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* Dipartimento di Patologia e Microbiologia Sperimentale, Università degli Studi di Messina, Messina, Italy; and
Dipartimento di Patologia e Medicina di Laboratorio, Sezione di Microbiologia, Università degli Studi di Parma, Parma, Italy
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In contrast, group B N. meningitidis (MenB), which is responsible for up to 80% of the total cases of disease in industrialized countries, has been resistant to any attempt to develop a capsule-based vaccine due to the poor immunogenicity of the CP even after protein conjugation. This is probably due to the similarity between the MenB CP and a polymer expressed on the neuronal cell adhesion molecule of mammalian tissues (6). Both polysaccharides are 
(2
8) homolinear polymers of N-acetylneuraminic acid (polysialic acid), although the bacterial CP is longer than human polysialic acid (
200 vs 50 residues, respectively). Therefore, the MenB CP is a self-Ag unable to stimulate an immune response and with the potential to induce autoimmunity if used as a vaccine.
A chemically modified MenB CP, obtained by removal of N-acetyl (N-Ac) and substitution with N-propionyl (N-Pr) groups, can elicit, after conjugation to a protein, bactericidal Abs that passively protect animals from experimental MenB infection (7). It is believed that the N-Pr CP somehow mimics antigenic determinants that are present on the bacterial surface, but are infrequently expressed on the purified N-Ac CP. The N-Pr CP, however, also elicits a small subset of autoreactive IgGs. Although the concentration and/or avidity of the latter are generally low (8), it may be difficult to prove that such autoantibodies are absolutely innocuous. By the use of mAbs raised against either the N-Pr or the N-Ac CP, at least two different classes of capsular epitopes have been defined, both of which can be found on the intact bacterial surface. One class, expressed mainly on N-Ac polysialic acid, is cross-reactive with human tissue, whereas the other, which predominates in N-Pr polysialic acid, is protective and non-cross-reacting (9, 10, 11).
It is well established that protein molecules, such as peptides and anti-idiotypic Abs, can mimic functional features of carbohydrate Ags, including their ability to induce the production of specific Abs (reviewed in Ref. 12). The ability of murine monoclonal anti-idiotypic Abs to function as surrogate vaccines against several different encapsulated pathogens has been shown in the past (13, 14, 15). More recently, it was shown that anti-idiotypic single-chain Ab fragments (scFv) are sufficient to induce protective, capsule-specific Ab responses (16). These recombinant fragments are attractive, because they can be easily manipulated for different purposes, including DNA vaccination and/or fusion with cytokines or immunogenic peptides. In the present study, using an appropriate mAb as a template, we generated scFv mimics of a protective, non-cross-reactive epitope of the MenB CP. These constructs were able to induce anti-meningococcal Ab responses in animals and therefore may be useful in the development of a safe MenB vaccine.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Three encapsulated group B N. meningitidis strains (2996, MC58, and 8047) and the unencapsulated M7 strain (17) were provided by M. Giuliani (Chiron, Siena, Italy). Purified N-Ac MenB, N-Pr MenB, and biotin-conjugated N-Pr MenB CPs, prepared as previously described (9, 18), were provided by A. Bartoloni (Chiron). Vaccines consisting of N-Ac MenB and N-Pr MenB CPs conjugated to tetanus toxoid (N-Ac MenB-TT and N-Pr MenB-TT, respectively) were prepared as previously described (9). Briefly, after partial acid hydrolysis of the polysaccharides, intermediate size (9–25 repeating units) oligosaccharides were separated by ion exchange chromatography, oxidized, and conjugated with TT (obtained from Calbiochem, La Jolla, CA). After purification by gel filtration, saccharide to protein ratios, determined as previously described (9), were 0.19 and 0.22 for N-Pr MenB-TT and N-Ac MenB-TT, respectively. Purified murine Seam 3 mAb was provided by M. Mariani (Chiron). The Seam 3 mAb is an IgG2b raised against N-Pr MenB that strongly reacts against encapsulated MenB to produce complement-mediated bacteriolysis and has no autoantibody reactivity (9). Rabbit MenB-specific typing serum was purchased from Difco (distributed by Voigt Global Distribution, Kansas City, MO).
