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

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

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

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

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

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)

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

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)

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

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

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

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

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

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

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

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

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

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

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

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

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