1. Vaccines for Otitis media: Where to next?
A/Prof Peter Richmond
Division of Paediatrics, School of Medicine, University of Western Australia
Vaccine Trials group & Ear Health Program, Wesfarmers Centre of Vaccine and Infectious Diseases, Telethon Kids Institute
Depts of Immunology and General Paediaitrics Princess Margaret Hospital for Children,
Perth Western Australia

2. Disclosures
Vaccine Scientific Advisory Groups / Boards (no personal remuneration)
Influenza Specialist Group (ISG)
GlaxoSmithKline - Pertussis, pneumococcal protein vaccine
Pfizer - Pneumococcal conjugate vaccines
Baxter - meningococcal C conjugate & Ross River Virus vaccines
CSL Ltd - adjuvanted influenza vaccine
MCRI - Rotavirus RV3 DSMB
Vaccine Research
Principal Investigator of industry sponsored multi-centre studies for Baxter, CSL, GSK, Medimmune, Merck, Pfizer, Sanofi, Novartis
Travel support to present trial data at scientific conferences
Sanofi, Pfizer, Baxter, GSK
Research funding for Investigator initiated studies
GSK, Merck, CSL, Novartis
Membership of Immunisation committees
Previous member Australian Technical Advisory Group on Immunisation,
Member ATAGI Pneumococcal working party
Member, NCIRS Scientific Advisory Committee
TAGI pnrumococcal working party

3. Talk Outline
Outlining current vaccine strategies and areas for improvement
Why is developing vaccines so difficult for otitis media?
Understanding the important issues in developing vaccines against OM
New vaccines on the horizon for OM
New strategies for vaccination

4. Impact of current vaccines on OM
Pneumococcal conjugate vaccines
Moderate efficacy (~70%) against vaccine type acute OM but small overall impact on AOM (6-9%) in RCT
Bigger impact on recurrent OM & need for surgery (~30%)
Significant impact on antibiotic resistance
Generally good coverage achieved (>90%)
Gains over time reduced by serotype & pathogen replacement
Influenza vaccines (recent meta-analysis)
4% reduction in influenza associated acute OM in 6 months following vaccination
15% reduction in antibiotic use associated with OM
Variable efficacy from year to year
Poor coverage in children in most countries
Australia <10%

5. Influenza vaccines for preventing AOM
Outcomes
Control
Incidence
Influenza vaccine
Relative effect (95% CI)
No. of subjects & studies
Quality
GRADE
≥ 1 episode AOM
264/1000
211/1000
RR 0.80
(0.67 to 0.96)
4736
(5 RCTs)
High
Courses of antibiotics
362 / 1000
216 /1000
RR 0.70
(0.59 – 0.83)
1223
(2 RCTs)
AOM by vaccine type
Trivalent Inactivated Vaccine(TIV)
117/226
170/356
RR 0.77
(0.48 – 1.23)
4154
(3 RCTs)
Moderate
LAIV
340/1508
469/2646
RR 0.79
(0.60 – 1.02)
582
Norhayati MN, Ho JJ, Azman MY.
Influenza vaccines for preventing acute otitis media in infants and children.
Cochrane Database of Systematic Reviews 2015,

6. 240 middle ear fluid samples from 143 children:
NTHi is predominant in nose and middle ear of PCV7-vaccinated children with recurrent OM
240 middle ear fluid samples from 143 children:
47% children had NTHi in middle ear
6% had S. pneumoniae in middle ear
Role for licensed PCV10 containing protein D?
Wiertsema et al, Vaccine 2011 6

7. 10V PHiD-CV & NTHi OM & Carriage studies
Trial
Design
Outcomes
Efficacy
(95% CI )
PCV7 USA, Finland
RCTs
All-cause AOM
6-9%
11V PHiD-CV
Czech Republic
RCT
NTHi AOM
NTHi Carriage
32.7%
35.5%
COMPAS
10V PHiD-CV
19.8% ( ) *
21.5% (-43.4% - 57)
7.4% (-15.1% %)
FinIP (nested study)
Cluster
6.4% (-5.5 – 17.2%)
No effect
Netherlands, PHiD-CV

