Factor H binding protein
Christoph Tang
Sir William Dunn School of Pathology
University of Oxford
TpmA
- elicits killing +/- functional antibodies
i)
ii)
iii)
iv)
v)
vi)

expressed by all strains
sequence is conserved
un...
fHbp- a key
meningococcal antigen

TpmA
The perfect meningococcal Antigen
- elicits killing +/- functional antibodies
i)
i...
fHbp- a key
meningococcal antigen

Tan L et al. N Engl J Med 2010
- expressed by all strains?

Yes, for > 99% of UK MenB isolates
923 UK isolates
ST-11 T366 frame shift
cc162 A650 frame sh...
- sequence conservation?

923 UK isolates
V1

101 fHbps

Family B

V2
Family A
Masignani et al., J Exp Med 2003
Fletcher e...
- sequence conservation?

PorA

fHbp
dark red is most conserved
blue is most variable going via grey

many different fHbps
- sequence conservation?

923 UK isolates
V1

101 fHbps

Family B

V2
Family A
Masignani et al., J Exp Med 2003
Fletcher e...
- sequence conservation?
Novartis

Pfizer

x1 fhbp
+ other antigens

x2 lipidated fhbps

1
144
110
238
312
89
2
78
306
61
...
- understand structure:function
CFH SCR6/7

Glu 304
Lys 351
His 371
Glu 341
Glu 283

Mascioni et al., J Biol Chem 2009
Can...
- understand structure:function

Ligand mimicry
Schneider et al. Nature 2009
- understand structure:function
GAGs*
CRP

C3b

1

CFH

2

3

4

5

6

7

GAGs*
Sialic acid
C3b/d

GAGs*
C3b

8

9

10

11...
- understand structure:function
GAGs*
CRP

C3b

1

2

3

4

5

CFH

6

7

GAGs*
Sialic acid
C3b/d

GAGs*
C3b

8

9

CFHR3
...
- understand structure:function
GAGs*
CRP

C3b

1

2

3

4

5

CFH

6

7

GAGs*
Sialic acid
C3b/d

GAGs*
C3b

8

9

CFHR3
...
- understand structure:function
GAGs*
CRP

C3b

1

2

3

4

5

6

7

GAGs*
Sialic acid
C3b/d

GAGs*
C3b

8

9

10

11 12

...
- inactivate function

fHBP

Hide immunogenic epitopes

Less opportunity for
‘blocking’ antibodies

SCR 6 and 7
Reduce imm...
- inactivate function
Amino acid substitutions generate
non-functional fHbps

Johnson et al. PLoS Pathog. 2012
- inactivate function
Amino acid substitutions generate
non-functional fHbps

Schneider et al.
Nature 2009

B

V1

double ...
- inactivate function

Host modification

human
mouse
human
mouse
human
mouse

fHbp

Murine SCR6/7
Human SCR6/7
- humanisi...
- inactivate function

X
C3b

Host modification
C3b

SAP/GAGs

mfH

mfH
SAP/GAGs

? C3b

? SAP/GAGs

extra
hfH

chimeric
f...
- easy to manufacture

Distribution of fHbps in MRF collection
2010-2012 (n=916)

10%

34%

v1

56%

v2

v3

no V2 fHbp co...
- easy to manufacture
fHbp V2 is inherently unstable
Trypsin digestion

Differential Scanning calorimetry (DSC)

V1

V2

V...
- easy to manufacture
fHbp V2 is inherently unstable
Trypsin digestion

Differential Scanning calorimetry (DSC)
Wild-type
...
- easy to manufacture
fHbp V2 is inherently unstable
5 of 10 single amino acid substitutions
de-stabilise the antigen
M6
R...
- ? elicits herd immunity

N. meningitidis
Temp. (°C)
CssA
fHbp

(alpha-2,3-sialyltransferase)

30

37

42

Temperature ac...
-

What next for fHbp?
Vaccine

enhanced immunogenicity
cross variant/family protection
non-functional fHbps

-

Serum bac...
-

What next for fHbp?
Vaccine

enhanced immunogenicity
cross variant/family protection
non-functional fHbps
Naturally occ...
-

