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*Ear Infections
The Journal of Immunology, 2003, 171: 1978-1983.
Copyright © 2003 by The American Association of Immunologists

The Fourth Surface-Exposed Region of the Outer Membrane Protein P5-Homologous Adhesin of Nontypable Haemophilus influenzae Is an Immunodominant But Nonprotective Decoying Epitope1

Laura A. Novotny and Lauren O. Bakaletz2

Division of Molecular Medicine, Department of Pediatrics, Columbus Children’s Research Institute, Ohio State University College of Medicine and Public Health, Columbus, OH 43205


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Nontypable Haemophilus influenzae is a major cause of otitis media and other mucosal infections. After natural disease in children and experimental disease in chinchillas, we found a hierarchical pattern of immunodominance among the four surface-exposed regions of the P5-homologous adhesin, with the greatest response directed to region 4. However, Ab to region 4 is not protective. When this natural but biased response was refocused to region 3 by immunization, augmented bacterial clearance and protection from ascending otitis media was observed. Collectively, the data indicate that region 4 contains a highly immunodominant but nonprotective decoying epitope, the presence of which dampens the immune response to a subdominant but protective epitope in region 3.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Nontypable Haemophilus influenzae (NTHI)3 is a predominant causative agent of chronic otitis media (OM) or OM with effusion as well as being responsible for roughly one-third of all cases of acute OM (1), a highly prevalent pediatric disease. Despite the fact that children do mount an immune response to NTHI during episodes of OM, these responses are generally highly strain specific, leaving the child susceptible to a new episode of OM upon acquisition of another of these diverse bacterial isolates. In fact, the recurrent nature of OM unequivocally demonstrates that naturally acquired infections of the middle ear do not induce the desired protective immune response (2, 3). Thus, it has been suggested that the immune systems of otitis-prone children tend to recognize only strain-specific heterologous surface or outer membrane proteins (OMPs) expressed by NTHI (such as the predominant OMP P2). Alternatively, otitis-prone children may not develop an adequate immune response to any Ag (3, 4).

This lack of protective immunity has also been observed in animal models where it has been found to be due to the highly strain-specific nature of the immune response that is induced (5, 6, 7, 8, 9, 10). Thereby, neither natural immunity nor immunization with any of several whole OMPs of NTHI in experimental models results in a protective response.

Nevertheless, several OMPs, other surface proteins, and lipooligosaccharides expressed by NTHI continue to be targets for development of vaccine candidates against OM (2). Due to the importance of adherence and colonization in the course of OM disease, we have focused our efforts in vaccine development on one of the several known adhesins expressed on the surface of NTHI, the OMP P5-homologous adhesin, or P5-fimbrin. This 36.4-kDa bacterial protein mediates adherence to human oropharyngeal cells and mucin (5, 11, 12), chinchilla Eustachian tube mucus (13), respiratory syncytial virus-infected A549 cells (14), and the carcinoembryonic Ag family of cell adhesion molecule-1-transfected Chinese hamster ovary cells (15). However, although partial homologous protection is afforded after immunization with isolated P5-fimbrin (5, 6), chinchillas are not protected against OM that is induced by heterologous challenge. Moreover, there is also a variable response to this adhesin in children with OM (16).

We thus concluded that due to the heterogeneity of NTHI OMPs and surface proteins, including P5-fimbrin (6, 17, 18), attempting to use this adhesin in its entirety as an immunogen would not likely result in the desired broad cross-strain protection. To overcome this obstacle, we have been attempting to more fully characterize the immune response to P5-fimbrin and to define a minimal protective epitope(s) of this NTHI adhesin. We have observed that Abs in the sera recovered from several children with acute OM due to NTHI and middle ear fluids (MEFs) collected from a single immunologically naive chinchilla with experimental NTHI-induced OM recognize the surface-exposed regions of P5-fimbrin in a similar manner (12). There are four predicted surface-exposed regions located in the N-terminal half of this bacterial surface protein (6, 17, 18), each of which displays variable degrees of sequence diversity. Although it is possible that each of these four epitopes is an immunoreactive domain, algorithmic analysis predicted relatively poor reactivity of regions 1 and 2 compared with regions 3 and 4 (6). These predictions have been supported by a study that showed very poor immunogenicity of a region 1-based immunogen (19) as well as another study that showed that regions 3 and 4 are indeed the primary targets of serum Ab as well as Abs present in MEFs (12). However, when immunogens based on these latter two focused regions were designed and tested in two animal models, it was observed that immunity to region 3 was highly protective whereas immunity to region 4 was not (6, 19, 20, 21).

