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The Journal of Immunology, 2003, 170: 4373-4379.
Copyright © 2003 by The American Association of Immunologists

Development of Peptide Mimotopes of Lipooligosaccharide from Nontypeable Haemophilus influenzae as Vaccine Candidates

Yingchun Hou and Xin-Xing Gu1

National Institute on Deafness and Other Communication Disorders, National Institutes of Health, Rockville, MD 20850


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Nontypeable Haemophilus influenzae (NTHi) is a common cause of otitis media in children and lower respiratory tract diseases in adults. So far there is no effective vaccine against NTHi. A major surface-exposed component of NTHi, lipooligosaccharide (LOS), is a virulence factor as well as a potential protective Ag. LOS is too toxic to be administered in humans. However, detoxified LOS is a T cell-independent small molecule and is poorly immunogenic in vivo, so we converted LOS into a nontoxic T cell-dependent Ag through the use of peptides that mimic the LOS by screening a phage-display peptide library with a rabbit Ab specific for NTHi LOS. Fifty-six phage clones were found to share LOS mimicry molecules. Among them, 22 clones were subjected to DNA sequencing, and four consensus sequences were identified as NMMRFTSQPPNN, NMMNYIMDPRTH, NMMKYISPPIFL, and NMMRFTELSTPS. Three of the four synthetic peptides showed strong binding reactivity to the rabbit anti-LOS Ab and also a mouse bactericidal monoclonal anti-LOS Ab in vitro, and elicited specific serum anti-LOS Abs in rabbits (27- to 81-fold) after conjugation with keyhole limpet hemocyanin. Passive immunization with the rabbit antisera resulted in a significantly enhanced pulmonary bacterial clearance in a mouse model. The enhanced bacterial clearance was eliminated if the rabbit serum was preabsorbed with NTHi LOS. These data indicate that the peptide mimotopes of LOS that we have identified might be potential components of peptide vaccines against NTHi.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Nontypeable Haemophilus influenzae (NTHi)2 is a common pathogen that causes otitis media (OM) in children and lower respiratory tract infections in adults, especially those with chronic obstructive pulmonary diseases (1, 2, 3). Prevention of NTHi infections is important because OM is a major cause of conductive hearing impairment in children who are at the critical age for speech and language development (4). The exacerbation of chronic obstructive pulmonary diseases in elderly is life threatening and is the fourth leading cause of death in the United States. In addition, repeated or inappropriate antibiotic treatment of OM and other respiratory diseases contributes to the emergence of antibiotic-resistant strains (5). Therefore, there is an urgent need to develop prophylactic vaccines to prevent these NTHi-caused diseases.

In the past years, significant progresses have been made toward identifying vaccine candidates based on NTHi surface Ags such as outer membrane proteins and lipooligosaccharide (LOS). These Ags are potential targets for humoral immunity and for bactericidal Abs that appear to be important in protecting against NTHi OM (6). Our strategy is to use LOS as a vaccine component because NTHi does not have a detectable capsular polysaccharide and its LOS is both a virulence factor (7, 8, 9) and a potential protective surface Ag (10, 11, 12). Previously, we chemically conjugated a relatively conserved detoxified LOS (dLOS) to proteins to form vaccines (13). These conjugates were immunogenic in mice and rabbits and conferred T cell-dependent immunological protection against experimental OM in chinchillas (14, 15).

The LOS can also be converted into a nontoxic T cell-dependent Ag through the use of anti-idiotype Abs (16, 17) or peptides that mimic the LOS (18, 19). A strategy based on the mimicry of saccharide Ags by anti-idiotype Abs is difficult and time-consuming. Recently, phage display has been used to identify ligands for a variety of target molecules by an affinity selection process called biopanning (20, 21, 22, 23, 24, 25, 26). Random peptide libraries displayed on bacteriophage outer proteins have been used successfully to screen for peptides that bind Abs as well as non-Ab molecules (27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37). These studies allow investigators to map the target sequences for monoclonal or polyclonal Abs that recognize both linear and conformational epitopes.

