Stringently Defined Otitis Prone Children Demonstrate Deficient Naturally Induced Mucosal Antibody Response to Moraxella catarrhalis Proteins

Moraxella catarrhalis (Mcat) is a prominent mucosal pathogen causing acute otitis media (AOM). We studied Mcat nasopharyngeal (NP) colonization, AOM frequency and mucosal antibody responses to four vaccine candidate Mcat proteins: outer membrane protein (OMP) CD, oligopeptide permease (Opp) A, hemagglutinin (Hag), and Pilin A clade 2 (PilA2) from stringently defined otitis prone (sOP) children, who experience the greatest burden of disease, compared to non-otitis prone (NOP) children. sOP children had higher NP colonization of Mcat (30 vs. 22%, P = 0.0003) and Mcat-caused AOM rates (49 vs. 24%, P < 0.0001) than NOP children. Natural acquisition of mucosal antibodies to Mcat proteins OMP CD (IgG, P < 0.0001), OppA (IgG, P = 0.018), Hag (IgG and IgA, both P < 0.0001), and PilA2 (IgA, P < 0.0001) was lower in sOP than NOP children. Higher levels of mucosal IgG to Hag (P = 0.039) and PilA2 (P = 0.0076), and IgA to OMP CD (P = 0.010), OppA (P = 0.030), and PilA2 (P = 0.043) were associated with lower carriage of Mcat in NOP but not sOP children. Higher levels of mucosal IgG to OMP CD (P = 0.0070) and Hag (P = 0.0003), and IgA to Hag (P = 0.0067) at asymptomatic colonization than those at onset of AOM were associated with significantly lower rate of Mcat NP colonization progressing to AOM in NOP compared to sOP children (3 vs. 26%, P < 0.0001). In conclusion, sOP children had a diminished mucosal antibody response to Mcat proteins, which was associated with higher frequencies of asymptomatic NP colonization and NP colonization progressing to Mcat-caused AOM. Enhancing Mcat antigen-specific mucosal immune responses to levels higher than achieved by natural exposure will be necessary to prevent AOM in sOP children.

inTrODUcTiOn Acute otitis media (AOM) is the most common infectious disease among children to cause parents to seek medical care for their child and receive antibiotics. Moraxella catarrhalis (Mcat) has been ranked as the third most common cause of AOM after Streptococcus pneumoniae (Spn) and non-typeable Haemophilus influenzae (NTHi) in children (1). However, the virulence of Mcat as an otopathogen may be increasing as evidenced by an increased occurrence of tympanic membrane (TM) rupture caused by the organism (Pichichero, unpublished observation).
Our recent studies on the prevalence of otopathogens show that Mcat has overtaken Spn and NTHi to become the most frequent cause of episodic and recurrent AOM in children (2). Similarly, Mcat was recently identified as the most common otopathogen in Finish children (3). AOM often recurs and poses a high burden on the quality of life of children, the health care system and the economy worldwide (4). There is no licensed vaccine for Mcat, and the development of an Mcat vaccine is currently moving from animal studies toward clinical trials. A number of antigens have been identified as potential Mcat vaccine candidates, of which outer membrane protein (OMP) CD, oligopeptide permease (Opp) A, hemagglutinin (Hag), and Pilin A clade 2 (PilA2) are promising representatives. OMP CD is a porin and adhesin and is highly conserved with exposed epitopes on the bacterial surface (5,6). OppA is a highly conserved oligopeptide binding protein mediating the uptake of peptides and fitness of the organism in the respiratory tract (7,8). Hag, also known as Moraxella IgD-binding protein (MID), is an adhesin, Hag, and autotransporter containing surface exposed and conserved epitopes (9). PilA2 is a conserved pilin involved in natural genetic transformation, biofilm formation, and adherence of the bacteria to human epithelial cells (10)(11)(12).
Otitis prone (OP) defines a health status of children who have recurrent AOM, with at least three episodes in 6 months or four episodes in a 12-month time span (13,14). To meet the definition of stringently defined otitis prone (sOP), a child must have every episode proven by a tympanocentesis-derived middle ear fluid (MEF) positive culture of an otopathogen while non-otitis prone (NOP) children are those with 0-2 episodes of AOM per year (14,15). Because sOP children are most vulnerable to AOM, they should be the focused population of vaccination against AOM. Evaluating naturally induced humoral immune responses to Mcat proteins may disclose the immunogenicity of these antigens and functional activity of the antibodies elicited by these proteins in the targeted age and most vulnerable population, especially sOP children. We have found that during nasopharyngeal (NP) colonization by Mcat all four antigens, OMP CD, OppA, Hag, and PilA2, are immunogenic in both sOP and NOP children (16). The age-dependent increase of naturally induced serum antibody ranked as OppA > Hag5-9 > OMP CD > PilA2 in both sOP and NOP children (Ren et al., unpublished). We also found that sOP children have deficient production of serum antibodies to OMP CD, OppA, Hag, and PilA2 at asymptomatic Mcat NP colonization and onset of AOM (Ren et al., unpublished), similar to our findings for protein vaccine candidates of Spn (17) and NTHi (18).
Moraxella catarrhalis is a mucosal pathogen and mucosal immunity plays an important role in host defense against Mcat infections. Here, we studied Mcat NP colonization, AOM frequency and mucosal antibody responses to four vaccine candidate Mcat proteins, OMP CD, OppA, Hag, and PilA2 in nasal washes from sOP compared to NOP children to identify differences in pathogenicity and mucosal immune responses.