8. 10V PHiD-CV & NTHi OM & Carriage studies
Trial
Design
Outcomes
Efficacy
(95% CI )
PCV7 USA, Finland
RCTs
All-cause AOM
6-9%
11V PHiD-CV
Czech Republic
RCT
NTHi AOM
NTHi Carriage
32.7%
35.5%
COMPAS
10V PHiD-CV
19.8% ( ) *
21.5% (-43.4% - 57)
7.4% (-15.1% %)
FinIP (nested study)
Cluster
6.4% (-5.5 – 17.2%)
No effect
Netherlands, PHiD-CV
Kilifi, Kenya
PHiD-CV
Observational
NTHi Carriage in <5yrs
Decrease from 54 to 40%
RR 0·62 (0·52–0·75)
Northern Territory PHiD-CV vs PCV13
NTHI carriage
NTHI ear discharge
PCV13 lower rate 63 vs 71%
PCV13 higher 64 vs 36%

9. Why is developing vaccines that are broadly protective against otitis media so hard? 90 Pneumococcal serotypes NTHi strains Moraxella Alloiococcus Pseudomonas Staph. aureus RSV, influenza, PIV, Rhinovirus

10. Issues for new OM Vaccines
Which disease(s) are we targeting?
Acute OM vs chronic OME vs CSOM
Polymicrobial infections common
Difficulties in diagnosing most important pathogen
Lack of clear correlates of protection
Need to understand development of natural immunity
Animal models vs human disease
Variable antigen expression
Impact on nasopharyngeal carriage
Bacterial load & transmission
Herd immunity vs pathogen replacement
Difficulties in getting to clinical trials
Is it possible to develop a polymicrobial vaccine?
Timing of vaccination vs onset carriage & OM in high-risk population

11. Early polymicrobial upper respiratory tract carriage and OM in Aboriginal and non-Aboriginal infants
Cumulative proportion
1.2
1.2
Otitis Media
Otitis Media 1 1
M. catarrhalis
0.8
0.8
H. influenzae
0.6
0.6
M. catarrhalis
S. pneumoniae
0.4
0.4
This data was published in 1994 and shows the very early onset of multiple bacterial colonisation. Colonisation with “any pathogen” (combining the three key species) predicts onset of otitis media in all infants by the age of 90 days.
Recurrent acute episodes led to perforation on 60% of infants by 12 months of age.
A comparison group of non-Aboriginal urban infants experienced much later single species bacterial colonisation and otitis media was transient and mild.
S. pneumoniae
0.2
0.2
H. influenzae 60
120
180
240 60
120
180
240
Age (days)
Urban non-Aboriginal
Aboriginal island community
Leach et al, PIDJ 1994

12. Multiple species co-aggregate within biofilms on the middle ear mucosa
Polymicrobial and intracellular biofilms are common in chronic OM
Intracellular bacteria always belong to the same species
Multiple species co-aggregate within biofilms on the middle ear mucosa
Animal model: bacterial species confer protection from host immune responses and antimicrobials1
More resistant than single species biofilms1
Universal, M catarrhalis , S. pneumoniae and Hoechst (nuclei )
Universal, S. pneumoniae , H. influenzae and Hoechst (nuclei )
Thornton, Coates et al, Unpublished

13. Correlates of protection against Otitis media : the blind men and the elephant
Neutrophils & OPA
CD4 Th17
Serum IgG
Serum Bactericidal antibody
Mucosal B-cell memory
Th1 & IFN
Mucosal IgA
Standardisation of assays
Role of human challenge models?