Libraries have been used to identify peptides that mimic the carbohydrate structures of bacteria, cancer cells, or viruses (27, 28, 29, 30, 38, 39, 40, 41). In the case of bacteria, several studies have reported success in isolating peptide mimetics that elicit an immune response against native bacterial saccharide structures in animal models. A study by Phalipon et al. (18) was an early example of immunogenic mimicry of bacterial saccharides by phage-displayed peptides. They used two mAbs specific for the O-Ag part of Shigella flexneri serotype 5a LPS to screen two phage-display nonapeptide libraries. Some of the selected phage clones could induce specific anti-O-Ag Abs in mice. The immune response selectively recognized the corresponding bacterial strains. Using a similar method, Pincus et al. (27) located peptides that bind to mAbs specific for type 3 capsular polysaccharide of group B streptococci (GBS). The peptide specifically blocked the binding of anti-GBS Abs to GBS and elicited an anti-GBS Ab response in mice when conjugated to protein carriers. Instead of using peptides from phage-display library, Westerink et al. (42) developed a peptide derived from anti-idiotype mAb. This peptide elicited a protective Ab of meningococcal group C polysaccharide. Table I summarizes reports of peptide mimetics of bacterial or fungal carbohydrate structures (18, 19, 27, 28, 29, 30, 31, 32, 42, 43, 44, 45, 46, 47). These studies reveal that some of the mimic peptides only show antigenicity in vitro, whereas others can be antigenic and immunogenic in vivo, indicating a new strategy for selection of immunogens for the development of anti-saccharide vaccines.


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  		Table I. Peptide mimetics of bacterial (fungal), capsular polysaccharide (CPS), LPS, or LOS

 
In this study, we have used a phage-display peptide library and a rabbit bactericidal polyclonal Ab specific for NTHi LOS to screen for a peptide mimic of NTHi LOS. Three peptides selected from the library were synthesized and conjugated to keyhole limpet hemocyanin (KLH). Immunogenicity of the peptide conjugates was evaluated in rabbits and the protective property of the conjugate-induced rabbit sera was further evaluated in a mouse challenge model.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Affinity purification of an Ab specific for NTHi LOS

LOS was extracted from NTHi strain 9274 by phenol-water extraction and then by column purification (13). A rabbit antiserum elicited by strain 9274 dLOS-tetanus toxoid (TT) with bactericidal activity (13) was purified through an LOS affinity column prepared by coupling the LOS to cyanogen bromide-activated Sepharose 4B (Pharmacia Biotech, Uppsala, Sweden). Sepharose 4B (2 g) was suspended in 1 mM HCl, transferred into a sintered glass filter, and washed with 1 mM HCl for 15 min. The washed 4B gel was mixed with 10 ml of coupling buffer (0.1 M NaHCO3, 0.5 M NaCl, pH 8.3) containing 20 mg of LOS in a tube and rotated overnight at 4°C. After washing with the coupling buffer, the gel was dried, suspended, and blocked with 0.1 M Tris-HCl (pH 8) for 2 h, followed by washing with 0.1 M acetate buffer (pH 4) and 0.5 M NaCl and then with 0.1 M Tris-HCl (pH 8) and 0.5 M NaCl. After repeating the washing step three times, the gel was washed with the coupling buffer, then 0.5% BSA in PBS (pH 7.4), and suspended in 0.5% BSA for 2 h. An LOS affinity column was prepared after washing with PBS and loaded with the rabbit antiserum. The column was washed with 0.5% BSA and PBS and eluted with 3.5 M MgCl2. Fractions with a protein peak at OD280 value, designated as the target anti-LOS Ab, were collected and immediately dialyzed against PBS at 4°C. The protein content of the Ab was determined with a Micro BCA kit (Pierce, Rockford, IL). The binding reactivity of the Ab to the LOS was tested with an LOS ELISA (13) using 1 µg/well of LOS as a coating Ag in a 96-well Nunc-Immuno plate (MaxiSorp surface; Nalge Nunc International, Roskilde, Denmark). The plate was then incubated with 1.4 µg/well of the target anti-LOS Ab or an irrelevant rabbit IgG Ab (Southern Biotechnology Associates, Birmingham, AL), followed by a goat anti-rabbit IgG (whole molecule) alkaline phosphatase conjugate at 1:3500 (Sigma-Aldrich, St. Louis, MO). ELISA OD values were obtained with a microplate autoreader (EL309; Bio-Tek Instruments, Winooski, VT) at A405 after addition of p-nitrophenyl phosphate for 30 min. This affinity-purified rabbit anti-LOS Ab would be used as the target Ab in each of the following biopanning round.