Although not yet fully understood, it has been recognized for some time that the immune system preferentially responds to only a few of the total available epitopes in a multideterminant Ag. These selective, epitope-specific primary responses also influence the hierarchy of immunodominance observed in later, secondary responses (22, 23). Whereas in the past we have described the ability of several immunogens derived from surface-exposed region 3 of the P5-homologous adhesin to confer augmented bacterial clearance or protection from development of experimental OM, we have not extensively characterized the quality of the induced protective Ab response in those animal models, nor have we compared that response to the nonprotective response induced by either experimental OM in chinchillas or natural OM in children. Thereby, in the present study, we further assessed the pediatric immune response to the noncontiguous surface-exposed epitopes of P5-fimbrin during an acute episode of OM caused by NTHI. This report also includes complementary studies examining sera from chinchillas that have been immunized with an individual epitope of P5 (6, 12, 24, 25) that elicited protective Abs.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Synthetic peptides

Synthetic peptides designed to map P5-fimbrin were synthesized and purified as previously described (12). The location of each of the peptides representing the four predicted surface-exposed regions of this adhesin can be seen in Fig. 1.



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  		FIGURE 1. Schematic diagram of synthetic peptides used to map P5-fimbrin. Numbering is based on the translated amino acid sequence of unprocessed P5-fimbrin.

 
Antisera and MEFs

MEFs were retrieved sequentially (every 3–4 days) from seven immunologically naive chinchillas during a 5-wk period of observation. These fluids were recovered by epitympanic tap of animals that had been both transbullarly and intranasally challenged with NTHI strain 86-028NP. Blood was also collected from these animals for serum 35 days after bacterial challenge. All animal studies were performed in concordance with institutional and federal guidelines and were conducted under an approved protocol.

Sera collected from a total of 19 children ages 3 mo–7 years who were undergoing tympanocentesis for an episode of acute OM were kindly provided to us by Dr. M. E. Pichichero (University of Rochester, Rochester, NY). The effusions present in the middle ears of each of these children were also recovered at the time of serum collection and confirmed to be culture positive for NTHI. In addition, sera were collected from four 2-mo-old infants with no known history of OM as well as from several adults. Blood was collected after obtaining informed consent and in concordance with all institutional and federal guidelines for the use of human subjects.

Blood for serum was also collected from nine chinchillas that had been immunized with a total of 30 µg of the synthetic chimeric peptide immunogen LB1 delivered in 200 µl of CFA or IFA as previously described (22) and detailed briefly below. Blood was obtained for serum 10 days after the final boosting dose of 10 µg was given.

Chinchilla model

As reported previously (25), nine adult chinchillas (Chinchilla lanigera) were immunized s.c. at monthly intervals with three doses of 10 µg of LB1. The primary immunization was delivered in CFA, whereas both subsequent boosting doses were delivered in IFA. Immune chinchillas were bled for serum 10 days after receipt of the final immunization. For passive transfer studies, these individual sera were pooled, and aliquots of the pooled hyperimmune serum pool were passively transferred by intracardiac perfusion (5 ml/kg) to cohorts of juvenile chinchillas 6 days after they had been challenged intranasally with adenovirus serotype 1. Twenty-four hours after passive transfer of the anti-LB1 serum pool, these juvenile chinchillas (data from 6 cohorts of 11 animals each are presented here) were challenged IN with ~108 CFU of either NTHI strain 86-028NP (a majority group 1 isolate), strain 1885 MEE (a minority group 2a isolate), or strain 1729 MEE (a minority group 3 isolate) (24).

Animals were blindly evaluated by otoscopy and tympanometry (EarScan, South Daytona, FL) daily or every 2 days from the time of adenovirus inoculation until 35 days after NTHI challenge. Signs of tympanic membrane inflammation were rated on a scale of 0 to 4+ (5, 26), and tympanometry plots were used to monitor changes in middle ear pressure, tympanic width, and tympanic membrane compliance (27, 28, 29). Tympanometry results indicated an abnormal ear if 1) a type B tympanogram was obtained, 2) compliance was <=0.5 or >=1.2 ml, 3) tympanic width was >150 daPa, or 4) middle ear underpressure was greater than -100 daPa. Clinical signs of viral respiratory tract infection (ruffling of fur, conjunctivitis, altered character of nasal/ocular secretions, wheezing, labyrinthitis, and cornering behavior) were recorded. Observers knew neither the antiserum received nor which animals formed a cohort group throughout the 35-day observation period after NTHI challenge.

For reference, when immunologically naive or sham-immunized juvenile chinchillas, with an ongoing AV infection, are challenged intranasally with NTHI, culture-positive OM begins to develop ~7 days after bacterial challenge with peak incidence of OM occurring ~12–14 days after NTHI challenge. OM typically resolves in all animals ~5 wk after challenge (24, 25).