What next for fHbp?
Vaccine

enhanced immunogenicity
cross variant/family protection
non-functional fHbps
-

What next for fHbp?
Vaccine

enhanced immunogenicity
cross variant/family protection
non-functional fHbps

Host suscept...
Susan Lea
Steven Johnson
Joe Caesar
Phil Ward
Rachel Everitt
Martin Maiden
Odile Harrison

- University of Oxford

Ray Bor...
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Factor H binding protein and vaccine approaches

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Professor Christoph Tang's presentation at Meningitis Research Foundation's 2013 conference, Meningitis & Septicaemia in Children & Adults

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  • Binding at SCR67 and E283/304Previous work in our lab has determined the crystal structure of fHbp shown here in turquoise, green and yellow in complex with fH shown in blueWe have shown that fHbp binds to a specific region of fH with high affinity, There are amino acid residues in fHbp which appear to be crucial, Glutamate at 283 and 304As fHbp binds to fH with high affinity, we hypothesised that this could affect its immunogenicity as a vaccine by (1) hiding immunogenic epitopes within the binding site and (2) also result in the development of autoantibodies as the immune system sees fH in complex with a foreign antigenI thus generated modified fHbps with mutations at these critical residues to assess whether these potentially non-fH binding proteins are immunogenic
  • Binding at SCR67 and E283/304Previous work in our lab has determined the crystal structure of fHbp shown here in turquoise, green and yellow in complex with fH shown in blueWe have shown that fHbp binds to a specific region of fH with high affinity, There are amino acid residues in fHbp which appear to be crucial, Glutamate at 283 and 304As fHbp binds to fH with high affinity, we hypothesised that this could affect its immunogenicity as a vaccine by (1) hiding immunogenic epitopes within the binding site and (2) also result in the development of autoantibodies as the immune system sees fH in complex with a foreign antigenI thus generated modified fHbps with mutations at these critical residues to assess whether these potentially non-fH binding proteins are immunogenic
  • Binding at SCR67 and E283/304Previous work in our lab has determined the crystal structure of fHbp shown here in turquoise, green and yellow in complex with fH shown in blueWe have shown that fHbp binds to a specific region of fH with high affinity, There are amino acid residues in fHbp which appear to be crucial, Glutamate at 283 and 304As fHbp binds to fH with high affinity, we hypothesised that this could affect its immunogenicity as a vaccine by (1) hiding immunogenic epitopes within the binding site and (2) also result in the development of autoantibodies as the immune system sees fH in complex with a foreign antigenI thus generated modified fHbps with mutations at these critical residues to assess whether these potentially non-fH binding proteins are immunogenic
  • Binding at SCR67 and E283/304Previous work in our lab has determined the crystal structure of fHbp shown here in turquoise, green and yellow in complex with fH shown in blueWe have shown that fHbp binds to a specific region of fH with high affinity, There are amino acid residues in fHbp which appear to be crucial, Glutamate at 283 and 304As fHbp binds to fH with high affinity, we hypothesised that this could affect its immunogenicity as a vaccine by (1) hiding immunogenic epitopes within the binding site and (2) also result in the development of autoantibodies as the immune system sees fH in complex with a foreign antigenI thus generated modified fHbps with mutations at these critical residues to assess whether these potentially non-fH binding proteins are immunogenic
  • Binding at SCR67 and E283/304Previous work in our lab has determined the crystal structure of fHbp shown here in turquoise, green and yellow in complex with fH shown in blueWe have shown that fHbp binds to a specific region of fH with high affinity, There are amino acid residues in fHbp which appear to be crucial, Glutamate at 283 and 304As fHbp binds to fH with high affinity, we hypothesised that this could affect its immunogenicity as a vaccine by (1) hiding immunogenic epitopes within the binding site and (2) also result in the development of autoantibodies as the immune system sees fH in complex with a foreign antigenI thus generated modified fHbps with mutations at these critical residues to assess whether these potentially non-fH binding proteins are immunogenic
  • Factor H binding protein and vaccine approaches