Preparation of Ab fragments
Preparation of Ab fragments was accomplished as previously described (16, 19), with minor modifications, using libraries of filamentous phages expressing murine scFvs on their surface. Briefly, female BALB/c mice (8 wk old) were s.c. injected on days 0 and 15 with 50 µg of mAb Seam 3 in 0.2 ml of CFA (Difco) and on days 21 and 28 with the same Ag dose in 0.2 ml of IFA (Difco). A final booster injection (50 µg of Ag in saline) was given i.p. on day 35, and 3 days later spleen cells were obtained. After incubating these cells on Seam 3-coated plates, RNA was extracted from adherent cells. After reverse transcribing purified mRNA with random hexadeoxyribonucleotides primers, a commercial system (Recombinant Phage Ab System; Amersham Pharmacia Biotech, Milan, Italy) was used to clone and express Ab fragments. Recombinant phages displaying scFv Abs on their tips were selected after absorption of the library with an irrelevant IgG2b mAb (mouse IgG2b MOPC 141; Sigma-Aldrich, Milan, Italy) as previously described (16, 19) by three rounds of panning on culture flasks that had been previously coated with mAb Seam 3. Screening of isolated colonies was performed by phage ELISA as previously described (16, 19). Seam 3-specific scFvs and the irrelevant H6 scFv (19, 20) were produced and purified as previously described (16, 19). Purity was checked using Coomassie staining of overloaded SDS-polyacrylamide gels as previously described (16). Under these conditions, only a single band with the expected molecular mass of 30 kDa was observed.
Restriction fingerprinting
To ascertain whether different clones from the same library shared a common ancestry, restriction analysis was performed on amplified DNA fragments. To amplify a region containing the scFv genes, 25 µl of a master PCR mix was prepared with the M13 sense primer 5'-GGTAACGCCAGGGTTTTCC-3' and the antisense M13 primer 5'-GGAATTGTGAGCGGATAAC-3'. A HotStart Taq DNA polymerase (0.25 U/tube; Qiagen, Milan, Italy) was used to perform the PCR assay, using isolated colonies directly transferred into PCR tubes. After a first cycle at 95°C for 15 min and 30 cycles at the annealing temperature of 50°C, the PCR products were digested for 3 h at 60°C with the frequently cutting restriction enzyme BstNI (5 U/PCR tube). The digested DNAs were then separated on a 3% agarose gel, and the fragment pattern was recorded.
Nucleotide sequence accession numbers
The DNA encoding for the G1 and B5 scFvs (see Results) was sequenced using the automated Alfexpress DNA Sequencer (Amersham Pharmacia Biotech). These sequences have been deposited with GenBank (accession no. AY374127 and AY374128).
Inhibition ELISA
To study the ability of anti-idiotypic scFvs to compete with N-Pr MenB CP for binding to MenB-specific Abs, an inhibition ELISA was used. Briefly, wells of microtiter plates (Nunc, distributed by M. Brunelli, Milan, Italy) were coated overnight at 4°C with hen’s egg avidin (ExtrAvidin; Sigma-Aldrich) at a concentration of 4 µg/ml in PBS (0.01 M phosphate and 0.15 M NaCl, pH 7.2). After washing, the wells were incubated for 2 h at 37°C with biotinylated N-Pr MenB CP (0.5 µg/ml) in PBS containing 1% BSA (PBS-BSA; Sigma-Aldrich). After washing with PBS containing 0.05% polyoxyethylene-sorbitan monolaurate, the plates were blocked with 1% gelatin in PBS for 3 h and fixed with a 10% sucrose and 4% polyvinyl-pyrrolidone solution. Test samples, consisting of mixtures of MenB-specific Abs and putative inhibitors, were added, and the plates were incubated for 2 h at 37°C. Positive and negative controls consisted of soluble N-Pr MenB and the group-specific polysaccharide of group B streptococci, obtained as previously described (21). After washing, bound Abs were detected by HRP-conjugated anti-mouse or anti-rabbit IgG Abs (Amersham Pharmacia Biotech), followed by 2% (w/v) 2,2'-azino-bis (3-ethylbenz-thiazoline-6-sulfonic acid) in 0.05 M citric acid. Reading was performed at 405 nm on a microplate reader. Percent inhibition was calculated by comparing the absorbance values of wells with and without the inhibitors.
Immunization with scFvs
Female BALB/c mice (6 wk old) or New Zealand rabbits were used for immunization with scFvs. In the case of mice, a first s.c. injection of 50 µg in 0.2 ml of CFA was followed, on days 21 and 42, by two additional s.c. injections of the same Ag dose in 0.2 ml of IFA. Rabbits underwent a similar immunization regimen, except that they received 500 µg of Ag for each immunization at multiple s.c. sites. Sera were obtained from the retro-orbital plexus (mice) and the saphenous vein (rabbits) at the indicated times and were tested as described below.
Control mice were immunized three times s.c. with N-Pr MenB-TT or N-Ac MenB-TT (4 µg of sialic acid/injection) in CFA (day 0) or IFA (days 21 and 42). In additional experiments mice were immunized with the G1 scFv conjugated with keyhole limpet hemocyanin (KLH; Pierce, distributed by Celbio, Milan, Italy). The scFv (5 mg) was mixed with KLH (20 mg) in phosphate buffer (0.1 M, pH 6.8) containing 0.5% glutaraldehyde (Sigma-Aldrich). The reaction was stopped after 30 min by the addition of 1 M glycine (Sigma-Aldrich), followed by extensive dialysis using 50,000 kDa cutoff membranes. The scFv-KLH conjugate was adsorbed to Al(OH)3 gel (alum; Sigma-Aldrich). A group of five mice was immunized s.c. on days 0 and 21 with 0.1 ml of the alum-adsorbed conjugate corresponding to 50 µg of the G1 scFv. Another group of five mice received 50 µg of the G1 scFv in CFA on day 0 and the same Ag dose in IFA on day 21.
