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Infection and Immunity, May 2005, p. 2790-2796, Vol. 73, No. 5
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.5.2790-2796.2005

Synthesis and Characterization of Lipooligosaccharide-Based Conjugate Vaccines for Serotype B Moraxella catarrhalis

Shengqing Yu and Xin-Xing Gu*

Vaccine Research Facility, National Institute on Deafness and Other Communication Disorders, Rockville, Maryland

Received 3 September 2004/ Returned for modification 18 October 2004/ Accepted 12 January 2005


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Moraxella catarrhalis is an important cause of otitis media in children and respiratory tract infections in the elderly. Lipooligosaccharide (LOS) is a major surface antigen of the bacterium that elicits bactericidal antibodies. Serological studies show that three major LOS types (A, B, and C) have been identified among clinical isolates. Our previous studies demonstrated that the type A LOS-based conjugates were immunogenic in animals. In this study, LOS from type B strain 26397 was detoxified and conjugated to tetanus toxoid (TT) or a cross-reactive mutant (CRM) of diphtheria toxin to form detoxified LOS (dLOS)-TT and dLOS-CRM, respectively, as vaccine candidates. The molar ratios of dLOS to TT and CRM in the conjugates were 43:1 and 19:1, respectively, while both weight ratios were around 0.9. The antigenicity of the conjugates was similar to that of the LOS, as determined by enzyme-linked immunosorbent assay using a rabbit antiserum to strain 26397. Subcutaneous immunization with each conjugate elicited a 180- to 230-fold rise of serum anti-LOS immunoglobulin G in mice and >2,000-fold rise in rabbits. In addition, both mouse and rabbit antisera showed elevated complement-mediated bactericidal activity against the homologous strain, and a representative rabbit antiserum showed bactericidal activity against nine of twelve clinical isolates studied. The bactericidal activity of the rabbit antiserum can be fully inhibited by the type B LOS but not the A or C LOS. These results indicate that the type B LOS-based conjugates can be used as vaccine components for further investigation.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Moraxella catarrhalis is a gram-negative diplococcus, currently the third leading cause of otitis media and sinusitis in children along with Streptococcus pneumoniae and nontypeable Haemophilus influenzae (4, 5, 15, 17). Up to 80% of children below 3 years of age will be diagnosed with at least one episode of acute otitis media, and 15 to 20% of these middle-ear infections are caused by M. catarrhalis (18, 43). Sinusitis, however, accounts for 5 to 10% of upper respiratory tract infections in early childhood (53). In addition, M. catarrhalis is a frequent cause of lower respiratory tract infections in the elderly, particularly in those with a compromised immune system or chronic obstructive pulmonary disease, where M. catarrhalis can cause severe infections such as pneumonia that can be life-threatening (44). Presently, treatment of the diseases has largely relied on antimicrobial agents. However, with growing antibiotic resistance observed in clinical isolates (34), attention has been focused on the possibility of vaccination against M. catarrhalis.

Efforts for the development of vaccines against M. catarrhalis have focused primarily upon two surface antigens, outer membrane proteins and lipooligosaccharide (LOS) (39), since there is no direct evidence for a capsular polysaccharide (3), while other surface antigens such as fimbriae and pili have not been found in all clinical isolates (36). Although there is insufficient information about the protective antigens or an in vitro correlate of immunity against M. catarrhalis in humans, as for other bacterial pathogens, serum or bactericidal antibodies appear to be involved in immunity against M. catarrhalis diseases (39, 42). Our rationale is that if a vaccine using conserved antigens can elicit immune responses involving antibodies that recognize surface antigens; bind to the organism; block the colonization of the organism to the host cells; neutralize toxicity of the organism; inhibit growth; or kill the organism by the mechanisms of complement-mediated bactericidal, opsonophagocytosis, or other pathways, such a vaccine candidate may be effective against the organism in human trials.

