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 Email: peter.richmond@uwa.edu.au

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, 2010-14 Member ATAGI Pneumococcal working party Member, NCIRS Scientific Advisory Committee TAGI pnrumococcal working party

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

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%

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,

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

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% (4.4-31.4) * 21.5% (-43.4% - 57) 7.4% (-15.1% - 25.5%) FinIP (nested study) Cluster 6.4% (-5.5 – 17.2%) No effect Netherlands, PHiD-CV

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% (4.4-31.4) * 21.5% (-43.4% - 57) 7.4% (-15.1% - 25.5%) 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%

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 http://trendsupdates.com

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

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

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 33342 (nuclei ) Universal, S. pneumoniae , H. influenzae and Hoechst 33342 (nuclei ) Thornton, Coates et al, Unpublished

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?

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

Aboriginal children have lower serum antibody to NTHi antigens P4 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

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

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

New Otitis media Vaccine candidates

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)

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

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?

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

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)

Murine Nanopatch Immunisation with PCV7 Serum OPA titres after im & transcutaneous PCV7 Pearson et al. Vaccine 2015 33:6675

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

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

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

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 3.9 95% CI 1.7- 9.1) 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

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?

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

Questions? Perth ISPPD April 2018

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

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

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

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

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,

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

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

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 33342 (nuclei stain – blue) Many bacteria unidentified but S. pneumoniae both IC and in biofilm, M catarrhalis throughout biofilm.

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

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

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): e1001657. https://doi.org/10.1371/journal.pmed.1001657 http://journals.plos.org/plosmedicine/article?id=10.1371/journal.pmed.1001657

Bacterial Respiratory Infectious Disease Group

Early nasopharyngeal carriage is associated with increased risk of OM 20 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

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