Biopanning to select phage-display peptides that mimic NTHi LOS

A Ph.D.-12 Phage Display Peptide Library was purchased from New England Biolabs (Beverly, MA) with a concentration of 1.5 x 1013 PFU/ml and complexity at 2.7 x 109 transformants. A biopanning protocol was executed following manufacturer’s instruction (version 2.5) by incubating the library of phage-display peptides with a 96-well Nunc-Immuno plate (Nalge Nunc International) coated with the affinity-purified target Ab (2.5 µg in PBS/100 µl/well), washing away the unbound phage and eluting the bound phage. The eluted phage was then amplified and taken through additional cycles of biopanning and amplification to enrich the phages with the highest affinity. After three rounds, an individual clone was selected, amplified, and characterized by a capture ELISA with the target anti-LOS Ab as a coating Ab. To overcome the difficulty in mimicking saccharide epitopes after several unsuccessful trials, three major modifications were made for each biopanning round. The library was absorbed with an irrelevant rabbit IgG at 3.75 µg in PBS/150 µl/well at room temperature (RT) for 1 h with agitation before incubation in another well coated with anti-LOS Ab. Before the eluting step with TBS (50 mM Tris-HCl (pH 7.5) and 150 mM NaCl) containing NTHi 9274 LOS (80 µg/150 µl/well), the well was eluted with the TBS containing the irrelevant Ab (10 µg/200 µl/well). For amplification of the selected individual clone, we made 1 ml of host bacteria in Luria-Bertani culture (OD600 = 0.5) with each selected plaque in a 14-ml Falcon tube (BD Biosciences, Franklin Lakes, NJ) instead of making 20 ml of host bacteria in Luria-Bertani culture in a 250-ml Erlenmeyer flask. The first two steps removed nonspecific binding phages while the third step retained high-affinity binding clones and reduced mutants.

ELISA for screening positive clones

A capture ELISA was performed for screening the positive phage clones described by the instructions of the peptide library kit with minor modifications. Briefly, the supernatant of the 1 ml amplified culture from each clone was used as the testing sample. A 96-well Nunc-Immuno plate was coated with the target anti-LOS Ab or the irrelevant Ab (8 µg/ml in PBS) at 110 µl/well overnight at 4°C. The plate was blocked with 3% BSA in TBS at 300 µl/well for 2 h at 4°C, followed by adding each duplicate sample suspended with 0.1% Tween 20 in TBS at 100 µl/well for 2 h at RT with agitation. An HRP anti-M13 mAb conjugate (Pharmacia Biotech) at 1:5000 with 3% BSA in TBS was added for 1 h at RT under agitation. A washing buffer with 0.5% Tween20 in TBS was used between steps, each for 8–10 times. The binding reactivity between the anti-LOS Ab and the selected phage clones was evaluated by OD405 values at 30 min at RT after addition of an ABTS (Sigma-Aldrich) substrate solution. The positive clones should show a blue-green color.