There is a list of potential outer membrane proteins and their related proteins for vaccine candidates (2, 20, 27, 39, 49). Based on their known features, they can be classified with the following properties: adherence (UspA1, CD, MID, and Mcap), hemagglutination (Hag), iron acquisition (LbpA/LbpB, TbpA/TbpB, CopB, and B1), serum resistance (UspA2), phase variation (UspA1/2), and high conservation (E, CD, and G1). Among them, UspA and CD have been studied frequently since both are relatively conserved among different strains and are able to generate bactericidal antibodies in animals (26, 57) and in humans for UspA (6). Immunization with UspA (7) or recombinant CD (40) enhanced pulmonary clearance of both homologous and heterologous strains in a murine challenge model. Other vaccine candidates such as TbpB and CopB elicit antibodies with bactericidal activity and promote clearance in the murine pulmonary model, however, they appeared in multiple serotypes (8, 25). Further study with mapping of a protective epitope of the CopB is encouraging since a CopB-related fusion protein elicited antibodies that reacted to both homologous and heterologous strains (1).

LOS is another prominent surface component of M. catarrhalis. It has been implicated as a potential virulence factor important in the pathogenesis of this organism (10, 19). Rahman et al. (41) reported that serum antibodies to LOS developed in patients with M. catarrhalis infections, while Tanaka et al. (48) discovered that the bactericidal activity of convalescent-phase anti-LOS immunoglobulin G (IgG) from patients was against M. catarrhalis. Our study showed that a specific anti-LOS mouse monoclonal antibody was bactericidal and able to inhibit M. catarrhalis adherence to human epithelia and promoted clearance in a mouse pulmonary model after an aerosol challenge (31). In addition, the serological properties of LOS in humans reveal a less variable structure among three serotypes of LOS accounting for 95% of clinical isolates (A, 61%; B, 29%; and C, 5%) (52). Like other nonenteric gram-negative bacteria, the M. catarrhalis LOSs contain an oligosaccharide linked to lipid A without an O-specific polysaccharide. Structural studies have shown that the LOSs from all three types are branched with a common inner core, and the lipid A portion is similar to that of other gram-negative bacteria (Fig. 1) (12, 13, 14, 37, 38). Thus, the LOS is becoming an attractive vaccine candidate.



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  		FIG. 1. Schematic structure of the LOS moieties on the surface of M. catarrhalis. Three main serotypes, A, B, and C, are shown with different R groups according to the published structural analysis (12, 13, 14, 37, 38). Abbreviations: Gal, galactose; Kdo, 3-deoxy-D-manno-octulosonic acid; Glc, glucose; GlcNAc, N-acetyl-D-glucosamine; p, pyranose.

 
We previously synthesized an immunogenic conjugate from serotype A LOS by detoxification of the LOS and conjugation of the detoxified LOS (dLOS) to protein carriers. Both mice and rabbits immunized with the conjugates developed anti-LOS IgG antibodies with bactericidal activity (21). Active or passive immunization with the conjugates or their antisera generated protection against homologous and heterologous strains in the mouse model with pulmonary clearance (30). Such protection was further demonstrated by mucosal immunization with the conjugates (32). Since three LOS serotypes are found in clinical isolation and the cross-reaction among different strains or serotypes, especially between A and B, is not sufficient, new serotype B conjugates were developed and characterized in this study.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Bacterial strains. Strains 26397 (B; an LOS source strain), 3292 (B), 26400 (B), 26395 (A), 26394 (A), 26391 (C), and 26404 (C; an LOS source strain) were obtained from the Culture Collection of the University of Goteborg, Department of Clinical Bacteriology, Goteborg, Sweden. Strains 8176, 8193, 23246, 25238 (A; an LOS source strain), 25239, 25240, 43167, 43618, 43627, 43628, and 49143 were purchased from the American Type Culture Collection, Manassas, Va. Strains O35E and TTA24 (51) were kindly provided by E. J. Hansen at University of Texas, Dallas, Tex. Ten other clinical isolates (M1 to M10) were kindly provided by G. Mogi at Oita Medical University, Oita, Japan.

Bacterial culture and LOS purification. Strains were grown overnight on chocolate agar plates at 37°C in 5% CO2 for 18 h, and four to five isolated colonies from each strain were transferred to new plates and incubated for 3.5 to 4 h (mid-logarithmic phase) for subsequent bactericidal and bactericidal inhibition assays. For LOS purification, bacteria were cultured, and the LOS was extracted from cells by phenol-water extraction as described previously (21). The yield of the purified LOS was 16 to 18 mg per liter of bacterial culture, and the protein and nucleic acid contents of the LOS were shown to be less than 2% (47, 55).

