Specific immune response to outer membrane protein of nontypable H.influenzae in otitis-prone children
P6 of nontypable H.influenzae is highly conserved among strains, and serves as a target for bactericidal antibody. Serum antibody response to P6 has been studied in our laboratory in the general population and both otitis-prone and non-otitis-prone children. Thirty of the subjects were classified as otitis-prone because they had experienced four or more episodes of otitis media in the first year of life or six or more episodes by the second year, or needed placement of tympanostomy tubes.
Anti-P6 anitibody levels in the general population were examined. At birth, anti-P6 IgGantibody was found in almost the same level as that of adults, whereas no IgM or IgA antibodies spcific for P6 were detected. Aniti-P6 antibody levels in the three isotypes studied were lowest at 6 months of age and rose significantly after two years ; IgG levels peaked at 10 years whereas IgM and IgA peaked at six y
ears. In every age-group, IgG antibody specific for P6 was in the highest concentration among the three isotypes. Anti-P6 IgG antibody was detected in all individuals in each age group, however, IgM antibody specific for P6 was detected in all individuals older than 6 year of age, and IgA antibody specific for P6 was detected in all individuals only after 10 years of age.
Anti-P6 antibody levels were measured longitudinally in 30 otitis-prone and 13 healthy children on 93 and 32 occasions, respectively. The age at time of sampling varied between one and 92 months. Antibody levels increased seven fold in the normal group over 36 months compared to less than three fold for the otitis-prone group over 48 months. The levels of antibody in the normal group were significantly higher than those in the otitis-prone group after the age of 18 months. In general, individual antibody levels in otitis-prone individuals did not demonstrate an age-dependent rise. The comparison of anti-P6 IgG antibody between otitis-prone children and healthy children under the age of 5 years was carried out. It is very interesting to note that 80% of otitis-prone children show the anti-P6 antibody level below 7 ug/ml (mean-2SD of ages 6 months to 6 years in healthy children). Furthermore, children who experienced two or more episodes of otitis media due to nontypable H.influenzae failed to manifest an anamnestic antibody response to P6. Immunoglobulin IgM and IgA antibody responses to P6 in otitis-prone children reached a plateau after 18 months of age, and anti-P6 IgM antibody level remained below the adult serum level even after 4 years of age. Difference between otitis-prone and healthy children were not statistically significant.
In our study, otitis-prone children were not unusually vulnerable to infections except otitis media This fact seems to refute the presence of a broad immunologic deficit in the children. However, children who experienced recurrent episodes of otitis media due to nontypable H.influenzae did not mount a normal response to P6 during otitis media and failed to develop a secondary immune response upon repeated challenge. The failure to recognize P6 as a specific immunogen may account for recurrent infections.
We also examined the local immune response to NTHI in middle ear fluids and in the nasopharyngeal secretions. The concentration of P6 antibody in the middle ear was inversely related to the number of viable bacteria present present in the middle ear fluid (r=0.62, p<0.05), suggesting a direct inhibitory effective antibody on the bacteria. Furthermore, the concentration of P6-specific IgG in the middle ear was directly related to the concentration of P6 antibody in the serum (r=0.89, p<0.001).These findings again emphasize the importance of specific serum bactericidal antibody of the IgG isotype directed against P6 as being responsible for at least one component in the immune resolution of NTHI in the middle ear. IgA and secretory IgA antibodies to P6 were common (96% and 95%, respectively) and in relatively high concentration in nasopharyngeal secretions. The concentration of P6 antibody in the nasopharynx was compared in children with and without NTHI.As seen in Figure, antibody concentrations were higher in the group without the bacteria. This result infers that local antibody in the nasopharynx redueed and/or prevented colonization with NTHI.There was no relationship between nasopharyngeal and serum levels of antibodies, suggesting that antibody in the nasopharynx is predominantly locally produced.
Our data suggest that otitis proneness may be related to specific immunological deficiencies. This defect may be present in both the local and systemic immune systems.
Preumococcal capsular polysaccharide-specific IgG subclass antibodies
S.pneumoniae is a common pathogenis organism causing respiratory and systemic infections including otitis media in adults and children. In order to assess specific antibody responses to pneumococcal capsular polysaccharide (PCP), polyvalent pneumococcal vaccine has been used as a coating antigen in ELISA and such assays are successful in evaluating specific antibodies to PCP antigens. A polyvalent pneumococcal vaccine (PneumovaX ; Merck Sharp & Dohme, West Point, PA,USA) co 23 serotypes : 1,2,3,4,5,6B,7F,8,9N,9V,10A,11A,12F,14,15B,17F,18C,19A,19F,20,22F,23F and 33F was used as coating antigen in ELISA.Pneumovax-specific IgG subclass antibodies was measured quantitatively by the ELISA technique described by Ishizaka et al. When measuring Pneumovax-specific IgG1 and IgG2 concentrations in each test serum, the plates were coated with a 1 : 10 dilution of Pneumovax, and the purified solution containing Pneumovax-specific antibodies was used as a reference standard in the ELISA method.