Sensor chip preparation for immobilization of synthetic peptides

Using a Biacore 2000 (Biacore AB, Uppsala, Sweden), reagent grade CM5 sensor chips were activated with 35 µl of 400 mM N-ethyl-N'-(3-diethylaminopropyl)carbodiimide-100 mM N-hydroxysuccinimide solution at a flow rate of 5 µl/min. Synthetic peptides representing each of the four surface-exposed regions of P5-fimbrin were suspended in 10 mM sodium acetate, pH 4.5, and injected to immobilize ~0.1–0.2 ng of peptide per mm2 of sensor chip surface. Excess ester groups were deactivated, and any loosely bound peptide was removed by injection of 35 µl of 1.0 M ethanolamine hydrochloride-NaOH.

Analysis of chinchilla middle ear fluids and both chinchilla and pediatric sera

To assay reactivity of sera or MEFs against synthetic peptides, 10 µl of each chinchilla MEF and serum sample or pediatric serum sample, diluted 1/5 in 0.01 M HEPES (pH 7.4), 0.15 M NaCl, 3 mM EDTA, 0.005% v/v surfactant P2, was exposed to the CM5-immobilized peptides. Before assay, MEFs were briefly centrifuged to remove cellular debris. The sensor chip surface was regenerated with a 5- or 10-µl pulse of 10 mM glycine-HCl after a dissociation period of ~95 s. For all sera and MEFs, biosensor assays were run against each synthetic peptide ligand a minimum of twice. Bars in each figure represent the average resonance unit (RU) value obtained for each individual animal or child.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Using MEFs collected sequentially every 3–4 days from seven naive chinchillas with experimental NTHI-induced OM and a biosensor-based assay system to analyze the relative reactivity of these fluids to the peptides representing the four surface-exposed regions of the P5 adhesin, we found that Ab within these effusions bound preferentially to peptides representing the third and fourth surface-exposed regions (Fig. 2A). Reactivity of these region-specific responses peaked between 7 and 28 days after NTHI challenge in individual animals. The semi-independent nature of the immune response in individual ears of a single animal can be seen in the responses of the left (L, peak on day 7) and right (R, peak on day 10) ears of animal 5; however, the overall pattern of reactivity is the same.



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  		FIGURE 2. Data obtained by biosensor analysis using middle ear fluids. These fluids were collected sequentially every 3–4 days during a 5-wk period of observation from seven immunologically naive chinchillas with active OM induced by NTHI challenge on the day that their overall response to these four peptides was greatest (A) and reactivity of sera from the same seven animals obtained 35 days after challenge with NTHI (B).

 
In addition to the reactivity of fluids that are depicted in Fig. 2A on the day that the RU values were greatest for the MEFs of any given animal, we also assayed fluids recovered sequentially from both ears of each of these animals on a total of 8 assessment days from 4 to 35 days after challenge (data not shown). An early and greater reactivity to the region 4 peptide over that to regions 1–3 was a consistent observation in MEFs recovered from immunologically naive chinchillas with ongoing experimental OM due to NTHI. On each of these days, three to seven unique MEFs were assayed (with the exception of day 21 on which a single MEF was recovered), and in all instances the average response in RU to the region 4 peptide exceeded that of the response to the region 1, 2, and 3 peptides by 2.1- to 6.9-fold; 1.9- to 19.2-fold, and 1.5- to 3.1-fold, respectively.

Biosensor data were also generated using sera collected from these animals after OM had resolved and NTHI had been cleared from the directly challenged middle ears, as evidenced by recovery of sterile lavage fluids (Fig. 2B). Whereas absolute RU values, which reflect relative Ab levels, were lower overall, the same pattern of hierarchical recognition is maintained, with greatest recognition demonstrated against region 4 followed by region 3. There was minimal recognition of peptides representing either surface-exposed region 1 or 2.

Sera collected from six children with an ongoing episode of acute OM due to NTHI were also strongly reactive in this assay system (Fig. 3A). Reactivity of sera from six children with acute OM was directed exclusively toward peptides representing the third and fourth surface-exposed regions of this adhesin for all six children (region 3, mean 264 ± 114 RU; region 4, mean 548 ± 259 RU). These findings were highly analogous to those obtained using both chinchilla MEFs and sera collected during experimental disease (Fig. 2). For comparison, RU values obtained using sera collected from adults (<107 RU), children with OM due to Streptococcus pneumoniae (<88 RU), or 2-mo-old infants with no history of OM (<11 RU) were largely unremarkable against any of these peptides (data not shown).