Detoxification of LOS. We detoxified 240 mg of LOS by anhydrous hydrazine as described (21). The resulting material was purified through a column of Sephadex G-50 (2.6 by 90 cm, Pharmacia LKB Biotechnology, Uppsala, Sweden) eluted with 25 mM ammonium acetate and monitored with a differential refractometer (Waters, Milford, Ma). The eluate was assayed for carbohydrate by the phenol-sulfuric acid method (11). The second peaks (major) of carbohydrate-containing fractions were pooled, freeze-dried five times to remove the salt, and designated dLOS.

Derivatization of dLOS. Adipic acid dihydrazide (ADH; Aldrich Chemical Co., Milwaukee, Wis.) was introduced to the carboxyl group of Kdo moiety of the dLOS to form adipic hydrazide (AH)-dLOS derivatives, using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide HCl (EDC) and N-hydroxysulfosuccinimide (Pierce, Rockford, Ill.) (21). dLOS (96 mg) was dissolved in 12 ml of 287 mM ADH (50 mg/ml, molar ratio of ADH to dLOS is 108 to 1 based on an estimated Mrof 3,000 for dLOS) (14). Other steps were described previously (21). The resulting AH-dLOS was measured for its composition using dLOS and ADH as standards (11, 33).

Conjugation of AH-dLOS to proteins. Tetanus toxoid (TT) (15 mg/ml) was obtained from Pasteur Merieux Connaught, Swiftwater, Pa (lot 45453), and CRM (23 mg/ml) was purified from Corynebacterium diphtheriae by J. B. Robbin's laboratory at the National Institute of Child Health and Human Development, Bethesda, Md (45). AH-dLOS was conjugated to TT or CRM as described previously (21) with modifications in lower pH, temperature, and EDC along with a dialysis process. Briefly, AH-dLOS (45 mg) was dissolved with 4.5 ml of 0.2 M NaCl to make a 10 mg/ml solution of AH-dLOS. For the conjugation reaction, each 20 mg of AH-dLOS solution (2 ml in volume) was mixed with 10 mg of TT (0.67 ml in volume) or a cross-reactive mutant (CRM) of diphtheria toxin (0.44 ml in volume). The initial concentration of AH-dLOS or TT in the TT reaction was 7.49 or 3.75 mg/ml, while in the CRM reaction, the initial concentration of AH-dLOS or CRM was 8.20 or 4.10 mg/ml. The molar ratio of AH-dLOS to TT (Mr 150,000) was 100 to 1, and to CRM (Mr 67,000) was 45 to 1. The pH was adjusted to 5.0~5.2 with 0.1 M HCl, followed by addition of 0.05 M EDC. The reaction was maintained at pH 5.0 to 5.2 for 4 h at 4°C, then adjusted to pH 7.0, dialyzed against 0.9% NaCl for 2 to 3 days, centrifuged, and passed through a Sephacryl S-300 column (2.6 by 90 cm) in 0.9% NaCl. Peaks that contained both protein and carbohydrate were pooled and designated as dLOS-TT or dLOS-CRM. Both conjugates were analyzed for their composition of carbohydrate and protein using dLOS and bovine serum albumin (BSA) as standards (11, 47).

Preparation of hyperimmune sera. Two New Zealand White rabbits (female, 2 to 3 kg) were injected subcutaneously three times at 4-week intervals with 109 bacteria from strain 26397 plus Ribi adjuvant (R-700, 250 µg each of monophosphoryl lipid A and synthetic trehalose dicorynomycolate, Corixa, Hamilton, Mont.). Each rabbit received 1 ml solution at two injective sites. Blood samples were collected before injections and 2 weeks after the third injection. Ten female BALB/c mice were injected subcutaneously three times at 3-week intervals with 108 bacteria from strain 26397 plus Ribi adjuvant (50 µg each component). Each mouse received 0.2 ml solution at two injective sites. Blood samples were collected 2 weeks after the third injection.

