM SPRHp5 TbpB WT 21 nM BLIHp5 TbpB Y93 A L3 TBD BLIHp5 TbpB Y117A L5 TBD BLIHp5 TbpB(e4535)
Y167A L8 40uM BLI
Hp5 TbpB W176A L8 TBD BLIHp5 TbpB(e4597)
Y167A,W176A
L8 NDB BLI
TBD – To Be DeterminedNDB – No Detectable BindingITC – isothermal titration calorimetrySPR – surface plasmon resonancesBLI – biolayer interferometry
FIGURE 22
Days of Survival
15
6
12
Control Native TbpB
Mutant TbpB
Porcillis Glasser
9
3
Mild or no symptoms or pathologyModerate to severe symptoms and pathology
Survival: Clinical Symptoms:
Transferrin Receptor-‐based Vaccine
Engineered mutant TbpB is a superior antigen
Mutant TbpBNative TbpBPorcillis GlasserControl
0 24 72 96 120 144 168 312 33648
Hours after challenge
% Survival
20
40
60
80
100
CEAC
AM1
infecteduninfected
wildtype
A B
huCEACAM1 N. meningitidis
Colonization of Humanized Transgenic Mice
DAPI
Scott Gray-‐Owen
Men C Conjugate Vaccine
Prevents ColonizationTbpB and TbpB C-‐lobe
Prevents Colonization
Factor H binding protein protects against sepsis but not colonization
PorA protects against sepsis but not colonization.
NHBA partially protects against sepsis but not colonization
NadA protects against sepsis and colonization
Prevention of Colonization is not a Common Feature of Protein Antigens
Invasive disease challenge with H44/76FHbp match
0 12 24 36 480
25
50
75
100
Time (h)
Per
cent
sur
viva
l
AdjuvantBexsero
Invasive disease challenge with SK016PorA match
0 12 24 36 480
25
50
75
100
Time (h)
Per
cent
sur
viva
l
AdjuvantBexsero
Invasive disease challenge with S3446NHBA match
0 12 24 36 480
25
50
75
100
Time (h)
Per
cent
sur
viva
l
AdjuvantBexsero
Invasive disease challege with BuFa 02/03NadA match
Time (h)
Per
cent
sur
viva
l
0 12 24 36 480
25
50
75
100AdjuvantBexsero
Bexse
ro
Adjuva
nt
10
100
1000
detection limit
CFU
per
ani
mal
3 day colonization challenge with H44/76FHbp match
Bexse
ro
Adjuva
nt
10
100
1000
detection limit
CFU
per
ani
mal
3 day colonization challenge with SK016PorA match
Bexse
ro
Adjuva
nt
10
100
1000
detection limit
CFU
per
ani
mal
3 day colonization challenge withS3446NHBA match
Bexse
ro
Adjuva
nt
10
100
1000
detection limit
CFU
per
ani
mal
3 day colonization challenge with BuFa 02/03 APgroup W, NadA match
Engineered TbpB Vaccines
Advantages: • Tf receptors are essential for survival on the mucosal surface (N.
gonorrhoeae, A. pleuropneumoniae) • Engineered TbpB antigens are superior in inducing a protective
immune response in the native host (H. parasuis challenge experiments)
• TbpB (and TbpB C-‐lobe) are capable of preventing colonization in a humanized mouse model (unlike antigens from commercial vaccines)
• Surrogate host-‐pathogen systems can provide proof of concept Question:
• Could a TbpB-‐based vaccine be used eradicate receptor-‐containing bacteria?
TbpB diversity transcends species barriers – single vaccine for the three porcine pathogens.
Phylogenetic groups defined by structural features and interaction with Tf
Cross-‐reactivity analysis predicts small number of engineered antigens required for broad cross-‐protection.
Porcine Pathogen TbpBs Sequence Diversity of TbpBs from Porcine Pathogens
Immunization Experiments in Pigs
From Strain H49
N-‐lobe induces more ‘Group/strain specific’ antibody response.
C-‐lobe induces broadly cross-‐reactive immune response.
Predicts that three representative mutant TbpBs would be capable of inducing a broadly cross-‐reactive and cross-‐protective response.
-‐ Group 3-‐ Group 3-‐ Group 2-‐ Group 1
Meningococcal Vaccine -‐ Sequence/Structural Diversity
Comprehensive evaluation of sequence diversity – intact TbpB & C-lobe.
Two TbpB isotypes (Group 1 vs Group 2-4).
Two antigens selected for immunization experiments (circled in red)
Representative variants selected for analysis of cross-reactivity (arrows).
Paul Adamiak
C-‐lobe Diversity
Intact TbpB Diversity
Sequence Diversity – Evaluation of Cross-‐reactivity
Immunization with TbpBs or C-lobes provides reactivity against diverse representative TbpBs.
Antigen:
Tbp Strain
Jamie Fegan
TbpB-based Meningococcal Vaccine
Can we eliminate important pathogens?
• TbpB present in some commensal Neisseria sp. and in N. gonorrhoeae -‐ potential reservoir for vaccine escape
• overall diversity of Neisseria TbpBs modestly greater than meningococcal TbpBs (i.e. limited number of engineered TbpBs/C-‐lobes required)
• commensal Neisseria with and without TbpB are similar, unlikely to be dependent upon TbpB for iron (eliminating TbpB won’t eliminate commensal Neisseria)
• require systematic approach for evaluating ability to induce complete cross-‐protection -‐ integrated vaccine evaluation pipeline
Global collection of disease isolates Bioinformatics
analysis of antigen diversity
Library of antigenic variants
Structural studies
Bacterial killing (SBA)
+ -‐
URT microbiome Cloning & Expression (SG)
ELISA Assay
immunize
serum
generate strain library
strain library
Engineered antigen
Challenge humanized mice
Integrated Vaccine Evaluation Pipeline
Commercialization
US Provisional Patent Applications
• Dec, 2013 • June, 2014 • October, 2014
PCT Patent Application
• Dec 2014
Exploring Partnerships/Licensing Agreements
Meningococcal Transferrin and Lactoferrin Receptors in
Colonization, Infection and Disease Prevention
ALMA Alberta Livestock
and Meat Agency Ltd.
Development of a Broad-‐Spectrum Porcine Vaccine for Bacterial Respiratory
Infections
University of Passo Fundo (Brazil) Rafael Frandoloso
University of Leon (Spain) Cesar Gutierrez-‐Martin Elias Ferri-‐Rodriguez
Sonia Martinez-‐Martinez
Genome Alberta ALMA
Disease Reduction in Cattle by Elimination of
Colonization by Pathogens