MaTerials anD MeThODs subjects and sampling
The samples collected and analyzed were obtained during a prospective study supported by the National Institute of Deafness and Communication Disorders, as previously described (19,20). Healthy children without previous episodes of AOM were enrolled at 6 months of age from a middle class, suburban sociodemographic pediatric practice in Rochester, NY, USA during years 2006-2016. For this study, we assessed a total of 628 children followed prospectively until 36 months of age. NP and oropharyngeal cultures were obtained seven times during the study period at 6,9,12,15,18,24, and 30-36 months of age. Whenever children experienced an AOM episode, a confirmatory tympanocentesis was performed, and MEF was microbiologically assessed (an essential component to the definition of "stringently defined" AOM since virtually all prior studies have relied on only a clinical diagnosis that is known to be variably accurate). NP, oropharyngeal, and MEF sampling was conducted as previously described (19,21), and the samples were analyzed to identify bacterial pathogens by using standard culture and PCR assay (19). The study was approved by the Rochester General Hospital Research Subjects Review Boards. Written informed consent was obtained for participation, and all procedures are in accordance with the Declaration of Helsinki.
Nasal wash samples were obtained by instilling 1 ml of sterile phosphate-buffered saline (PBS) into each nare and then aspirating from each nare yielding approximately 2 ml of material for each subject. The nasal wash solution was subsequently centrifuged at 3,000 rpm (1,100 × g) at 4°C for 10 min, and the supernatant was then stored at −80°C until used for quantification of mucosal antibody by using enzyme-linked immunosorbent assay (ELISA).

enzyme-linked immunosorbent assay
Recombinant Mcat proteins OMP CD, OppA, Hag5-9 (truncated Hag protein), and PilA2 were expressed and purified as previously described (7,9,11,22). Protein-specific antibody concentrations were determined by ELISA using purified recombinant proteins. Three hundred eighty-four well Greiner Microlon plates were coated with 1-3 µg/ml of individual proteins (20 µl/well) in PBS (pH 7.4) and incubated at 37°C for 1 h. After five washes, the plates were blocked with 10% fetal bovine serum in PBS (pH 7.4) at room temperature for 1 h (40 µl/well). After washing, 20 µl of nasal wash twofold serially diluted in a buffer of PBS, 0.5% BSA, with 0.005% Tween was added to each well. Carimune (CSL Behring AG, Bern, Switzerland) for Data are represented as percentage ± 95% confidence interval. Fisher's exact test was used to compare the difference of rates or percentages between sOP and NOP children. *P < 0.05, **P < 0.01, and ****P < 0.0001 comparing sOP vs. NOP children.
sera for IgA assays were used as references and in-house control sera with high and low titers were run on each plate. The plates were incubated at room temperature for 30 min followed by the addition of affinity-purified goat antihuman IgG/IgA antibody conjugated to horseradish peroxidase (Bethyl Laboratories, Montgomery, TX, USA) as a secondary antibody. The reaction products were developed with TMB Microwell Peroxidase Substrate System (KPL, Gaithersburg, MD, USA), stopped by addition of 1.0 M phosphoric acid and read by a Spectramax 340PC plate reader (Molecular Devices, Sunnyvale, CA, USA) using a 450-nm filter.
To provide quantitative results on antibody concentrations, the level of the specific antibody present in the unknown sample was determined by comparing to an internal reference serum (Carimune for OMP CD IgG and Gammagard for OppA IgG, Hag IgG, and PilA2 IgG or in-house human IgA sera). The levels of IgG and IgA in the reference serum were quantitatively measured by using a human IgG or IgA ELISA quantitation kit (Bethyl laboratories). A four-parameter logistic-log function was used to form the reference and sample curves. This ELISA was validated according to the International Council for Harmonization Guidance. The inter-assay coefficient of variation was ≤30% for all antigens and secondary antibody combinations.