ELISA. The antigenicity of the dLOS and conjugates was tested by enzyme-linked immunosorbent assay (ELISA), using the rabbit hyperimmune serum against strain 26397. ELISA was performed as described (21), with minor modifications. Briefly, after overnight coating with a 100 µl sample of conjugates at 10 µg/ml (carbohydrate) in phosphate-buffered saline (PBS, pH 7.4) or of LOS at 10 µg/ml in PBS containing 10 mM MgCl2, the plate was blocked with 1% bovine serum albumin for 2 h, and the rabbit serum (1/8,000) was added for 2 h followed by alkaline phosphatase-conjugated goat anti-rabbit IgG for 1.5 h. The reactions were read by using a microplate auto-reader at A405 after 1 h with a substrate.

Immunization with conjugates. Immunogenicity of the conjugates was examined in mice and rabbits. Five-week-old female BALB/c mice, 8 per group, were injected subcutaneously in two sites with dLOS-TT, dLOS-CRM (5 µg of carbohydrate), or a mixture of dLOS, TT, and CRM (5 µg for each) in 0.2 ml of 0.9% NaCl with or without Ribi adjuvant. The injections were given three times at 3-week intervals, and the mice were bled 2 weeks after every injection.

New Zealand White rabbits, two per group, were injected subcutaneously three times at 4-week intervals with 50 µg of dLOS-TT or dLOS-CRM (carbohydrate), or a mixture of dLOS, TT and CRM (50 µg for each) in 1 ml of 0.9% NaCl, with or without Ribi adjuvant. The rabbits were bled before injection and 2 weeks after each injection.

Serum anti-LOS levels were expressed as ELISA units using 26397 LOS as a coating antigen. As references, the mouse hyperimmune sera were assigned values of 19,683 and 81 U/ml for IgG and IgM, and the rabbit hyperimmune serum assigned values of 177,147 and 81 U/ml for IgG and IgM.

Bactericidal assay. Rabbit pre- and postimmune sera were inactivated at 56°C for 30 min and tested for bactericidal activity against M. catarrhalis by a microbactericidal assay (21) with modifications. The sera were diluted at 1:5 and then twofold continuous dilution with Dulbecco's PBS containing calcium, magnesium, and 0.1% gelatin (DPBSG). Each well of a 96-well plate contained 50 µl of serial diluted sera. The bacteria were adjusted to 6 x 103 CFU/ml in DPBSG with 70% transmission at 540 nm against viable counts, and 30 µl of bacterial suspension was added. Rabbit complement sera (1:8, 20 µl per well, Sigma, Saint Louis, Mo) were used as a source of complement. After incubation at 37°C for 60 min, 50 µl of the mixture was plated onto chocolate agar plates. The plates were incubated at 37°C under 5% CO2 overnight, and the colonies were counted. The highest serum dilution causing >50% killing was expressed as the reciprocal bactericidal titer. For mouse sera, guinea pig sera (1:5, 20 µl per well, Calbiochem, La Jolla, Ca) were used as a source of complement. Each serum sample was performed for three times.

Bactericidal inhibition assay. Each 1:5 diluted rabbit antiserum (25 µl) elicited by dLOS-TT was incubated with equal volumes of LOS or lipopolysaccharide (LPS) inhibitors from strain 25238 (A), 26397 (B), 26404 (C), or Salmonella enterica serovar Minnesota Ra (Sigma) at concentrations of 25, 12.5, 6.3, 3.2, 1.6, and 0 µg/ml, at 37°C for 60 min. After reaction, 30 µl of strain 26397 and 20 µl of rabbit complement were added. Other steps are the same as in the bactericidal assay. Inhibition was calculated as follows: [(CFU from serum with LOS inhibitor – CFU from serum without inhibitor)/(CFU from complement only – CFU from serum without inhibitor)] x 100.

Limulus amebocyte lysate assay. LOS and dLOS were tested for endotoxin reactivity by the Limulus amebocyte lysate pyrogent plus reagent (24 single test vials, Biowhittaker, Inc. Walkersville, Md.). The sensitivity of the Limulus amebocyte lysate assay is 0.12 endotoxin unit (EU)/ml.

SDS-PAGE and silver staining. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and silver staining were performed as described (50).