DNA sequencing on positive clones

To prepare DNA sequencing templates, each 700 µl of the supernatant of the 1 ml amplified culture was transferred into a 1.5-ml tube, and 300 µl of polyethylene glycol/NaCl (20% polyethylene glycol 8000 and 2.5 M NaCl) was added. The mixture was kept at RT for 10 min, spun, and the supernatant was discarded. The pellet was suspended in 100 µl iodide buffer (10 mM Tris-HCl (pH 8), 1 mM EDTA, 4 M NaI), followed by the addition of 250 µl of 70% ethanol. The mixture was kept at RT for 10 min, spun, and the supernatant was discarded. The pellet was washed with the ethanol and suspended in 30 µl of TE buffer (10 mM Tris-HCl (pH 8) and 1 mM EDTA) for DNA sequencing (Veritas, Rockville, MD). Peptide sequences were deduced from DNA sequences.

Synthetic peptides and their binding reactivity to the target anti-LOS Ab

Four peptides were synthesized with a spacer (GGGS) at each C terminus (Genemed Synthesis, South San Francisco, CA). Binding reactivity of the synthetic peptides to the target anti-LOS Ab was tested with a peptide ELISA using Nunc NucleoLink Strips (Fisher Scientific, Pittsburgh, PA). Following the instructions of the product with modifications, the strips were coated covalently with its amino groups to the C-terminal carboxyl groups of the synthetic peptides by a coupling reagent, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide HCl (EDC; Pierce). Briefly, peptides and 0.1 M 1-methyl-imidazole (MeIm; Sigma-Aldrich) at pH 7 were precooled on ice, and a 100 µl individual peptide solution with 5 µg of peptide in 10 mM MeIm was prepared in a well of strips on ice. This peptide solution (75 µl) was mixed with 25 µl of freshly prepared 0.2 M EDC in 10 mM MeIm in another well of the strips. The strips were sealed and incubated at 50°C for 5 h. After removing the reaction mixture, the strips were soaked with washing buffer (freshly prepared 0.25% SDS in 0.4 N NaOH and warmed at 50°C) for 5 min and emptied. This step was performed three times, followed by another three washings without the 5-min soaking. After removing the washing buffer, the strips were washed thoroughly with deionized water. The following steps were the same as described in the LOS ELISA (13). After blocking with 1% BSA in PBS, the target anti-LOS Ab was added (0.4 µg/well) followed by a goat anti-rabbit IgG alkaline phosphatase conjugate. The binding reactivity of the synthetic peptides to the target anti-LOS Ab was evaluated by OD405 values at 30 min after adding a substrate.

Synthetic peptide conjugates and their antigenicity in vitro and immunogenicity in rabbits

Three peptide-KLH conjugates were synthesized covalently using sulfosuccinimidyl 4-(N-maleimido-methyl) cyclohexane-1-carboxylate (SMCC) as a linker for the coupling reaction (Genemed Synthesis). A peptide spacer, GGGSC, was introduced at each peptide’s C terminus and was conjugated to KLH. The ratio of the synthetic peptides to KLH was ~20% by weight. Antigenicity of these conjugates in vitro was examined in ELISA using conjugates or KLH as coating Ags (2 µg/well in 0.1 M Tris buffer, pH 8) and the target rabbit anti-LOS Ab (0.1 µg/well). The conjugates (5 µg/well) were also tested for their binding reactivity with a mouse bactericidal monoclonal anti-LOS Ab 6347C11 at 1:500 of ascites (11). Other steps of this ELISA were the same as described in the LOS ELISA except goat anti-mouse IgM (µ-chain-specific) alkaline phosphatase conjugate (Sigma-Aldrich) was used at 1:3500 for detecting the mAb. In addition, each peptide was also conjugated with BSA as a coating Ag (10 µg/well) for the following ELISA to detect rabbit Abs against peptides without interference from the existing KLH Abs.

To study the immunogenicity of the peptide conjugates, New Zealand White female rabbits 3.5 mo of age, two for each group, were s.c. immunized with 500 µg of each conjugate (~100 µg of peptide content) suspended in 0.5 ml of PBS (Genemed Synthesis). The immunization was repeated three times with 20-day intervals. Each injection volume was 1 ml in which the first dose was mixed with 0.5 ml of complete Freund adjuvant and the boosters with 0.5 ml of incomplete Freund adjuvant. A control rabbit immunized with 500 µg KLH along with the above adjuvants was also included. Blood samples were collected before and 10 days after the fourth injection to detect serum Abs against synthetic peptides or NTHi LOS in ELISA using peptide-BSA conjugates or LOS as coating Ags (13). The ELISA was performed by incubation with the rabbit sera, followed by addition of a goat anti-rabbit IgG alkaline phosphatase conjugate. The Ab endpoint titer was defined as the highest dilution of a serum sample giving an OD value >0.5 at A405 after addition of a substrate for 20 min.

