Streptococcus pneumoniae Evades the
Immune System by Subverting Host Defense:
Developing a New Vaccination Target
Rotation Talk – Janoff LabKristin Shotts
November 23, 2011
Infections Caused by Streptococcus pneumoniae
• Colonizatio
n
• Otitis media
• SinusitisPneumonia
• Meningitis
• Endocarditis
• Arthritis
• Bacteremia/sepsis
Mucosal
Systemic
• IgA1 antibodies are generated in the mucosa that bind specifically to the polysaccharide capsule of S. pneumoniae
• S. pneumoniae bacteria have over 90 different serotypes, which are the current targets for vaccinations
Host response to S. pneumoniae by IgA
Ref 1
Mechanism of Killing by IgA1-Mediated Opsonization
Ref 3
Macrophage
Ref 2
KILL
Ref 3
Macrophage
Ref 2
KILL
• Highly conserved protease across pneumococcal serotypes
IgA1 Protease as a Vaccination Target
IgA1PRef 4
Enhanced Adherence to and Colonization of Lung Epithelium
Ref 3
IgA1 protease
Macrophage
Ref 2
KILL
IgA1PRef 4
NO KILL
Macrophage
Hypothesis:
Ref 3
Macrophage
Ref 2
KILL
IgG (from patient sera)
4
Capsule-specific IgG in patient sera will bind to IgA1 protease to inhibit IgA1 cleavage
• Optimize an ELISA assay to detect IgA1 cleavage using IgA1 protease purified from H. influenzae and four strains of S. pneumoniae.
• Determine the frequency of IgA1 protease-specific IgG generation in 23 patients’ sera after acute and 2-4 weeks post S. pneumoniae infection vs. 46 control subjects.
Specific Aims
Immunoglobulin Structure
Ref 5
kappa
CH3
CH2
CH1
Fc region
ELISA to Detect IgA1 Cleavage
IgA-CH3 specific Capture Ab
Cleaved IgA1+ IgA1 ProteaseAPAPAP
Kappa specific-AP detector AB
Uncleaved IgA1
kappa
CH3
IgA1 Cleavage by H. influenzae Protease
Detected using SDS-PAGEProtease Conc. (ug/mL)
Heavy chain
Cleaved Heavy chains
IgA1 alone 42.8 14.3 4.28 1.43 0.428 0.143 Protease alone
2025
37
5075
150250
15
100
Light chain
Optimized ELISA Detects IgA1 Cleavage
• IgA1 concentration decreases 25 fold in the presence of IgA1 protease, as detected by the ELISA• Addition of IgG does not interfere with the assay
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
kappa-AP
IgA1
Con
cent
ratio
n (O
D)
Detector Antibody
Cleavage of IgA1 by H. influenzae proteaseIgA1 alone
IgA1 + protease
IgA1 + protease + IgG
Concentrations:
IgA1: 400 ug/mL
IgA1P: 42.8 ug/mL
IgG: 2240 ug/mL
S. Pneumoniae Bacterial Strain Information
• Characterize the phenotype of the S. pneumoniae strains using PCR and gel electrophoresis
Wild Type or Mutant
Strain Number F2 + rev Fragment length
F3 + revFragment length
IgA1P+ 98.086 None 850 bp
IgA1P- 98.087 1700 bp None
IgA1P+ 98.088 None 850 bp
IgA1P- 98.089 1700 bp None
IgA1P+ (purified DNA)
D39 None 850 bp
REV
F2
F3
erm
iga
IgA1 protease gene diagramMade by: Ron Gill
Determination of Strain Phenotype by PCR
151712001000800700600
500400
300
200
100
DNA ladder
86(+)
87(-)
88(+)
89(-)
D39 (+)
86(+)
87(-)
88(+)
89(-)
D39 (+)
Lanes 6-10F2 + rev primer set
Lanes 12-16F3 + rev primer set DNA
ladder
• The F2 + rev primer set generated IgA1P- fragments in 2 strains and the F3 + rev set generated IgA1P+ PCR fragments in 2 strains and the positive control.•There are two mutant and two wild type S. pneumoniae strains.
REV
F2
F3
erm
iga
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
98.086 (+) 98.087 (-) 98.088 (+) 98.089 (-)
IgA1
Con
cent
ratio
n (O
D)
Bacteria Strain
Cleavage of IgA1 by S. pneumoniae Protease Detected by Kappa-AP Conjugate
IgA1 alone
IgA1 + protease
ELISA Detects IgA1 Cleavage by Wild Type IgA1P S. pneumoniae
Strains
Concentrations:
IgA1: 40 ug/mL
IgA1P: 1/10 dilution
• Wild type (IgA1P+) strains show a 4- 7 fold reduction in optical density (OD), indicating IgA1 is being cleaved• The OD for mutant (IgA1P-) strains remains constant
Neutralization using IgG Purified from Patient Sera
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
IgA1 alone IgA1 + P 1/625 1/125 1/25 1/5
IgA1
Con
cent
ratio
n (O
D)
Patient IgG Dilution
Neutralization Assay with Acute and Conv. Patient Purified IgG using Kappa-AP Conjugate
92.077 Acute
92.085 Conv
95.30 Acute
95.112 Conv
• Inhibition of IgA1 cleavage is seen by protease-specific IgG purified from acute and 2-4 weeks post infection sera samples drawn from the same patient• The other patient appears to be a non-responder
• The optimized, neutralization ELISA can detect inhibition of IgA1 cleavage in the presence of IgA1P-neutralizing IgG purified from patients infected with S. pneumoniae.
• The next steps are to analyze the rest of the patient samples using the neutralization assay as well as to express IgA1 protease in E. coli, in order to characterize where the patient IgG binds to the protein to inhibit cleavage of IgA1.
Conclusions
Acknowledgements
• Dr. Edward Janoff, M.D.• Dr. Ronald Gill, P.H.D.• Dr. Dan Frank, P.H.D.• Jeremy Rahkola• Claire Gustafson• Leah Feazel• Diana Ir• Jana Palaia
References
1. http://www.chori.org/Principal_Investigators/Test_Samuel_T/test_research.html2. http://www.immunopaedia.org.za/index.php?id=7503. http://en.wikipedia.org/wiki/File:Macrophage.png4. http://www.sayers.staff.shef.ac.uk/igap/5. http://www.biology.arizona.edu/immunology/tutorials/antibody/structure.html