As previously described, evidence from early studies that human antibodies to polysaccharide antigens were largely restricted to IgG2 suggested that the susceptibility to pneumococcal infection might be caused by a selective deficiency of IgG2 antibody. In response to reports that IgG2 deficiency is more common than was previously appreciated, the serum concentration of IgG2 is often used clinically as a marker for susceptibility to infection. In our series of otitis-prone children, however, only 7% of otitis-prone children show serum IgG2 level below 80mg/ml(2SD below the mean for age of 1 year) as shown in Figure. This result brings about skepticism regarding a simple relationship between low serum IgG2 concentrations and susceptibility to infection including otitis media. The study of Pneumovax-specific IgG2 antibody demonstrated that 79% of otitis-prone children had subnormal concentrations of the specific antibody. It is very interesting to note that over 90% of the children with subnormal Pneumovax-specific IgG2 antibody level showed normal serum concentrations of IgG2.
Taken together with data of P6-specific antibody in otitis-prone children, the above result clearly indicates that pathogen-specific IgG or IgG2 antibodies are highly reliable immunological paramenters for the otitis-prone condition. Therefore, in a child who is being examined for recurrent otitis media, measurement of the response to pneumococcal capsular polysaccharide and P6 of nontypable H.influenzae should be useful in determining whether immunological deficiency may contribute the otitis-prone condition.
New strategies for the prevention of otitis media-Preventive vaccine for otits media
The pathogen causing otitis media originates mainly from the adenoid or nasopharyngeal space. The resolution of otitis media by immunological mechanisms within the middle ear space is most likely the result of pathogen-specific IgG antibodies and complement which reaches the middle ear from the serum. Secretory IgA,on the other hand, may be responisible for coating the bacteria and preventing its attachment to the middle ear mucosa. Efforts to reduce the otitis-prone condition will include augmenting the production of systemic specific IgG antibody and local specific secretory IgA antibody.
Systemic vaccination with specific outer membrane proteins of nontypable Haemophilus influenzae or P6 or the use of specific fimbrial proteins that may be conserved in NTHI, may be used to stimulate specific antibodies that may be bactericidal. Regarding mucosal immunity, an effort to slicit local specific secretory IgA antibody has just atarted. Oral immunization may stimulate gut precursors of B cells in Peyer's patches that may seed the upper respiratory tract and produce specific IgA in the nasopharyngeal secretion. This has been demonstrated for both nontypable Haemophilus influenzae and Pneumococcus in the experimental animal modell. Furthermore, there is evidence that oral immunization with bacterial vaccines may activate macrophage by stimulating metabolic and functional properties in macrophages that are characteristic of the activated atate and are important for host defense. Recently the evidence showing the existence of nose-associated lymphoid tissue (NALT) has been accumulating and NALT may play important roles in immune responses of the upper respiratory tract. The nasal mucosa will be one of the highly efficient parts for the induction of specific mucosal immunity. Although an equivalent organ of NALT in human has not been cleared yet, nasal immunization may stimulate nasal precursors of B cells in NALT-equivalent organ that may seed immunocometent cells in the upper respiratory tract and distribute memory cells to the systemic immune organs. The cells will produce immunogen-specific IgA in the nasopharygeal secretion and IgG in sera.
We designed animal experiments to investigate whether the intranasal immunization of P6 with cholera toxin B subunit subunit (CTB) would induce specific mucosal immunity against nontypable H.influenzae. Intranasal immunization of P6 with every 2 days interval for 2 weeks efficiently elicited P6-specific secretory IgA antiboby. A good IgG antibody response to P6 was also found in this approach. It is clinically very important whether these P6-specific antibodies will protect infections of H.influenzae. Our study showed that the antibodies significantly prevented the colonization of nontypable H.influenzae in the nasopharynx of mice. Taken together with our results and the fact that P6 is a common antigen among strains of nontypable and type b H.influenzae, intranasal immunization of P6 is one of promising strategies for the prevention of infections caused by H.influenzae including otitis media. Less