statistical analysis
Fisher's exact test was used to analyze the difference of rates for Mcat NP colonization, AOM and NP colonization progressing to AOM. The Mann-Whitney test was used to compare differences in Mcat protein-specific IgG or IgA concentrations, normalized by total IgG or IgA concentration in nasal wash, respectively. P values of <0.05 were considered significant.  (Figure 1). Mcat NP colonization rates were consistently higher in sOP vs. NOP children: 33 vs. 27% at age 6 months, 31 vs. 25% at age 9 months, 34 vs. 26% at age 12 months, 26 vs. 23% at age 15 months, 26 vs. 23% at age 18 months, 36 vs. 22% at age 24 months (P = 0.010), 23 vs. 11% at age 30-36 months (P = 0.006), and overall 30 vs. 22% at age 6-36 months old (P = 0.0003, Figure 1A). The relative risk of Mcat NP colonization for sOP:NOP children was 1.7 at age 24 months old, 2.1 at age 30-36 months old, and overall 1.3-fold higher for age 6-36 months old.
Thirty seven of 75 (49%) sOP children with AOM had Mcatcaused AOM, which is significantly higher than 62 of 255 (24%) NOP children with AOM (P < 0.0001, Figure 1B). The relative risk of Mcat-caused AOM for sOP:NOP was thus twofold higher.

