STUDY OF THE PATHOGENESIS IN RECURRENT OTITIS MEDIA AND THE DEVELOPMENT OF PREVENTIVE VACCINE

对中耳炎易发儿童非分型流感嗜血杆菌外膜蛋白的特异性免疫应答p6在菌株间高度保守，可作为杀菌抗体的靶点。在我们的实验室研究了普通人群以及中耳炎易发和非中耳炎易发儿童对P6的血清抗体反应。其中30名受试者被归类为中耳炎易发人群，因为他们在出生的第一年经历了四次或更多的中耳炎发作，第二年经历了六次或更多的中耳炎发作，或者需要放置鼓室造口管。检测普通人群的抗p6抗体水平。出生时，抗P6 IgGantibody的水平与成人几乎相同，而未检测到针对P6的IgM或IgA抗体。三种同型犬的抗p6抗体水平在6月龄时最低，2年后显著升高；IgG水平在10岁时达到峰值，而IgM和IgA水平在6岁时达到峰值。在各年龄组中，P6特异性IgG抗体的浓度均最高。各年龄组均检测到抗P6 IgG抗体，6岁以上均检测到P6特异性IgM抗体，10岁以上均检测到P6特异性IgA抗体。在30名中耳炎易发儿童和13名健康儿童中分别测定了93次和32次抗p6抗体水平。抽样时的年龄在1至92个月之间。正常组的抗体水平在36个月内增加了7倍，而中耳炎易发组在48个月内增加了不到3倍。18个月后，正常组的抗体水平明显高于中耳炎易发组。一般来说，中耳炎易发个体的抗体水平没有表现出年龄依赖性的上升。比较5岁以下中耳炎易发儿童与健康儿童的抗p6 IgG抗体水平。非常有趣的是，80%的中耳炎易感儿童显示抗p6抗体水平低于7 ug/ml（6个月至6岁健康儿童平均- 2sd）。此外，由于非分型流感嗜血杆菌而经历两次或两次以上中耳炎发作的儿童未能表现出对P6的遗忘抗体反应。中耳炎易发儿童对P6的免疫球蛋白IgM和IgA抗体反应在18个月后达到平台期，抗P6 IgM抗体水平在4岁后仍低于成人血清水平。中耳炎易发儿童与健康儿童的差异无统计学意义。在我们的研究中，除了中耳炎外，中耳炎易感儿童对感染的易感程度并不高，这一事实似乎反驳了儿童普遍存在免疫缺陷的说法。然而，由于非分型流感嗜血杆菌而经历中耳炎反复发作的儿童在中耳炎期间对P6没有正常的反应，并且在反复攻击后未能产生二次免疫反应。不能识别P6作为特异性免疫原可能是反复感染的原因。我们还检查了中耳液和鼻咽分泌物对NTHI的局部免疫反应。中耳液中P6抗体浓度与存在的活菌数呈负相关（r=0.62, p<0.05），提示抗体对细菌有直接抑制作用。中耳P6特异性IgG浓度与血清P6抗体浓度直接相关（r=0.89, p<0.001）。这些发现再次强调了针对P6的IgG同型特异性血清杀菌抗体的重要性，至少在中耳NTHI的免疫解决中起着一个作用。针对P6的IgA和分泌性IgA抗体较为常见（分别为96%和95%），且在鼻咽分泌物中浓度较高。比较NTHI患儿和非NTHI患儿鼻咽部P6抗体的浓度。如图所示，无细菌组的抗体浓度较高。这一结果推断鼻咽部的局部抗体减少和/或阻止了NTHI的定植。鼻咽部和血清抗体水平之间没有关系，提示鼻咽部抗体主要是局部产生的。我们的数据表明，中耳炎的易感性可能与特定的免疫缺陷有关。这种缺陷可能存在于局部和全身免疫系统。肺炎球菌荚膜多糖特异性IgG亚类抗体。肺炎是一种常见的病原体，可引起呼吸道和全身感染，包括成人和儿童中耳炎。为了评估对肺炎球菌荚膜多糖（PCP）的特异性抗体反应，在ELISA中使用多价肺炎球菌疫苗作为包被抗原，这种检测方法成功地评估了针对PCP抗原的特异性抗体。ELISA采用多价肺炎球菌疫苗（PneumovaX; Merck Sharp & Dohme, West Point， PA，USA）共23种血清型：1、2、3、4、5、6B、7F、8、9N、9V、10A、11A、12F、14、15B、17F、18C、19A、19F、20、22F、23F和33F。采用Ishizaka等人描述的ELISA技术定量检测肺炎病毒特异性IgG亚类抗体。