14. Indigenous children do not have lower salivary antibody titres to NTHi protein antigens
Protein D *
Salivary IgA (AU/mg protein)
Have significantly higher mucosal IgA antibody titre for P4.
Higher levels of mucosal antibody likely reflect colonisation/infection with NTHi and suggest that the mucosal compartment is capable of mounting a robust response.
Healthy controls
Non-Aboriginal cases
Aboriginal cases
Thornton et al Clin Vaccine Immunol. 2017

15. Aboriginal children have lower serum antibody to NTHi antigensP4 P6
Protein D
*** **
Serum IgG (AU)
Have significantly lower Protein D and P4 systemic IgG titres.
As Protein D is included in the Synflorix® Vaccine, could vaccination be more effective in this sub-population in reducing disease: await Previx Study results
Healthy controls
Non-Aboriginal cases
Aboriginal cases
Thornton et al Clin Vaccine Immunol. 2017

16. Natural development of NTHI Protein D serum IgG antibodies with age
Age (yrs)
Healthy
Non-Indigenous OM
Indigenous OM

17. OM pathogens are not always able to be targeted by antibody
Bacterial biofilm in the middle ear – protects bacteria from antibodies by covering them with slime
Thornton PLoSONE 2013
Immune mechanisms other than antibody may be important in getting rid of chronic otitis media infection
Mucosal epithelial cells containing live bacteria in the middle ear fluid of a child with recurrent ear infections

18. New Otitis media Vaccine candidates

19. Potential NTHi vaccine candidates
Adhesins
Haemophilus adhesin protein (Hap)
HMW1, HMW2
H. influenzae adhesin (Hia)
Protein E (also binds IgD & involved in resistance to complement)
P5 fimbrin
Protein F (also ABC transporter)
Type IV pilus (PilA; Tfp) (also role in biofilm transformation)
Other proteins
Protein D (glycerophosphodiesterase)
D15 protein (?nucleotidyltransferase)
HtrA (Heat shock protein)
P2 porin
P4 protein (Acid phosphatase)
OMP 26 (Skp family of translocation proteins)
P6 OMP (Peptidoglycan-associated lipoprotein)
Detoxified lipooligosaccharide
Combinations of above protein eg ChimV4 (L. Novotny Tues 5.46pm)

20. Combination NTHi vaccine Phase 1 (GSK)
Contains Protein D, Protein E & PilA
Safe & well tolerated as 2 dose schedule
Modest antibody response to unadjuvanted proteins in healthy adults
Antibody & CMI responses improved with use of adjuvants AS01 & AS04
Now progressing to Phase II studies in COPD patients with AS01
AS01adjuvant is a liposome based adjuvant containing MPL & saponin QS21used in GSKs inactivated zoster vaccine and RTS malaria vaccine
G. Leroux-Roels et al. Vaccine 2016; 34:3156–63

21. Improved immune responses to NTHi combination vaccine in older adults with adjuvants
3 dose schedule
2 dose
Anti-Protein E antibody responses
Anti-Protein E Cell mediated immune responses to various vaccine formulations
AS01 adjuvant provides strongest Ab & CMI (mainly TH1) responses
Will this vaccine be evaluated in children for OM & carriage?

22. Potential Moraxella vaccine candidates
Adhesins
MID/Hag (also binds IgD)
MchA1,2, MhaB1,2
McmA A1
McaP
UspA1, UspA2/2H/2V (also binds complement, hCEACAM, vitronectin)
OMP CD (binds mucin)
Type IV pilus (PilA) (also role in biofilm transformation)
OlpA
ABC transporters: OppA, CysP, SBP2, AfeA
Other proteins
Lactoferrin binding protein
OMPE (Fatty acid transport)
MSP75
M35 porin
MSP22 lipoprotein (binds Heme)
OMPG1a,1b
Detoxified lipooligosaccharide
T. Murphy Tues 5.22pm
Perez Vaccine 2017

23. Other vaccine approaches & pathogens
Intranasal vaccines
Adjuvanted proteins
Commensal bacteria (H. hemolyticus) may elicit both immunity and compete for same niche (L. Kirkham Thurs 8.45)
Oral vaccines
bacterial mixture containing otopathogens (Bronchovaxom®)
Oral whole-cell NTHi (Bronchostat)
Transcutaneous vaccines
Topical application NTHi - “Bandaid approach” (L Bakaletz Thurs 9.15am)
Nanopatch (Mark Kendall UQ) – microprojection based skin patch dry coated with vaccine antigen
Maternal immunisation
RSV vaccines (maternal and infant)