Statistical analysis. Antibody levels are expressed as the geometric mean ELISA units or titers (reciprocal) of n independent observations ± standard deviation. Significance was determined with the two-tailed independent student t test, and P values smaller than 0.05 were considered significant.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Characterization of dLOS. To examine the residual LOS in dLOS preparations, SDS-PAGE followed by silver staining for carbohydrates was performed. The results revealed that 25 ng of LOS showed a single band, but not as much as 20 µg of dLOS (Fig. 2), suggesting that its residual LOS was less than 0.13%. In addition, the LOS showed 10,000 EU/µg, whereas the dLOS showed <0.12 EU/µg, an over 80,000-fold reduction of toxicity by the Limulus amebocyte lysate assay.



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  		FIG. 2. Silver-stained SDS-PAGE patterns of LOS and dLOS from M. catarrhalis strain 26397. Lanes 1 and 2 contain 200 ng each of Salmonella enterica serovar Minnesota LPSs Ra and Rc. Lanes 3 through 6 contain 200, 100, 50, and 25 ng, respectively, of LOS; and lane 7 contains 20 µg of dLOS from M. catarrhalis strain 26397.

 
Characterization of AH-dLOS and the conjugates. The molar ratio of AH to dLOS in AH-dLOS was 0.30, and the yield, on the basis of carbohydrate content, was 84.4%. Both conjugates showed similar weight ratios and yields but different molar ratios due to different sizes of the protein carriers (Table 1). Both conjugates showed similar antigenicity by ELISA. Both conjugates (2 µg each of dLOS content) were analyzed by SDS-PAGE and silver staining to examine any unconjugated carbohydrate or protein. The result showed a single conjugate band on the top of the gel.


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  		TABLE 1. Composition, yield, and antigenicity of conjugates

 
LOS antibodies in mice. A mixture of dLOS, TT, and CRM did not elicit a significant rise of anti-LOS antibodies (Table 2). In contrast, the conjugates showed a 180- to 230-fold rise of anti-LOS IgG after the third injection compared to that of the first injection (185 and 234 versus 1, P < 0.01). Formulation of both conjugates with Ribi adjuvant significantly enhanced their immunogenicity: there was a 20-fold rise of anti-LOS IgG after three injections with dLOS-TT and a 26-fold rise with dLOS-CRM (compared to three injections without Ribi, P < 0.05). Both conjugates elicited similar levels of anti-LOS IgG after three injections. For IgM, conjugates elicited low levels of anti-LOS after each injection.


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  		TABLE 2. Murine antibody responses to M. catarrhalis serotype B LOS elicited by conjugates

 
LOS antibodies in rabbits. A mixture of dLOS, TT, CRM, and Ribi elicited low levels of anti-LOS IgG or IgM antibodies after three injections (Table 3). In contrast, both dLOS-TT and dLOS-CRM elicited a significant rise of anti-LOS IgG in rabbits after three injections (2,187- to 6,561-fold above the preimmune serum levels). Formulation of both conjugates with the Ribi enhanced their immunogenicity: there was a 46-fold rise of anti-LOS IgG after three injections in dLOS-TT and a 139-fold rise in dLOS-CRM compared with each group without Ribi. Both conjugates elicited similar levels of anti-LOS IgG after three injections. For IgM, both conjugates elicited low levels of anti-LOS antibodies after three injections.


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  		TABLE 3. Rabbit antibody responses to M. catarrhalis serotype B LOS elicited by conjugates

 
Bactericidal activity of mouse and rabbit antisera. In the mouse model, as many as 50 to 62.5% mouse sera immunized with the conjugates plus Ribi showed bactericidal activity against the homologous strain (Table 4). In the rabbit model, all rabbits immunized with the conjugates showed bactericidal activity against the homologous strain with a titer range of 1:10 to 1:160 (Table 5). There is an increasing tendency of bactericidal activity for the antisera elicited by the conjugates plus Ribi versus that without Ribi.


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  		TABLE 4. Bactericidal activity against serotype B M. catarrhalis with mouse antisera elicited by conjugates

 

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  		TABLE 5. Bactericidal activity against serotype B M. catarrhalis with rabbit antisera elicited by conjugates

 
The bactericidal activity of a rabbit antiserum elicited by dLOS-TT formulated with Ribi was further assayed with several known serotype strains (Table 6). Besides the homologous strain 26397, the rabbit antiserum also demonstrated bactericidal activities against the other serotype B strains and one serotype A strain but not to strains 25238 (A) and 26404 (C). In addition, strains 26394 (A) and 26391 (C) were complement sensitive, such that the complement itself killed the strains without the presence of the antiserum.


