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