Passive protection with rabbit antisera elicited by peptide conjugates in a mouse model

The passive protection study was performed in accordance with National Institutes of Health guidelines under Animal Study Protocol 1009-01. Female BALB/c mice at the age of 8 wk were immunized i.p. with 1 ml rabbit presera (40%) or postsera (8 and 40%) elicited by peptide-KLH conjugates (rabbit number 7819, conjugate 1; 7850, conjugate 2; and 7822, conjugate 3) diluted in normal saline 17 h before NTHi challenge. Presera were pooled and injected only with 40% for the bacterial challenge. Two positive antisera included a rabbit antiserum elicited by NTHi 9274 whole cells and a rabbit antiserum elicited by NTHi 9274 dLOS-TT. Antiserum 7819 elicited by conjugate 1 was also pretreated with 9274 LOS to determine whether the Abs elicited by the peptide conjugate could be removed by the LOS Ag. The 40% serum 7819 was incubated with 9274 LOS (100 µg/ml) at 37°C for 1 h and then at 4°C overnight with gentle rotation. The preabsorbed antiserum along with other diluted sera were low centrifuged and sterile filtered before use. Ten mice as a group were used for each rabbit serum, followed by an aerosol challenge in an Inhalation Exposure System (Glas-Col, Terre Haute, IN) in which freshly harvested strain 9274 bacteria in log-phase growth were suspended (108 CFU/ml) in 10 ml of PBS containing 0.1% gelatin, 0.15 mM CaCl2, and 0.5 mM MgCl2 PBSG as the challenge bacteria (48). Six hours postchallenge, blood samples were collected for Ab measurements and mouse lungs were removed for bacterial counts and Ab measurements. The lungs were homogenized in 10 ml of PBSG for 1 min using a tissue homogenizer. Each homogenate was diluted serially in PBS and 50 µl of the homogenate, and the diluted samples were plated on chocolate agar plates. The plates were incubated at 37°C with 5% CO2 overnight, and the bacterial colonies were counted. The minimal detection level of bacterial counts was 200 CFU per mouse. Both serum and lung homogenate samples were also detected for the rabbit anti-LOS IgG passively immunized in mice before the challenge by the LOS ELISA as previously described.

Statistical analysis

Bacterial colonies were expressed as the mean CFU of an independent observation ± SD. Significance was determined using Mann-Whitney nonparametric analysis. Linear regression and correlation analysis was done between the bacterial count in each mouse lung and its corresponding IgG level in serum or lung homogenate.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Specificity of a target anti-LOS Ab

A rabbit antiserum elicited by NTHi 9274 dLOS-TT conjugate was purified through an LOS affinity column. The binding specificity of the anti-LOS Ab to NTHi 9274 LOS was analyzed by an LOS ELISA. The rabbit anti-LOS Ab bound strongly to the 9274 LOS with an OD value of 1.57, a 17-fold greater binding than for an irrelevant rabbit Ab with an OD value of 0.09 (data not shown). This rabbit anti-LOS Ab was used as a target Ab for each of the following biopanning rounds.