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  		TABLE 6. Cross-bactericidal activity of a representative rabbit serum elicited by serotype B dLOS-TT conjugate

 
For the other 10 strains from the American Type Culture Collection and 12 clinical isolates from Japan and the United States, 10 (45%) were complement sensitive (8176, 8193, 23246, 25240, 43167, 43618, 43628, M2, M5, and M8). With the remaining 12 strains, the rabbit antiserum demonstrated bactericidal activities to three American Type Culture Collection strains and six clinical isolates, at titers ranging from 1:20 to 1:160 (Table 6).

Bactericidal inhibition assay. To further characterize the specificity of the bactericidal activity of the rabbit antiserum, inhibition with purified LOSs from the homologous strain 26397 (B) and strains 25238 (A) and 26404 (C) was performed (Fig. 3). LOS from an unrelated Salmonella enterica serovar Minnesota strain (Ra) was also included. The bactericidal activity was fully inhibited by the homologous 26397 LOS, 20% by type A LOS and 30% by type C LOS, at a concentration of 6.3 µg/ml, but not with S. enterica serovar Minnesota LOS.



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  		FIG. 3. Inhibition of bactericidal activity of a rabbit antiserum elicited by serotype B dLOS-TT with Moraxella catarrhalis LOSs, serotype A (25238), serotype B (26397), and serotype C (26404), and Salmonella enterica serovar Minnesota LPS (Ra). Inhibition was calculated as follows: [(CFU from serum with LOS inhibitor – CFU from serum without inhibitor)/(CFU from complement only – CFU from serum without inhibitor)] x 100.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We used anhydrous hydrazine (mild alkali) to treat serotype B LOS from strain 26397 to remove ester-linked fatty acids while preserving amide-linked fatty acids of lipid A (24). The resulting dLOS showed more than a 80,000-fold less toxicity than the original LOS by a Limulus amebocyte lysate assay, which makes it clinically acceptable (56). We further conjugated the dLOS with two protein carriers to form the conjugates dLOS-TT and dLOS-CRM. Both conjugates showed good antigenicity in vitro and elicited high levels of serum anti-LOS IgG in both mouse and rabbit models. The immunogenicity of the conjugates was better in rabbits than in mice and was enhanced with the addition of an adjuvant. These data are consistent with our previous studies with M. catarrhalis serotype A, meningococcal, and nontypeable Haemophilus influenzae LOS-based conjugate vaccines (21, 23, 24).

To investigate the protective capacity of the antisera elicited by the conjugates, a complement-mediated bactericidal assay was performed, using guinea pig or rabbit sera as the source of complement for testing the mouse and rabbit antisera. We found that the source and the quantity of the complements were critical for successful assays. For instance, the rabbit complement sera were not suitable for testing the mouse antisera. Less complement resulted in a false negative reaction, while more produced bacterial killing without addition of the test antisera. Our current bactericidal assay revealed that 50 to 65% of the BALB/c mouse antisera elicited by the conjugates plus Ribi adjuvant showed bactericidal activity against the homologous type B strain. This result is partially consistent with type A dLOS-protein conjugates, where 45% of outbred mouse antisera and up to 100% of BALB/c mouse antisera showed bactericidal activity (21, 29). It is not clear if the difference is due to different LOS serotypes, protein carriers, mouse strains, or the assays themselves. We believe that the current assay is more reliable and specific since a higher baseline was set up using a starting serum dilution of 1:5 for the bactericidal assay instead of using a starting dilution of 1:2 (21). Future comparison studies will be performed using different serotype strains and different serotype antisera from varied animal species to confirm our finding.

Similar to type A dLOS-protein conjugates, all type B conjugate-induced rabbit sera showed bactericidal activity against the homologous strain. A representative rabbit antiserum also showed bactericidal activity against nine of twelve testable clinical isolates without complement self-killing. Of the nine, seven were assigned as serotype B strains, and two were serotype A/C strains (Table 6). For eight known serotype strains, the rabbit antiserum revealed bactericidal activity to all three type B strains and also to a type A strain. A bactericidal inhibition study further suggested that the bactericidal activity of the rabbit antiserum elicited by the type B conjugate was mainly specific to type B LOS, while there were some cross-reactions among B and A or C LOSs. Such cross-reactions may be explained by the structural similarity among three serotypes (Fig. 1).