24. Murine Nanopatch Immunisation with PCV7
Serum OPA titres after im & transcutaneous PCV7
Pearson et al. Vaccine :6675

25. Chronic & recurrent OM in high risk populations
Host Susceptibility, Exposures, Environment, Microbes
Exposure to OM Pathogens
LOW DOSE
LATE
EARLY
HIGH DOSE
Clearance
Chronic Nasal Discharge
Normal carriage
Protective immunity
?Immune tolerance
Persistent carriage
Timing of Vaccination likely to be important for impact on OM especially recurrent and chronic disease:
A role for maternal vaccination?
Acute Disease: OM, Pneumonia & Invasive Diseases
Chronic Bacterial
Mucosal Disease

26. Rationale for maternal vaccination schedules in high-risk populations
Success of maternal pertussis & influenza vaccination in preventing infant disease
Protective antibody levels in first 3 months
Reduce maternal infections & carriage
Prevent early carriage through direct & indirect effects
Benefits through breast feeding as well
Applicable for polysaccharide and protein bacterial vaccines as well as viral vaccines

27. PneuMum Study Impact of Maternal PPV on OM at 7 months of age in Indigenous infants
Controls
(n=77)
Pregnancy PPV
(n=75) VE
(95% CI)
Post-Partum
PPV
Otitis media
71%
63%
12%
(-12 to 31%)
76%
-6%
(-30 to 13%)
Pnc VT Carriage
26%
18%
30%
(-34 to 63%)
29%
OM + VT carriage
27%
13%
51%
(-2 to 76%)
17%
39%
(-19 to 69%)
No significant differences in Ear Disease or VT carriage between infants of PPV recipients and Controls
Binks et al Vaccine 2015

28. Uncertainties for Maternal Vaccination
No published studies with licensed PCVs
Maternal PCV13 study ongoing in the Gambia
No studies planned for PCV10
Interference with infant vaccine responses (PCV)1
Serotype or pathogen replacement disease1
9V PCV increased risk AOM in first 6 months (RR % CI ) but carriage not examined
Role of Pnc & NTHi protein vaccines
Requirement for doses in subsequent pregnancies
Regulatory, liability & legal issues
Is neonatal vaccination an option?
Daly Vaccine 2014; 32:6948

29. Summary
Pneumococcal conjugate and influenza vaccines have important roles in prevention of OM
Impact of protein D containing vaccine on NTHi carriage and OM appears limited but may have some benefits on a population basis
The polymicrobial nature of OM and bacterial persistence in biofilms and intracellular communities makes vaccine development difficult
Understanding the ontogeny of immune responses to otopathogens is important when considering vaccine candidates and programs and may be antigen and population specific
Development of standardised assays and outcome measures will be important for early phase clinical studies
Promising new vaccine candidates for NTHi may be able to specifically target biofilm
Maternal immunisation may need to be considered where early acquisition of pathogens
RSV vaccines may also impact on acute OM
Need to characterise the MEE of Indigenous children
Which bacteria are actually present in the middle ears?

30. WA Ear Health Team Epidemiology, Health Empowerment & Community
Pathogenesis
Deborah Lehmann
Lea-Ann Kirkham
Victoria Stroud
Ruth Thornton
Rose Walley
Janessa Pickering
June Doyle
Karli Corscadden
Glen Pearson
Caitlyn Granland
Michael Wright
Selma Wierstma
Sharon Weeks
Maxine Janka
Cheryl Kickett-Tucker
Tulia Mateus
Darren Westphal
Camilla de Gier
Hannah Moore
Andrew Currie
Anke Hoskins
Dino Tan
Clinical Trials
ENT Collaborators
Jennifer Kent
Harvey Coates
Stephanie Jeffares
Shyan Vijaysekeran
Ruth Monck
Francis Lannigan
Peter Jacoby
Kelvin Kong