    1. 1. Factor H binding protein Christoph Tang Sir William Dunn School of Pathology University of Oxford
    2. 2. TpmA - elicits killing +/- functional antibodies i) ii) iii) iv) v) vi) expressed by all strains sequence is conserved understand structure:function inactivate its function elicits herd immunity easy/cheap to manufacture
    3. 3. fHbp- a key meningococcal antigen TpmA The perfect meningococcal Antigen - elicits killing +/- functional antibodies i) ii) iii) iv) v) vi) expressed by all strains sequence is conserved understand structure:function inactivate its function elicits herd immunity easy/cheap to manufacture MimA Most interesting meningococcal Antigen vaccine antigen pathogenesis host susceptibility
    4. 4. fHbp- a key meningococcal antigen Tan L et al. N Engl J Med 2010
    5. 5. - expressed by all strains? Yes, for > 99% of UK MenB isolates 923 UK isolates ST-11 T366 frame shift cc162 A650 frame shift cc286 fHbp deletion 4 have frame shifts 3 have a deletion
    6. 6. - sequence conservation? 923 UK isolates V1 101 fHbps Family B V2 Family A Masignani et al., J Exp Med 2003 Fletcher et al., Infect Immun 2004 Brehony et al. Microbiology 2009 V3 many different fHbps
    7. 7. - sequence conservation? PorA fHbp dark red is most conserved blue is most variable going via grey many different fHbps
    8. 8. - sequence conservation? 923 UK isolates V1 101 fHbps Family B V2 Family A Masignani et al., J Exp Med 2003 Fletcher et al., Infect Immun 2004 Brehony et al. Microbiology 2009 V3 many different fHbps
    9. 9. - sequence conservation? Novartis Pfizer x1 fhbp + other antigens x2 lipidated fhbps 1 144 110 238 312 89 2 78 306 61 236 276 37 249 254 295 318 4 5 313 303 298 62 14 309 220 307 258 10 299 13 311 297 292 86 87 210 305 15 68 118 310 109 302 19 83 119 16 106 296 308 24 25 301 95 147 23 104 22 202 34 21 102 300 30 174 29 293 212 294 45 185 59 85 94 84 160 47 304 10 5 8 11 18 22 23 32 35 60 103 162 167 174 198 213 226 254 269 364 461 865 1157 41/44 N/A V1 V2 V3 0 10 20 30 40 50 60 70 80 90 100 110 2008 data Family B Family A 120 many different fHbps some immunological cross reactivity within families Lucidarme et al. Clin Vacc Immunol 2010 some frequent peptides/correlate with ST
    10. 10. - understand structure:function CFH SCR6/7 Glu 304 Lys 351 His 371 Glu 341 Glu 283 Mascioni et al., J Biol Chem 2009 Cantini et al., J Biol Chem 2009 fHbp Schneider et al. Nature 2009
    11. 11. - understand structure:function Ligand mimicry Schneider et al. Nature 2009
    12. 12. - understand structure:function GAGs* CRP C3b 1 CFH 2 3 4 5 6 7 GAGs* Sialic acid C3b/d GAGs* C3b 8 9 10 11 12 13 14 15 16 17 Negative complement regulator - co-factor for fI cleavage of C3b - accelerates decay of C3bBb - binds fB multiple partners, multiple roles GWAS highlights CFH and CFHRs CFHRs antagonise CFH Understand molecular basis of host susceptibility 18 19 20
    13. 13. - understand structure:function GAGs* CRP C3b 1 2 3 4 5 CFH 6 7 GAGs* Sialic acid C3b/d GAGs* C3b 8 9 CFHR3 10 11 12 CFHR1 13 14 15 CFHR4 16 17 CFHR2 CFH locus multiple partners, multiple roles GWAS highlights CFH and CFHRs CFHRs antagonise CFH Understand molecular basis of host susceptibility Davilla et al. Nat Genet 2010 18 19 20 CFHR5
    14. 14. - understand structure:function GAGs* CRP C3b 1 2 3 4 5 CFH 6 7 GAGs* Sialic acid C3b/d GAGs* C3b 8 9 CFHR3 10 11 12 CFHR1 13 14 15 CFHR4 16 17 18 19 20 CFHR2 CFHR5 CFH locus multiple partners, multiple roles GWAS highlights CFH and CFHRs CFHRs antagonise CFH Understand molecular basis of host susceptibility Goicoechea de Jorge et al., PNAS 2013
