Comparative Analysis of the Humoral Immune Response to Moraxella catarrhalis and Streptococcus pneumoniae Surface Antigens in Children Suffering from Recurrent Acute Otitis Media and Chronic Otitis Media with Effusion

INTRODUCTION

Otitis media (OM) is an important upper respiratory tract disease of early childhood and the primary reason for young children to visit a physician. The disease has a considerable negative impact on the quality of life during childhood and causes much concern to parents. OM encompasses a spectrum of conditions, including acute otitis media (AOM) and otitis media with effusion (OME), with approximately 80% of children having experienced an episode of AOM by the age of 3 years. Up to one-third of these children will have experienced recurrent infections, with many of these episodes being facilitated by a bacterial infection (3, 37). In fact, bacteria may be isolated from the middle ear fluid (MEF) of approximately 80% of children with AOM and 30 to 50% of chronic middle ear effusions obtained from children presenting with OME (12). In many countries, OM is a common reason to prescribe antibiotics or to undergo surgery for the insertion of ventilation tubes, resulting in a significant burden on health care systems (21, 25, 29). This means that the direct costs associated with OM are substantial (2) and that the prevention of OM disease via alternative methods such as vaccination offers a promising approach to reduce the burden of OM disease and its economic consequences.

Traditionally, Streptococcus pneumoniae has been reported to be the predominant bacterial species cultured in AOM disease, followed by Haemophilus influenzae and Moraxella catarrhalis. However, H. influenzae tends to predominate in OME disease, followed to a lesser extent by S. pneumoniae and M. catarrhalis (7, 9, 32). Further, although these common OM-related bacterial species may be cultured from the middle ear of children during OM episodes, either as single pathogens or as cocultures (28), research has also shown the importance of (frequently culture-negative) bacterial biofilm formation in the development of middle ear disease (22). Finally, the introduction of a conjugated heptavalent pneumococcal vaccine (PCV7) for use in children in the community has resulted in a significant reduction in the overall proportion of S. pneumoniae isolates and vaccine serotypes observed in AOM. Indeed, the success of vaccination against S. pneumoniae means that H. influenzae is now becoming the predominant pathogen isolated from children suffering from persistent AOM disease (6, 10).

Children are frequently colonized with bacterial pathogens at an early age, and the pattern of nasopharyngeal colonization is an important determinant for OM disease (15, 16). Further, research has also indicated that, as well as the presence of particular bacterial species, both the adaptive and innate immune systems, Eustachian tube dysfunction, viral load, and genetic and environmental factors all may be involved in the pathogenesis of OM (19, 23, 30, 31, 33, 38).

The recent recognition of M. catarrhalis as an important human pathogen has stimulated active investigation into the molecular mechanisms of its pathogenesis. An essential step in colonization and infection is bacterial adherence to the mucosal epithelium of the respiratory tract. A growing number of adhesins have been identified in M. catarrhalis, and most of these proteins are highly conserved and immunogenic and express distinct epitopes on the bacterial surface. This means that they may be suitable as potential M. catarrhalis vaccine candidates (27). However, relatively little is known regarding the development of the natural humoral immune response to these potential vaccine candidates in children. As yet, no licensed vaccine has been marketed against M. catarrhalis, and to date, none of the putative vaccine candidates so far described in the literature have actually progressed to clinical trials.

On the other hand, vaccination against S. pneumoniae infection is already established, for example, via the introduction of the PCV7 vaccine. PCV7 was primarily used to prevent invasive pneumococcal disease (meningitis and other pneumococcal infections such as pneumonia) in children, with the introduction of PCV7 having led to a noticeable reduction in the incidence of S. pneumoniae vaccine strains in the etiology of AOM (13). However, an increase in the carriage of nonvaccine serotypes has been reported, as well as a consequent increase in invasive disease by these nonvaccine serotypes, which could reduce or even negate the benefits initially obtained through vaccination with PCV7 (11, 26). In fact, S. pneumoniae serotype replacement and subsequent vaccine failure in PCV7-vaccinated children have become serious concerns in recent years, with most of these problems assigned to the serotype-specific nature of the PCV7 vaccine. Currently, several pneumococcal surface proteins, either alone or in combinations, have been suggested as putative vaccine candidates (1, 13, 20) and could serve as more effective vaccines than those currently available, providing broad coverage against most pneumococcal serotypes. However, similar to the situation faced by vaccine candidates of M. catarrhalis, relatively little is known about the development of the humoral immune response to S. pneumoniae protein-based vaccine candidates in children.

With this study, we provide insights into the anti-M. catarrhalis and antipneumococcal humoral immune response in a cohort comprising Dutch children exhibiting recurrent (recurrent acute otitis media [rAOM]) and chronic (chronic otitis media with effusion [COME]) episodes of OM. Currently, there is a common perception that rAOM tends to be caused by recurrent episodes of AOM that are associated with bacteria and/or viral infections. In contrast, COME is generally considered a sterile inflammation. Studying the immune response and the accompanying pathogens associated with rAOM and COME will allow us to better distinguish the types of bacterial and immune factors that are involved in the pathogenesis of rAOM and COME and lead to a better understanding of the pathogenesis of these 2 clinical presentations of OM disease.