Screening for positive clones by the biopanning procedure

A modified biopanning protocol was performed to screen a phage-display peptide library for positive clones with the target anti-LOS Ab. After three biopanning rounds, 56 positive phage clones were identified from 108 individual clones. (Table II). These clones showed specific binding to the target anti-LOS Ab with the ELISA OD values of 2- to 1510-fold higher than that of an irrelevant Ab. Twenty-two positive clones, most with high ELISA values, from different biopanning rounds were selected for DNA sequencing. As comparison, four negative clones were also selected randomly for DNA sequencing. Twenty-two peptide sequences were deduced and analyzed. Four consensus sequences with a frame of NMMXXXXXP(S)XXX were identified in Table III. Each sequence also shows a consensus aromatic group (F or Y) at the fifth position of the N terminus. As controls, no consensus peptide sequence was found from negative clones (TQARATPFQSFT, SDTKLTTSSKWV, KPFHSHTGATTP, and AYSPPTPAEAPI).


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  		Table II. Positive clones and their ELISA binding reactivity to a target anti-LOS Ab

 

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  		Table III. Four consensus peptide sequences derived from 22 positive clones

 
Binding specificity of synthetic peptides and synthetic peptide conjugates to anti-LOS Abs

Because the N terminus of each phage-displayed peptide was free during biopanning while its C terminus with GGGS fused to the phage membrane, four peptides and a negative peptide were synthesized with a spacer sequence (GGGS) added to each C terminus. Binding reactivity of peptides to the target anti-LOS Ab was examined in an ELISA coated with the peptides (Table III). Peptides 2, 3, and 4 showed higher binding reactivity to the Ab than did peptide 1 or peptide 5 (controls). Therefore, peptides 2, 3, and 4 were further conjugated to KLH to form conjugates 1, 2, and 3 to enhance the immunogenicity of the peptides in vivo. Fig. 1 shows the ELISA binding reactivity of the synthetic peptide conjugates to the rabbit anti-LOS Ab (target Ab) (Fig. 1A) or to a mouse monoclonal anti-LOS Ab 6347C11 (Fig. 1B). All three synthetic peptide conjugates bound strongly to either the rabbit or the mouse anti-LOS Ab with the best binding reactivity in conjugate 1. In contrast, the KLH alone showed lower binding reactivity relative to the conjugates.



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  		FIGURE 1. Binding reactivity of synthetic peptide conjugates to the target rabbit anti-LOS Ab (A) or a mouse anti-LOS Ab 6347C11 (B) by ELISA. Antigenicity of the synthetic peptide conjugates was examined in ELISA using three synthetic peptide conjugates or a carrier KLH as coating Ags, followed by incubation with either the target anti-LOS Ab or the monoclonal anti-LOS Ab. The binding reactivity of the synthetic peptides to both Abs was evaluated by average OD405 values of each duplicate sample after incubation with a goat anti-rabbit IgG (or anti-mouse IgM) alkaline phosphatase conjugate, and then addition of a substrate. Conjugates 1, 2, and 3 were synthesized from peptides 2, 3, and 4.

 
Immunogenicity of the synthetic peptide conjugates in rabbits

The immunogenicity of the synthetic peptide conjugates was evaluated in rabbits. All peptide conjugates elicited high levels of anti-peptide Abs in rabbit serum as measured by ELISA using peptide-BSA as a coating Ag at a titer of >=1:10,000 (data not shown). Importantly, these rabbit antisera showed strong binding cross-reactivity with LOS as measured by the LOS ELISA using 9274 LOS as a coating Ag at average titers of 1:810 to 1:2430 (27- to 81-fold rise when compared with presera) (Fig. 2). Among the three conjugates, conjugate 1 elicited the highest anti-LOS Ab titers. In contrast, presera or antiserum elicited by KLH showed lower anti-LOS levels (<=1:90)



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  		FIGURE 2. Rabbit serum Ab responses elicited by synthetic peptide conjugates. New Zealand White female rabbits, two for each group, were s.c. immunized with 500 µg of each conjugate (~100 µg of peptide content) mixed with complete Freund adjuvant for the first injection or incomplete Freund adjuvant for three boosters with 20-day intervals. A control rabbit immunized with 500 µg of KLH along with the previously described adjuvants was also included. Blood samples were collected 10 days after the last booster to detect serum IgG Abs against LOS by ELISA, using NTHi 9274 LOS as a coating Ag. Rabbit anti-LOS IgG Ab levels were expressed as reciprocal of ELISA titers.