    15. 15. - understand structure:function GAGs* CRP C3b 1 2 3 4 5 6 7 GAGs* Sialic acid C3b/d GAGs* C3b 8 9 10 11 12 13 14 15 16 17 18 19 20 fHBP CFH CFHR3 CFHR1 CFHR4 CFHR2 CFH locus multiple partners, multiple roles GWAS highlights CFH and CFHRs CFHRs antagonise CFH Understand molecular basis of host susceptibility CFHR5
    16. 16. - inactivate function fHBP Hide immunogenic epitopes Less opportunity for ‘blocking’ antibodies SCR 6 and 7 Reduce immunogenicity
    17. 17. - inactivate function Amino acid substitutions generate non-functional fHbps Johnson et al. PLoS Pathog. 2012
    18. 18. - inactivate function Amino acid substitutions generate non-functional fHbps Schneider et al. Nature 2009 B V1 double Glu Beernik et al. J Immunol 2010 Johnson et al. PLoS Pathog 2012 Arg-Ser 8 Ala subs. V2 14 Ala subs. V3 9 Ala subs. Jongerius et al. PLoS Pathog 2013 Rossi et al. Vaccine 2013 (two subs.) A one sub. Johnson et al. PLoS Pathog. 2012
    19. 19. - inactivate function Host modification human mouse human mouse human mouse fHbp Murine SCR6/7 Human SCR6/7 - humanising the binding site insufficient to allow murine fH to bind fHbp - SCR 6 and 7 have distinct orientations in mfH and hfH Johnson et al. PLoS Pathog. 2012
    20. 20. - inactivate function X C3b Host modification C3b SAP/GAGs mfH mfH SAP/GAGs ? C3b ? SAP/GAGs extra hfH chimeric fH fHbp + fHbp Non-functional fHbp as or more immunogenic than w/t fHbps Johnson et al. PLoS Pathog. 2012 Beernik et al. J Immunol. 2011
    21. 21. - easy to manufacture Distribution of fHbps in MRF collection 2010-2012 (n=916) 10% 34% v1 56% v2 v3 no V2 fHbp containing vaccine
    22. 22. - easy to manufacture fHbp V2 is inherently unstable Trypsin digestion Differential Scanning calorimetry (DSC) V1 V2 V3 no V2 fHbp containing vaccine Crystal structure
    23. 23. - easy to manufacture fHbp V2 is inherently unstable Trypsin digestion Differential Scanning calorimetry (DSC) Wild-type M6 M5 M4a M4b no V2 fHbp containing vaccine stable V2 fHbps
    24. 24. - easy to manufacture fHbp V2 is inherently unstable 5 of 10 single amino acid substitutions de-stabilise the antigen M6 R85A L135A V136A A204P V211A E222A T225A E252A no V2 fHbp containing vaccine stable V2 fHbps non-functional stable V2 fHbps
    25. 25. - ? elicits herd immunity N. meningitidis Temp. (°C) CssA fHbp (alpha-2,3-sialyltransferase) 30 37 42 Temperature acts as a danger signal Lst RmpM ? less fHbp at lower temp in the nasopharynx ? less effect of vaccine on carriage Oriente et al., J Bact 2010 Loh et al. Nature 2013
    26. 26. - What next for fHbp? Vaccine enhanced immunogenicity cross variant/family protection non-functional fHbps - Serum bactericidal activity
    27. 27. - What next for fHbp? Vaccine enhanced immunogenicity cross variant/family protection non-functional fHbps Naturally occurring variants Seib et al., Infect Immun 2009 Jongerius et al., PLoS Pathog 2013 Hybrid fHbp Scarselli et al., Sci Trans Med 2011
    28. 28. - What next for fHbp? Vaccine enhanced immunogenicity cross variant/family protection non-functional fHbps
    29. 29. - What next for fHbp? Vaccine enhanced immunogenicity cross variant/family protection non-functional fHbps Host susceptibility fHbp:fH interactions fHbp:CFHR interactions interactions with polymorphic proteins
    30. 30. Susan Lea Steven Johnson Joe Caesar Phil Ward Rachel Everitt Martin Maiden Odile Harrison - University of Oxford Ray Borrow Jay Lucidarme - Meningococcal Reference Laboratory Matthew Pickering - Imperial College London Elena Goicoechea de Jorge Hayley Lavender Rachel Exley Ilse Jongerius Stijn van den Veen Edmund Loh

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