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  		FIGURE 3. Reactivity of sera from six children with acute OM (A) and from immune chinchillas (B) against peptides representing the four surface-exposed regions of this bacterial adhesin.

 
We then assayed serum collected from animals that had been parenterally immunized with a region 3-based synthetic peptide immunogen (LB1) (6). As might be expected, the Ab response in chinchillas immunized with the region 3-based peptide made a robust response to the immunizing Ag with little or no reactivity to the other three surface-exposed peptides (Fig. 3B). Chinchilla immune sera showed significant reactivity (608 ± 322 RU) against only the region 3-derived peptide. These findings were corroborated via ELISA wherein the median reciprocal titers against peptides representing regions 1, 2, and 4 were 500, whereas that for the region 3 peptide was 50,000.

These data showed that high titered Ab to region 3 could be induced when animals were immunized with a vaccine candidate that lacked the highly immunodominant region 4. These sera were then pooled and used in a passive transfer experiment in chinchillas (25). Solid protection is observed in animals challenged with strain 86-028NP (a majority group 1 isolate) when both augmented clearance of NTHI from a colonized nasopharynx (not shown) and protection from development of ascending OM (Fig. 4) were used as outcome measures. This particular finding was perhaps not unexpected given that the amino acid sequence of region 3, on which the immunogen LB1 is based, is well conserved. Seventy-six percent of a panel of 99 NTHI isolates examined had the consensus sequence found in the third region of P5-fimbrin expressed by strain 86-028NP (24, 25). However, chinchillas are also protected from induction of OM when challenged with either of two heterologous isolates. Strains 1885 MEE (a minority group 2a isolate) and 1728 MEE (a minority group 3 isolate) are clinical isolates with 15 amino acid substitutions or 6 substitutions plus 9 fewer amino acids in toto, respectively, than did strain 86-028NP in terms of diversity within region 3 of the P5 adhesin.



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  		FIGURE 4. Percentage of ears with OM within cohorts of adenovirus-compromised chinchillas after intranasal challenge with homologous and heterologous NTHI strains (25 ). Data demonstrate the cross-strain protection afforded by passive transfer of antiserum from chinchillas that had been immunized with a region 3-based immunogen (LB1) compared with cohorts of chinchillas that received a sham immunization. Each cohort is composed of 11 juvenile chinchillas.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have shown in the present study that children with acute OM and chinchillas experiencing an active middle ear infection due to NTHI preferentially recognized the third and fourth predicted surface-exposed regions of mature native P5-fimbrin, with greatest absolute recognition directed toward the latter two of these epitopes. These findings suggested that whereas both region 3 and region 4 elicit the production of Abs, the fourth region is the most immunodominant epitope of this adhesin as presented by NTHI in the environment of the middle ear during active disease. Nevertheless, as previously mentioned, it has been shown that despite the presence of high titered and specific Abs to NTHI in sera and MEFs, chinchillas, like children, are fully susceptible to reinfection with another strain of NTHI (3, 7). Thus, in terms of the selective immunodominance of the four surface-exposed regions of P5-fimbrin, the immune response described here that occurs in children or chinchillas during active NTHI-induced OM is not a protective one. Moreover, despite the clear natural immunodominance of surface-exposed region 4, immunization with vaccine candidates derived from this epitope are ineffective in animal models (17, 18), unlike the highly protective response that can be elicited via immunization with vaccine candidates derived from the qualitatively subdominant region 3 (24, 25).

These combined findings suggested to us that surface-exposed region 4 might be an immunological decoy, as Ab directed to this epitope appears to have little or no role in either ability to promote bacterial clearance from the NP or ability to protect against ascending OM and thus fits the paradigm of a decoying epitope (23), one that has evolved as part of an immune evasion strategy. By polarizing the immune response toward production of nonprotective Abs, this partisan recognition of the fourth epitope may serve as a means of protecting the more functionally important but less immunodominant epitope(s) in surface-exposed region 3. For bacteria for which the preferential ecological niche is the pediatric nasopharynx, one could theorize that by presenting a highly immunodominant epitope on the bacterial surface that elicits the production of Ab of limited or no protective function (i.e., clearance, opsonization, ability to fix complement, etc.), NTHI could protect other more critical but subdominant epitope(s). Thus, this immune evasion technique may provide a mechanism for NTHI to maintain its characteristic, long term colonization state in the nasopharynx.