 
Passive protection with rabbit antisera elicited by peptide conjugates in a mouse challenge model

BALB/c mice were passively immunized with rabbit presera and antisera elicited by the peptide conjugates, whole cells, or dLOS-TT at 17 h before an aerosol challenge of NTHi strain 9274 (Table IV). Six hours postchallenge the mice were sacrificed to determine bacterial counts in lungs and passively transferred rabbit anti-LOS Ab levels in lungs and sera. The result indicated that 40% of rabbit antisera 7819, 7850, and 7822 elicited by conjugate 1, 2, or 3 (group 1, 4, or 6) significantly enhanced the bacterial clearance from mouse lungs by 35–44% when compared with that of preserum (group 8, p < 0.05). Evidence of an Ab dose-dependent effect on bacterial clearance was found in conjugate 1 or 2. The 40% of rabbit antiserum 7819 or 7850 elicited by conjugate 1 or 2 significantly enhanced the bacterial clearance from mouse lungs by 32% when compared with that of the 8% sera (group 1 vs 3 or group 4 vs 5, p < 0.05). When rabbit antiserum 7819 was pretreated with 9274 LOS, it showed no enhanced bacterial clearance in the mouse lungs (group 2). Similar reductions of 41% and 50% were also obtained from two positive rabbit antisera elicited by dLOS-TT (group 9) and whole cells (group 10), respectively (p < 0.05). There was no statistical significance for rabbit sera elicited by three peptide conjugates or by whole cells or dLOS-TT.


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  		Table IV. Effect of passive immunizaiton with rabbit antisera elicited by peptide conjugates on bacterial clearance in mouse lungs challenged with NTHi strain 9274a

 
The passively transferred rabbit anti-LOS IgG levels in mouse lung homogenates and sera were measured and consistent with the injection dose of each rabbit serum (data not shown). The correlation between bacterial counts in mouse lung homogenates and the Ab levels of rabbit IgG in the mouse lung homogenates or sera was analyzed in all mice except for two negative controls (inhibition group 2 and preserum group 8). The results indicated that there was negative correlation between the bacterial counts and the rabbit IgG levels in mouse lung homogenates (r = -0.47, p < 0.0001) and sera (r = -0.45, p < 0.001).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Using phage-display libraries, immunogenic peptide mimics of bacterial or fungal polysaccharides have been identified in Candida albicans, GBS, group A meningococci, serotype 4 pneumococci, and S. flexneri (Table I). In the case of mimicry of bacterial endotoxin (LPS or LOS), the biggest advantage of using a peptide library is that the peptide mimetics are nontoxic and can be easily synthesized. However, attempts to identify mimotopes of bacterial endotoxins have met with only limited success. Peptide mimetics of Brucella LPS elicited weak anti-O-specific polysaccharide Abs of the LPS, whereas peptide mimetics of meningococcal LOS could elicit a 2- to 4-fold anti-LOS IgG Ab in mice. Also, there is a successful report of mimic O-specific polysaccharide of S. flexneri LPS. Using a similar method but with an affinitive purified rabbit anti-LOS polyclonal Ab, we have identified three mimotope peptides that mimic the LOS of NTHi through a 12-mer random peptide library.