MATERIALS AND METHODS

RESULTS

DISCUSSION

Studies have shown that M. catarrhalis and S. pneumoniae colonize the nasopharynx soon after birth, and frequent colonization with M. catarrhalis and S. pneumoniae has been reported to increase the risk of OM (15). The adaptive immune system and the innate immune system are also important factors in the pathogenesis of OM (31). The immune response to pathogens develops rapidly during the first few years of life, as children become exposed to an increasing number of microbial species and strains. The high incidence and high rate of spontaneous recovery from OM suggests that recovery is a natural phenomenon and part of the gradual maturation of the child's immune system, though a defective or immature antibody response to OM pathogens may explain the increased susceptibility of some children to OM (36).

Differences in the presence/absence and levels of pathogen-specific IgG and IgM at particular body sites may be one of the mechanisms by which OM pathogens are able to facilitate the development of OM disease. In this study, no significant differences in anti-M. catarrhalis and anti-S. pneumoniae serum and MEF IgG levels were observed between the rAOM or COME groups for all antigens tested, suggesting that differences in IgG levels between body sites do not play a significant role in the development of rAOM or COME disease. Interestingly, however, a strong correlation was observed between IgG antibody levels in serum and IgG antibody levels in MEF, for both M. catarrhalis and pneumococcal proteins for the whole study cohort. This is an interesting finding, as it indicates that serum antibody levels are predictive of the presence of local (middle ear) IgG antibody (14, 17) and raises the possibility that the measurement of OM pathogen-specific IgG antibodies in the middle ear may be achieved by simply measuring OM pathogen-specific IgG antibody levels in serum. At this moment in time, however, it is not known whether OM pathogen-specific IgG is actually produced locally in the middle ear or transudes into the middle ear from the general circulation (4, 14). From our results, it appears more likely that OM pathogen-specific IgG actually transudes into the middle ear rather than being locally produced, where it potentially provides some protection against OM disease. No significant differences in anti-M. catarrhalis and anti-S. pneumoniae MEF IgM levels were observed for either the rAOM or COME cohort in this study.

The local production of secretory antibody is an important immunological defense at epithelial surfaces, including the surfaces of the upper respiratory tract and middle ear. Secretory IgA has been shown to inhibit S. pneumoniae adherence and reduce nasopharyngeal bacterial colonization (18), and it is therefore possible that children with recurrent OM might lack OM pathogen-specific secretory IgA. In this study, the MFI levels of antigen-specific IgA showed extensive interindividual variability over time, with IgA levels to all M. catarrhalis and S. pneumoniae OMPs being relatively low throughout the study period. However, though not significant, S. pneumoniae-negative/COME-positive children showed higher MEF IgA levels against the pneumococcal IgA-1 protease protein than S. pneumoniae-positive/COME-positive children. In fact, many mucosal pathogens, including S. pneumoniae, express an IgA-1 protease that cleaves IgA molecules, thereby circumventing the protective effects of IgA production (40). Our results suggest that IgA antibody directed against anti-IgA-1 protease may have a restorative effect by helping to neutralize the effect of secreted anti-IgA-1 protease, thereby facilitating the binding of intact IgA to bacterial cells and promoting pathogen recognition and clearance by the immune response. However, it should be noted that the possibility exists that the differences in antibody levels observed in S. pneumonia-positive and -negative COME MEFs are simply a product of antibody binding to S. pneumoniae bacterial cells, thereby making the antibodies inaccessible to measurement using our Luminex assay. Further research will be required in order to determine whether IgA-1 protease antibodies actually provide protection in patients suffering from OM disease.

Studying the immune response and pathogens associated with rAOM and COME disease indicated that COME is not in fact a sterile inflammation as is generally considered the case. Importantly, investigation into the immune response to potential vaccine candidates of M. catarrhalis and S. pneumoniae indicated that there may be a lack of a characteristic immune response profiles that will allow clinicians to distinguish between rAOM and COME. Our data correlate with the findings of Stol et al. (submitted), who investigated bacterial colonization and infection in the nasopharynx and middle ear of the same prospective cohort of children and found no clear-cut differences in the microbial flora present in middle ear fluids obtained during rAOM and COME disease.

In summary, the levels of antigen-specific serum IgG and MEF IgG/IgM/IgA showed extensive interindividual variation in our rAOM and COME cohorts of children, with no significant differences between the 2 groups. Both rAOM and COME children possessed a range of antibodies (IgG, IgA, and IgM) against a variety of novel M. catarrhalis and S. pneumoniae recombinant proteins, and there were no distinguishing immune response profiles observed between the 2 groups. Factors such as Eustachian tube dysfunction, viral load, and genetic and environmental factors may play a more important role in the pathogenesis of rAOM and COME disease than the humoral immune response to OM-associated bacterial pathogens.