Mucosal antibody levels against Mcat Proteins in sOP and nOP children
We analyzed mucosal IgG and IgA antibodies in nasal wash samples from 238 healthy visits of 59 sOP children and 270 healthy visits of 75 NOP children age 6-36 months old. sOP children had significantly lower levels of mucosal IgG to OMP CD (P < 0.0001), OppA (P = 0.018), and Hag5-9 (P < 0.0001) than NOP children (Figure 2A). Similarly, sOP children had significantly lower levels of mucosal IgA antibody to Hag5-9 (P < 0.0001) and PilA2 (P < 0.0001) than NOP children ( Figure 2B). We did not find a significant difference of mucosal IgG and IgA to the four Mcat proteins among different age time points 6,9,12,15,18,24, and 30-36 months in either sOP or NOP children (P > 0.05). Mucosal IgG levels to four proteins could be ranked as OMP CD = OppA = Hag5-9 > PilA2 (P < 0.0001) in sOP and NOP children altogether. Mucosal IgA levels to the four proteins could be ranked as Hag5-9 > OMP  Figure S1 in Supplementary Material). We did not find a significant difference of mucosal antibody levels between these two groups of children (P > 0.05, Figure S1 Figure 4A). In contrast, NOP children displayed higher mucosal IgG to OMP CD (P = 0.0070, Figure 4C) and Hag5-9 (P = 0.0003, Figure 4C) and IgA to Hag5-9 (P = 0.0067, Figure 4D) at Mcat NP colonization than that at onset of AOM. There was a significantly higher rate of Mcat NP colonization progressing to AOM in sOP children (28 in total of 107 Mcat NP colonization positive visits, 26%) than that in NOP children (4 in total of 130 Mcat NP colonization positive visits, 3%) (P < 0.0001, Figure 5). The relative risk of asymptomatic colonization progressing to AOM for sOP was 8.5-fold higher than NOP children.  Studies have shown that AOM has a multifactorial etiology (4). The mechanism of OP has been attributed to many pathogenic factors, such as preceding upper respiratory infection, immature Eustachian tube, social and environmental risk factors (day care environment, parent smoking, multiple siblings at home, etc.) (4), and importantly the delayed maturation of the child's immune systems as identified by our group (15). In this study, we observed decreased mucosal IgG and IgA responses to all four Mcat proteins in sOP children at healthy visits, which is Age of the child and preexisting antibody levels are important covariates in predicting an antibody response to NP colonization. Our prior studies showed varied serum IgG responses during NP colonization of Spn and NTHi vs. non-NP colonization in children age 6-30 months old (19,20,24). Spn and NTHi also display differences from Mcat in producing acute responses to AOM (29). Thus, different pathogens may evoke different patterns of antibody responses in relation to NP colonization in young children, reinforcing the need for systematic study of each organism in different hosts. However, consistent with our prior observations on antibody responses to Spn and NTHi, sOP children tend to produce lower mucosal antibody responses to natural exposure to Mcat proteins than NOP children suggestive of a more neonatal-like, delayed immune maturation response.
Mucosal antibodies to OMP CD (22,30) and Hag (9, 31) have been detected in healthy adults and in those with chronic obstructive pulmonary disease. Mucosal antibodies to Hag have also been observed in young children (32). The current results show that all four Mcat protein antigens are immunogenic with regard to mucosal antibodies in young children. The mucosal IgG levels to four Mcat proteins could be ranked as OMP CD = OppA = Hag5-9 > PilA2 while mucosal IgA levels to four Mcat proteins could be ranked as Hag5-9 > OMP CD > OppA > PilA2 in both sOP and NOP children. The current mucosal data support our prior serum data that OMP CD, OppA, and Hag5-9 exhibit promising immunogenicity for Mcat vaccine candidates (16).
Nasopharyngeal colonization of pathogens induces naturally acquired serum and mucosal antibodies, which may confer host defense against infections. The current mucosal results are consistent with our prior serum data that high antibodies to Mcat proteins are associated with reduced Mcat carriage, suggesting a protective effect of naturally induced antibodies against NP colonization. The observation of reduced mucosal antibodies in the current study is in line with our prior data of decreased serum antibodies to Mcat proteins in sOP children during asymptomatic colonization, suggesting that sOP children had a deficient immune responses to Mcat. Stenfors and Raisanen found that there are less secretory IgA (sIgA)-coated bacteria in the nasopharynx of OP children, which may be responsible for the otitis-prone condition (33). Our results of lower naturally acquired mucosal Mcat-specific IgA support Stenfors' findings in OP children. Impaired mucosal IgA production could lead to less inhibition of Mcat adherence to NP epithelial cells and higher NP colonization in sOP children, which was observed in both the current and prior studies (24,34).
High frequency of NP colonization of otopathogens Spn, NTHi, and Mcat is associated with increased development of AOM (35). Among the three otopathogens, early colonization with Mcat is associated with the greatest risk of AOM and otitis media with effusion (35). Our results had consistent findings for sOP children of lower production of mucosal IgG and IgA to Mcat proteins upon NP colonization and a higher rate of progressing to Mcat-caused AOM. On the other hand, NOP children had higher mucosal antibodies to Mcat proteins and lower rates of NP colonization progressing to AOM (>8 times lower than sOP children). Mucosal immunity is the frontline of host defense against bacterial infections. Enhanced bactericidal effect of IgG and coating of bacteria with sIgA to inhibit bacterial adherence to respiratory epithelia may account for the protection against AOM observed in this study.

acKnOWleDgMenTs
The authors are grateful to Dr. Anthony A. Campagnari and Dr. Nicole Luke-Marshall at University at Buffalo, The State University of New York for their kindness to provide the expression plasmid and bacterial strains for production of PilA2 recombinant protein. The authors are indebted to Jill Mangiafesto and Konnor Shares for their ELISA work. The authors thank Dr. Janet Casey, the nurses and staff of Legacy Pediatrics, the collaborating pediatricians from Sunrise Pediatrics, Westfall Pediatrics, Lewis Pediatrics, and Long Pond Pediatrics, and the parents who consented and the children who participated in this challenging study. The authors also thank the support of materials and methods from R01 DC012200 to TM.

FUnDing
This work was supported by NIH NIAID R03 AI113649 to DR and NIDCD R01 DC008671 to MP. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.