A peptide mimic of carbohydrates is difficult to demonstrate, as there are only a few successful cases. Most are mimics of polysaccharides and the mechanisms of peptide mimetics are unclear although considerable progress has been made recently (49). We speculate that it would be even more difficult to mimic a much smaller molecule of LOS (around 4 Kd), particularly for oligosaccharides with a big portion of hydrophobic lipid A molecule. The only attempt with meningococcal LOS mimetics resulted in weak to medium anti-LOS Ab in mice (19). In our initial studies, we failed to identify mimetics of NTHi LOS from a 7-mer-linear phage display peptide library or a 7-mer-circle phage display peptide library. After switching to the 12-mer-linear phage display peptide library, along with several modifications of the biopanning protocol, we were able to find 56 positive colonies from three individual biopanning processes. Twenty-two colonies were sequenced, resulting in four consensus sequences (mimotopes) with a possible motif of NMM. We were unable to identify the repeating aromatic groups in our peptide mimetics found in peptides that are antigenic mimics of cryptococcal polysaccharide (28) or that are immunogenic mimics of the Lewis Y Ag (50) and meningococcal group C polysaccharide (42). However, there is one aromatic group (phenylalanine (F) or tyrosine (Y)) at the fifth position of the N terminus of each mimotope peptide. Furthermore, all mimotope peptides possess arginine (R), lysine (K), or asparagine (N) at the fourth position of the N terminus. These three residues can form hydrogen bonds in their side chains. The terminal NMM and the aromatic group along with RKN might play a major role in peptide binding to anti-LOS Ab as an Ag or for peptide eliciting LOS Ab as an immunogen in vivo. However, other amino acids may interfere with the LOS mimicry because peptide 1 did not show good antigenic mimic of the LOS (Table III). The precise interactions between the peptide mimotopes and the Ab paratopes may be revealed in the crystallographic structure of the Ab in complex with the mimotopes in future studies (49, 51, 52).

Three synthetic mimotope peptides were shown to bind strongly to the target anti-LOS Ab and a monoclonal anti-LOS Ab in vitro. This indicates that the mimotope peptides share the antigenic mimicry of LOS. To evaluate the immunogenicity of the peptide mimotopes, rabbits were immunized with the peptide mimotope conjugates. High levels of anti-LOS Abs were found in sera of immunized rabbits. Passive immunization of mice with the rabbit antisera showed protection against NTHi, comparable to that seen with rabbit antiserum against whole cells or dLOS-TT. The protection in most animals was in an antiserum dose-dependent pattern, and when this antiserum was preabsorbed by LOS, the protection was inhibited. These data suggest that the mimotope peptides can also specifically mimic the LOS immunogenically and induce protective immune responses against NTHi.

The strategy we have reported is the basis for the development of a new type of anti-NTHi LOS vaccines. The use of peptide mimetics as vaccines offers several advantages in terms of safety, cost, stability, and relative ease of production. Needless to say, peptide mimetics have other potential advantages over LOS for immunogenicity, such as effective infant immunization, secondary immune response, and long-lasting immunity. In our case, this approach has the greatest utility because NTHi LOS is toxic and difficult to purify. To produce an experimental vaccine candidate for human use, these promising peptides need to be conjugated to clinically acceptable carriers, such as TT, and conventionally tested in animal models, such as in active protection models of mouse pulmonary clearance and chinchilla OM by systemic or mucosal administration. Meanwhile, these peptides can be rationally designed with multivalent LOS mimotopes, multiple Ag peptides, or to contain additional structures, including Th cell epitopes and built-in adjuvants (53, 54, 55, 56). These applications might further improve immunogenicity of these LOS mimotopes in vivo. In addition, DNA vaccine candidates based on these LOS mimotopes are contemplated to further extend the present strategy or open a new strategy for vaccine development (57).


   Acknowledgments
 
We thank Takashi Hirano and Xinan Jiao for their assistance in the protection study, Kezuo Wu for his assistance in coating peptides on Nunc NucleoLink strips, and Wei-Gang Hu for his advice in biopanning protocol and manuscript preparation.


   Footnotes
 
1 Address correspondence and reprint requests to Dr. Xin-Xing Gu, National Institute on Deafness and Other Communication Disorders, National Institutes of Health, 5 Research Court, 2A31, Rockville, MD 20850. E-mail address: guxx{at}nidcd.nih.gov Back

2 Abbreviations used in this paper: NTHi, nontypeable Haemophilus influenzae; LOS, lipooligosaccharide; dLOS, detoxified LOS; OM, otitis media; GBS, group B streptococci; KLH, keyhole limpet hemocyanin; TT, tetanus toxoid; RT, room temperature. Back

Received for publication October 1, 2002. Accepted for publication February 4, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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