31. Questions?
Perth
ISPPD
April 2018

33. PneuMum PPV Antibody levels in Serum & Breast milk: serotype 10A

35. Aboriginal children do not have lower serum antibody titres to S
Aboriginal children do not have lower serum antibody titres to S. pneumoniae protein antigens
PspA1
Ply
CbpA
PspA2 * **
Serum IgG (AU)
In the GROMIT study - Children with a history of rAOM had signif higher GM serum IgG levels to NTHi proteins compared to healthy controls, while there was no difference against Pnc protein antignes.
Have significantly higher systemic antibody titres for several of the antigens.
May reflect constant colonisation/infection with the pathogen.
Healthy controls
Non-Aboriginal cases
Aboriginal cases

36. Aboriginal children do not have lower salivary antibody titres to S
Aboriginal children do not have lower salivary antibody titres to S. pneumoniae protein antigens
PspA1
PspA2
Ply ** *
Salivary IgA (AU/mg protein)
Appear to mount a robust mucosal immune response which again is likely to reflect persistent or recurrent colonisation/infection with S. pneumoniae.
* CbpA IgA data is not available in saliva as it binds secretory component.
Healthy controls
Non-Aboriginal cases
Aboriginal cases

37. Development of Protein Vaccines for otitis media
Needs for protein vaccines
Expand coverage and decrease replacement
Broaden efficacy at mucosal level: OM, pneumonia; colonisation
Simpler and lower cost to manufacture
Back up if PCV poorly effective eg serotype 3
Serum institute Ph1 later this year
Pnc protein vaccines
Protein vaccines that induct antibody or T-cells
Whole killed vaccines
Functional Ab : OPA, prevent adhesion: inhibit invasion; inactivate host defence eg stopping complement deposition invaders; neutralisation of toxin
Challenges
Opsonic assay but because coverage by CPS
Need to demonstrate that opsonises in animal models with heavily encapsulated
TH17 – safe, will it work against colonisation
Will it work against invasive disease
Candidates
GSK
Sanofi PcpA, PhtD PlyD1
Nasvax 8 B cell targets
7 Easy steps
Demonstrate mechanism of protection
Develop potency test that links to mechanism
Develop functional assays
Demonstrate biological effects OPA or reduction in colonisation (precursor to mucosal disease; required for transmission and herd immunity; easy to measure
Phase 3 demonstrate in trial with PCV, reduction in colonisation for NVT of >= 50%
Perform Phase 4 RCT of PCV10 + PCV + protein
If effect strong enough then consider stand alone of protein vs PCV

38. IgG antibody levels to Haemophilus protein antigens are significantly higher in young children with rAOM
Cases
Controls
* Cases have significantly higher levels of H. influenzae protein antibodies compared to controls
Corscadden et al,

39. Antibody assays Cytokine assays Antimicrobial peptides
• Measure IgA, IgG, IgG1 and IgG2 (~60µl serum, 100µl saliva)
– Pneumococcal proteins – Ply, CbpA, PspA 1 and 2
– Pneumococcal polysaccharides -1,3,4,5,6B,7F,9V,14,18C,19A,19F,23F,6A
– NTHi proteins – Protein D, P6, P4, rsPilA, ChimV4, P26
– GAS protein (Pfizer) - ScpA
• MOPA to 13 serotypes (~120µl serum)
• Ply functionality (~60µl serum)
• Protein D functionality assay – under development
Cytokine assays
• Pro-inflammatory (IL1, IL6, IL8, TNFa, IL-1β), Th1 (IFNg), Th2 (IL-4, IL-5, IL-10,
IL-13) and Th17 (IL-17, IL-23, TGFβ)
Antimicrobial peptides
• LL-37, β-defensins 1,2 & 3, Human Defensin-5, Lactoferrin and Lysozyme

40. Infant antibody levels after Maternal PCV9
Serotype Specific IgG (µg/mL)
Maternal PCV infants GMCs were significantly higher for all 9 serotypes at birth and significantly lower at 7 mths for all PCV7 types
Daly Vaccine 2014; 32:6948

41. Biofilm and Intracellular infection can be seen on the mucosa of children with OM
Child with rAOM
FISH : EUB338 (yellow), M catarrhalis (green), S. pneumoniae (pink) and Hoechst (nuclei stain – blue)
Many bacteria unidentified but S. pneumoniae both IC and in biofilm, M catarrhalis throughout biofilm.