Whereas multiple examples exist in the literature of such a strategy used by viral and protozoal pathogens which fit with the concept of original antigenic sin (22, 23, 30, 31, 32), to date few such examples exist for bacteria. This deceptive strategy may, however, be one that is used rather globally by H. influenzae to evade the host’s immune response as immunodominant regions of another predominant OMP (P1) also induce a nonprotective response (33). Additionally, whereas loop 5 of P2 is the major immunodominant target of type-specific Abs directed against this NTHI OMP, it is also the most sequence variable of the eight surface-exposed regions (34). Interestingly, peptides derived from the less dominant, more conserved loop 6 were capable of eliciting bactericidal antisera capable of binding 14 of the 15 NTHI strains assayed (35). Hence, by locking the immune response onto a dominant decoying epitope of a multideterminant Ag, this group of microorganisms could ensure that, via the mechanisms of deceptive imprinting, the host would continue to mount and expand an ineffective immune response to that epitope even when reinfected with a different but antigenically related member of the same microbial family (22).

Importantly however, our data also showed that we were able to bypass this naturally immunodominant but nonprotective response to region 4 of P5-fimbrin by refocusing the host’s immune response toward a subdominant but protective epitope in region 3. Sera collected from chinchillas immunized with the region 3-based LB1 (a synthetic chimeric peptide) demonstrated a highly distinct pattern of recognition of peptides representing the four surface-exposed regions of P5-fimbrin. As anticipated, in each of these nine animals, there was a strong preferential response to the region 3 peptide. Chinchillas that were immunized with this serum pool by passive transfer were significantly protected from the development of OM after challenge with NTHI isolates that are either homologous or heterologous in terms of their specific sequence diversity in region 3 (24, 25). Thus, Abs elicited by immunization with this synthetic peptide immunogen are clearly able to recognize the native OMP P5-homologous adhesin sufficiently well to induce significantly earlier clearance of NTHI from the upper airway of the chinchilla host and in doing so confer protection from OM (25).

The key question remaining is which is most important to augmented clearance and subsequent protection from OM, a certain threshold amount of region 3-specific Ab or the absence of Ab to the decoying epitope in region 4? Whereas the data presented here do not directly answer this question, data amassed during the past 10 years collectively address the first half of this question. To date, using published as well as unpublished studies conducted in our laboratory wherein LB1 was mixed successfully or not with various adjuvants as reference, it appears to us that reciprocal Ab titers of 50K or greater against the third surface-exposed region of the P5-adhesin are highly and consistently protective in chinchilla models (24, 25, 36), whereas those formulation that induced titers <=10K against region 3 have not been. Studies designed to answer the latter portion of this question regarding the absence of Ab to the decoying region 4 epitope are ongoing and will hopefully contribute to our better understanding of the mechanisms of a protective response to the P5 adhesin.

In summary, to develop effective vaccines against heterogeneous pathogens that cause chronic or recurrent disease, such as OM, it will be necessary to identify those epitopes presented by the microorganism that are able to induce a broadly cross-protective immune response (30). Ultimately, the epitope(s) of greatest interest may be neither the most immunodominant nor the least diverse ones presented (30). Moreover, it may be necessary to use a strategy of targeted immunodampening to minimize or eliminate a dysfunctional, natural immune response that is directed toward either a nonprotective but strain cross-reactive immunodominant epitope, or toward an immunodominant and protective but strain-specific Ag (such as any of several NTHI OMPs when used in their entirety; Refs. 5, 6, 7, 8, 9, 10). This could be achieved by deletion, an approach being used to bypass the hypervariable V3 region of HIV envelope glycoprotein (31); by refocusing, the approach we have used here relative to the OMP P5-homologous adhesin of NTHI (24, 25) or other methods (23, 30) and may ultimately prove to be an effective strategy for the development of vaccine candidates for OM.


   Acknowledgments
 
We thank Jennifer Neelans for preparation of the manuscript and are grateful to both Robert S. Munson, Jr. and Caroline C. Whitacre for helpful comments and suggestions.


   Footnotes
 
1 This study was supported by National Institutes of Health-National Institute on Deafness and Other Communication Disorders Grant R01 DC02830. Back

2 Address correspondence and reprint requests to Dr. Lauren O. Bakaletz, Division of Molecular Medicine, Department of Pediatrics, Ohio State University, College of Medicine and Public Health, Columbus Children’s Research Institute, Room W302, 700 Children’s Drive, Columbus, OH 43205-2696. E-mail address: BakaletL{at}pediatrics.ohio-state.edu Back

3 Abbreviations used in this paper: NTHI, nontypable Haemophilus influenzae; MEFs, middle ear fluids; OM, otitis media; OMP, outer membrane protein; RU, resonance units. Back

Received for publication February 19, 3002. Accepted for publication June 10, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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