测定各检测血清中Pneumovax特异性IgG1和IgG2浓度时，将Pneumovax按1:10稀释后包被，含Pneumovax特异性抗体的纯化溶液作为ELISA法的参比标准品。如前所述，早期研究的证据表明，人对多糖抗原的抗体主要局限于IgG2，这表明对肺炎球菌感染的易感性可能是由IgG2抗体的选择性缺乏引起的。有报道称IgG2缺乏症比以前认为的更常见，因此血清IgG2浓度在临床上常被用作感染易感性的标志。然而，在我们的中耳炎易发儿童系列中，只有7%的中耳炎易发儿童血清IgG2水平低于80mg/ml（低于1岁平均水平2SD），如图所示。这一结果引起了对低血清IgG2浓度与中耳炎等感染易感性之间的简单关系的怀疑。pneumovax -特异性IgG2抗体的研究表明，79%的中耳炎易感儿童特异性抗体浓度低于正常水平。值得注意的是，在肺炎链球菌特异性IgG2抗体水平低于正常水平的患儿中，超过90%的患儿血清IgG2浓度正常。结合中耳炎易发儿童p6特异性抗体的数据，上述结果清楚地表明，病原体特异性IgG或IgG2抗体是中耳炎易发条件下高度可靠的免疫学参数。因此，在接受复发性中耳炎检查的儿童中，测量对肺炎球菌荚膜多糖和非分型流感嗜血杆菌P6的反应应有助于确定免疫缺陷是否可能导致中耳炎易发状况。预防中耳炎的新策略——预防中耳炎的疫苗引起中耳炎的病原体主要来自腺样体或鼻咽间隙。中耳炎通过中耳腔内的免疫机制解决，很可能是病原体特异性IgG抗体和补体从血清中到达中耳的结果。另一方面，分泌IgA可能负责包裹细菌并防止其附着在中耳粘膜上。努力减少中耳炎易发条件将包括增加生产全身特异性IgG抗体和局部特异性分泌IgA抗体。系统接种非分型流感嗜血杆菌或P6的特异性外膜蛋白，或使用可能在NTHI中保守的特异性毛膜蛋白，可用于刺激可能具有杀菌作用的特异性抗体。关于粘膜免疫，筛选局部特异性分泌IgA抗体的努力刚刚开始。口服免疫可刺激Peyer’s patches中B细胞的肠道前体，这些前体可进入上呼吸道并在鼻咽分泌物中产生特异性IgA。这在实验动物模型中已被证实为非分型流感嗜血杆菌和肺炎球菌。此外，有证据表明，细菌疫苗的口服免疫可以通过刺激巨噬细胞的代谢和功能特性来激活巨噬细胞，这些特性是活化物的特征，对宿主防御很重要。近年来，越来越多的证据表明鼻相关淋巴组织（NALT）的存在，NALT可能在上呼吸道的免疫应答中发挥重要作用。鼻黏膜将是诱导特异性粘膜免疫的高效部位之一。虽然人体中NALT的等效器官尚未被清除，但鼻腔免疫可刺激NALT等效器官中B细胞的鼻腔前体，从而在上呼吸道播下免疫功能细胞，并将记忆细胞分布到全身免疫器官。这些细胞在鼻咽分泌物中产生免疫原特异性IgA，在血清中产生免疫原特异性IgG。我们设计了动物实验来研究用霍乱毒素B亚单位（CTB）鼻内免疫P6是否会诱导对非分型流感嗜血杆菌的特异性粘膜免疫。P6的鼻内免疫每2天间隔2周，有效地激发P6特异性分泌IgA抗体。在这种方法中还发现对P6有良好的IgG抗体反应。这些p6特异性抗体是否具有保护流感嗜血杆菌感染的作用，在临床上具有重要意义。我们的研究表明，抗体显著阻止非分型流感嗜血杆菌在小鼠鼻咽部的定植。结合我们的研究结果，以及P6在非分型型和b型流感嗜血杆菌株中是一种常见抗原的事实，鼻内免疫P6是预防流感嗜血杆菌感染（包括中耳炎）的一种有希望的策略。少

Specific immune response to outer membrane protein of nontypable H.influenzae in otitis-prone childrenP6 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 … More 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 antibodiesS.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 mediaThe 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