42. Biofilms and intracellular bacteria are also present in the middle ear effusion
MEE from non-Indigenous children
Bacteria within 92% of MEEs tested.
Most bacteria are alive and many of the effusions were culture negative
Live bacteria in microcolonies associated with DNA strands
95% of middle ear fluid were positive for bacteria using FISH
Bacterial clusters were frequently polymicrobial
Biofilm - 81%, Intracellular bacteria – 50% C
NETs - Active immune mechanism
Important in confining infection to site
Activated cells consisting of DNA backbone embedded with antimicrobial peptides and enzymes
Distinguished from necrotic neutrophils by lack of cytoplasmic markers (ie actin)
Some otopathogens have been shown to resist NET killing
NETs are formed as part of an active immune response by neutrophils and are thought to act to confine infections to a site. It is basically when the neutrophil actively spits out its DNA embedded with antimicrobial peptides so as to capture, contain and kill bacteria, however it also known that many of the otopathogens can actually resist NET killing and that it can actually enhance biofilm production. One of the major differences between NETs and necrotic neutrophils is the absence of cytoplasmic markers such as actin in NETs and this is what we stained for to determine if the DNA was NET derived.
- 80% positive for more than one type tested
Modified from Thornton et al, PLoS ONE 2013

43. Vaccine efficacy of PHiD-CV against first or all AOM episodes (Compas study)
Footnote: PP: N = 3010 (PHiD-CV); N = 2979 (Control); ITT:
N = 3602 (PHiD-CV); N = 3612 (Control).
Error bars depict 95% confidence interval;
N, number of children in PP or ITT cohort;
PP, per-protocol; ITT, intent-to-treat;
AOM, acute otitis media; C-AOM, clinically confirmed AOM;
B-AOM, bacteriologically confirmed AOM;
Pn, S. pneumoniae; VT, vaccine type;
NVT, non-vaccine non-vaccine-related type;
NTHi, non typeable H. influenzae.
*Vaccine efficacy against first C-AOM in per-protocol analysis was assessed
as secondary confirmatory objective.
Xavier Sáez-Llorens; et al; Human Vaccines & Immunotherapeutics  2017 ePub Ahead of Print

44. Efficacy of PHiD-CV against first acute otitis media episodes (end-of-study analysis).
Tregnaghi MW, Sáez-Llorens X, López P, Abate H, Smith E, et al. (2014) Efficacy of Pneumococcal Nontypable Haemophilus influenzae Protein D Conjugate Vaccine (PHiD-CV) in Young Latin American Children: A Double-Blind Randomized Controlled Trial. PLOS Medicine 11(6): e

45. Bacterial Respiratory Infectious Disease Group

46. Early nasopharyngeal carriage is associated with increased risk of OM20 40 60 80
100 1 2 3 4 5 6 7 8 9 10 11 12
Papua New Guinea
Australian Aboriginal
Alabama
Costa Rica
Buffalo
Age (months)
Colonization (%)
Colonization of the upper respiratory tract:
median age 17 days in PNG infants
100% by 3 months of age
Cumulative carriage of 50% documented at 1 month of age in PNG, 2 months in Aboriginal children in TOP END and 6 months in the Alabama study by Gray. Other populations acquire pneumococci later – mainly due to difference in exposure, rather than immune/host factors.
Amanda Leach, Menzies School of Health Research
Persistence of Pnc carriage into adulthood
- 43% in PNG compared to 1-13% in European countries

47. PHiD-CV Impact on NTHi carriage
Age (yrs)
Healthy
Non-Indigenous OM
Indigenous OM