The three antigenic LOS serotypes contain a common oligosaccharide inner core and a terminal tetrasaccharide, {alpha}-D-Galp-(1->4)-ß-D-Galp-(1->4)-{alpha}-D-Glcp-(1->2)-ß-D-Glcp-(1->) at the branch substituting position 6 of the trisubstituted Glc residue. The differences among them are mainly limited to the branch substituting position 4 of the trisubstituted Glc residue; type B contains {alpha}-D-Glcp, while types A and C contain {alpha}-D-GlcpNAc in its place (28). Significant cross-reactivity between types by a serological ELISA inhibition assay using rabbit antisera was observed previously, especially between type A and C (52). The same study also revealed cross-reactivity between type B and C since five type B strains gave a 60 to 80% inhibition of the homologous reaction of one type C strain. In addition, the cross-reactivity between type B and A showed that some type A or B strains gave a 20 to 43% inhibition of one type B or A strain. This phenomenon was supported by monoclonal antibody systems, where one monoclonal antibody was reactive to all serotype LOS strains while Ramman et al. reported that monoclonal antibodies generated from serotype A or C LOS were reactive to all three serotypes while monoclonal antibodies generated from serotype B LOS were type B specific (40a, 41a). Therefore, future LOS-based conjugate vaccines with two LOSs, type A and B or C and B, will be sufficient enough to cover the majority of the clinical isolates after investigation with all three serotype conjugate vaccines.

Serum bactericidal LPS or PS antibodies confer immunity to many pathogens in humans including H. influenzae type b, Neisseria meningitidis, Vibrio cholerae, and Shigella sonnei (9, 42). In the case of otitis media, vaccination with pneumococcal PS for children older than 2 years of age (35) or administration to otitis-prone children with hyperimmune human immunoglobulin (46), which contains antibodies to pneumococcal PS, resulted in a decrease in the incidence of pneumococcal otitis media. Children with nontypeable Haemophilus influenzae otitis media lack bactericidal antibodies before their infection and develop strain-specific bactericidal antibody following infection (16). Previously, we showed that systemic immunization with dLOS from nontypeable Haemophilus influenzae conjugated to proteins confers protection to experimental otitis media in chinchillas (22).

For M. catarrhalis infections, the role of anti-LOS antibodies in conferring protective immunity in humans has not been elucidated (41), however, the convalescent-phase anti-LOS IgG from patients demonstrated bactericidal activity against M. catarrhalis strains (48). In addition, our studies demonstrated that both monoclonal and polyclonal anti-LOS antibodies for serotype A LOS showed bactericidal activity and rapid pulmonary clearance in a mouse model (30, 31). We suggest that dLOS-TT and dLOS-CRM induced serum anti-LOS antibodies (IgG) with bactericidal activity can transude to the mucosal surfaces of the nasopharynges and lyse the inocula of M. catarrhalis causing otitis media and respiratory diseases (22, 54). For further evaluation of the dLOS-protein conjugates as potential vaccines for human use, animal protection studies in a mouse model of pulmonary clearance are planned with the combination of type A or C and B conjugates.


   ACKNOWLEDGMENTS
 
We thank John C. McMichael (Wyeth Vaccines, New York) for kindly supplying some strains and LOS material.


   FOOTNOTES
 
* Corresponding author. Mailing address: 5 Research Court, Room 2A31, Rockville, MD 20850. Phone: (301) 402-2456. Fax: (301) 402-5354. E-mail: guxx{at}nidcd.nih.gov. Back

Editor: D. L. Burns


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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3. Ahmed, K., N. Rikitomi, A. Ichinose, and K. Matsumoto. 1991. Possible presence of a capsule in Branhamella catarrhalis. Microbiol. Immunol. 35:361-366.[Medline]
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Infection and Immunity, May 2005, p. 2790-2796, Vol. 73, No. 5
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.5.2790-2796.2005




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