1. Antibody response against NTHi in the Junbo mutant mouse
Derek Hood
Mammalian Genetics Unit
MRC Harwell Institute
United Kingdom

2. Junbo otitis media mutant mouse
NTHi infection model
single intranasal inoculation with the human otopathogen non-typeable Haemophilus influenzae (NTHi) results in efficient middle ear infection
Potential to utilize the model to investigate means to treat or prevent disease
+/+
Jbo/+
What is the role for host adaptive and innate immune components in NTHi survival in the Junbo mouse?
-relevant to both middle ear infection and translational investigations

3. Prevention of NTHi infection - vaccination
Immunize Junbo mice with NTHi bacteria (162sr, 176sr, 375sr)
cohort A: immunized with NTHi 162sr (sera 162IMs)
cohort B: immunized with NTHi 176sr (sera 176IMs)
cohort C: immunized with NTHi 375sr (sera 375IMs)
cohort D: immunized with PBS (sera pbsIMs)
Following immunization infect with NTHi 162sr

4. NTHi infection rate in immunized Junbo mouse
Jbo/+ mice immunized with:
NTHi 162sr (cohort A)
NTHi 176sr (cohort B)
NTHi 375sr (cohort C)
PBS (cohort D)
All mice infected with
NTHi 162sr
ME – middle ear
NP - nasopharynx
With respect to infection rate, there is protection with homologous
but not heterologous NTHi strain immunization

5. IgG in Junbo mouse immune sera
lysate of NTHi 162sr (1010 CFU in 0.5 ml)
mouse
cohort A
162IMs B
176IMs C
375IMs D
pbsIMs
homologous
heterologous
control
Peggy Simple Western System

6. Role of antibody in the infected Junbo mouse
Two main antibody killing mechanisms:
Opsonin to promote uptake and killing of NTHi by host immune cells
– opsonophagocytosis assay (OPA)
Bactericidal role in complement mediated killing of NTHi
- serum bactericidal assay (SBA)

7. Opsonophagocytic killing of NTHi 162sr by neutrophils (NP) derived from bone marrow of Junbo (Jbo) or wild type (WT) mouse in the presence or absence of immune mouse serum
nMS Jbo
A12 Jbo
nMS WT
A12 WT
NP:NTHi=50:1
nMS-non-immune mouse serum
A12-immune mouse serum
nMS Jbo
A12 Jbo
nMS WT
A12 WT
NP:NTHi=1:1

8. Serum bactericidal assay (SBA)
PBS (+ Mg2+, Ca2+ and glucose )
Mouse test serum (MS) - antibody
Baby rabbit serum (BRS) - complement
500 NTHi bacteria
Incubate mixture at 37ºC for 45 mins
Plate and enumerate surviving bacteria
Calculate % survival with respect to a decomplemented control
Calculate titre of test serum (inverse of dilution giving 50% bacterial killing)

9. NTHi sensitivity to baby rabbit serum (BRS)
375sr
162sr

10. NTHi sensitivity to immune mouse sera
NTHi 162sr v 176IMs
NTHi 375sr v 176IMs
avLD50: 631
avLD50: 560
NTHi 162sr v 375IMs
NTHi 375sr v 162IMs
avLD50: 1420
avLD50: 920
Killing by sera from mice immunized with heterologous strain

11. SBA titres for NTHi strains 162sr and 375sr
LD50
serum
162sr
375sr
162IMs1
8000
2500
375IMs1 10
2780
162IMs2
495
250
375IMs3
1000
162IMs3
307
300
375IMs4
1100
4000
162IMs5
138
375IMs5
162IMs6
4500
830
375IMs6
1362
3529
162IMs7
480
560
375IMs7
1632
1432
162IMs8
2000
375IMs8
1533
4300
162IMs9
100
375IMs9
720
2490
162IMs10
341
average
1420
4315
162IMs11
pbsIMs2
800
162IMs12
pbsIMs3
2624
920
pbsIMs4
160
176IMs1
288
809
pbsIMs5 5
200
176IMs2
1600
191
pbsIMs6 50
176IMs3
350
120
pbsIMs7
269
176IMs4 25
229
pbsIMs8
219
176IMs5
308
pbsIMs9
107
1400
176IMs6
167
pbsIMs10
282
166
176IMs7
310
pbsIMs11
144
176IMs8 79
631 80
420

12. NTHi 176sr sensitivity to Baby Rabbit Serum
BRS alone
BRS+10mMEGTA
BRS concentration

13. NTHi 176sr sensitivity to Baby Rabbit Serum
BRS alone
BRS+100mM D-man
BRS+10mMEGTA
BRS concentration
Mannose Binding Lectin (MBL) accounts for a substantial proportion of
NTHi 176sr killing by BRS

14. Killing activity of mouse immune serum against NTHi 176sr in the presence
of 100 mM D-mannose
LD50
579
1857
1778
8000
205
6128
895
2772
176sr
LD50
93.75 43
162.5
379
800
500
329.84
2772
329
homologous sera – 176IMs
control sera - pbsIMs

15. Killing of NTHi 176sr strains by baby rabbit serum
baby rabbit serum concentration

16. Killing of NTHi 176sr strains by baby rabbit serum
baby rabbit serum concentration

17. Comparison of LPS from NTHi 176sr and 176sr1c - using LPS-specific monoclonal antibodies
mAb
strain
TPEC15
12D9
L6A9
LLA9
176sr +
(+)
+++++
176sr1c
++++ ++
mAb TEPC15 specifically detects the presence of PCho

18. LPS structure of NTHi 176sr
Neu5Ac
Glc
Gal
HexNAc
Hep
Kdo
LipA
PEtn
PCho
P...PEtn
1,5
1,2
1,2
1,3
1,4 6 4
1,3
Hex
2,3
Phosphocholine (PCho) is phase variably expressed

19. PCho expression in bacteria isolated after infection of Junbo mouse
162sr ME NP
176sr
375sr
PCho +/-
4.2
2.4
2.1
5.6
7.5
1.3
PCho ++
92.1
93.9 87
85.4
87.6
93.4
PCho +++
3.7
10.9
9.1
4.9
ME – middle ear
NP - nasopharynx

20. summary
Junbo mouse facilitates investigation of host-microbial interactions relevant to otitis media and NTHi infection
Junbo mouse can be utilized in NTHi immunization and protection studies
SBA is a useful tool for estimating the bactericidal activity of antibodies in sera raised against NTHi
Details of the components used in the assay can be important for the outcome
Choice of NTHi strain can be crucial to the study
Variants of the same strain can influence the study in alternative ways
Opsonophagocytosis may be a major killing mechanism in the mouse middle ear

21. Acknowledgements
MRC Harwell Institute Mary Lyons Centre MGU Ward 4 Jiewu Yang Lisa Ireson Tom Purnell Chris Brett Pratik Vikhe Tamzin Osborne Sebastian de Kruijf Lucie Vizor Michael Cheeseman Quarantine Steve Brown Chris Jones Protein Core Julie Roberts Helen Hilton Sara Wells Chris Esapa