N-Pr MenB CP and whole bacteria ELISA
Abs in sera from scFv-immunized animals were assayed for binding to N-Pr MenB CP or to whole bacteria using ELISA. Microtiter plates were coated with avidin, followed by biotinylated N-Pr MenB, as described above (see Inhibition ELISA). To sensitize plates with whole bacteria, strains 2996, MC58, 8047, and M7 were grown at 37°C in 5% CO2 in Muller-Hinton broth to the early exponential phase, washed in PBS, heat-inactivated (56°C for 30 min), and lyophilized. Five micrograms of lyophilized bacteria per well were used to sensitize the plates. After the addition of diluted mouse or rabbit sera, bound Abs were detected with HRP-conjugated polyvalent anti-mouse or anti-rabbit IgG (Amersham Pharmacia Biotech). Isotype-specific HRP-conjugated Abs (Vector Laboratories, distributed by Labtek, Milan, Italy) were used to detect mouse IgM, IgG1, IgG2a, IgG2b, and IgG3.
Bactericidal and opsonophagocytosis assays
Bactericidal activity was assayed in serum samples from scFv-immunized animals using 96-well microtiter plates, with modifications of a previously described procedure (22). Group B N. meningitidis (2996 strain) was grown overnight on chocolate agar plates (Oxoid, Milan, Italy). Approximately five colonies were inoculated into Muller-Hinton broth, and test organisms were grown to an A620 of 0.24 (early log phase). After washing, 1000 CFU in 25 µl of PBS-BSA were added to 25 µl of undiluted, freshly thawed baby rabbit complement (Cedarlane, distributed by Celbio) or heat-inactivated complement and to 25 µl of diluted test sera. After a 1-h incubation with shaking, an aliquot (10 µl) of the reaction mixture from each well was transferred onto chocolate agar plates, and CFU were counted after an overnight incubation at 37°C in 5% CO2 and 95% humidity. Each assay was conducted in triplicate. Bactericidal activity was expressed as the percent variation in CFU numbers observed in the presence of fresh complement relative to heat-inactivated complement.
Opsonophagocytic activity was measured in serum samples in an identical way, except that 5 x 103 human polymorphonuclear leukocytes (PMNs) were added to the mixtures containing serum dilutions, bacteria, and complement. To isolate PMNs, the peripheral blood from adult healthy donors was centrifuged on a Polymorphoprep gradient (Sentinel CH, Milan, Italy), as described by the manufacturer.
Detection of cross-reactive Abs
Ab binding to mammalian polysialic acid was assayed in sera from immunized mice using the human neuroblastoma CHP 212 cell line (provided by A. Pession, Università di Bologna, Bologna, Italy), which expresses high levels of polysialic acid (23). Cells from nearly confluent cultures were collected, washed in PBS, and counted. One aliquot was incubated with exoneuraminidase (Sigma-Aldrich; 150 U/107 cells/ml) at 30°C for 2 h to cleave surface polysialic acid. The cells from each aliquot were then dispensed into the wells of microtiter plates (105 cells/well) and fixed for 1 h at 20°C with 2% paraformaldehyde (Sigma-Aldrich) in PBS. After blocking with 1% gelatin (1 h at 37°C) and washing with PBS-BSA, diluted serum samples were added to the wells, and the ELISA was developed as described above. The Seam 26 mAb (9), known to react against mammalian polysialic acid, was used as a positive control.
Passive immunization with sera
To study the protective effects of passively administered sera from immunized animals, a neonatal mouse model of systemic meningococcal infection was used. Neonatal (<48-h-old) BALB/c mice were randomly assigned to control or experimental groups (30/group), marked, and kept with the mother. Pups were inoculated s.c. with 30 µl of preimmune or immune serum pools and 4 h later were challenged s.c. with 4 and 34 x 105 CFU of strain 2996 corresponding, respectively, to 50 and 90% lethal doses. Mortality was assessed every 24 h for 5 days. Deaths rarely occurred after this time. In selected experiments, MenB was confirmed as the cause of death by blood cultures. Preliminary experiments showed that this model correlated well with the infant rat model in assaying the protective activities of mouse sera.
Statistical analysis
Differences between Ab concentrations and functional activities of sera were analyzed for statistical significance using one-way ANOVA and the Student-Keuls-Newman test. Differences in lethality were analyzed with Fisher’s exact test.
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Results |
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To produce anti-idiotypic scFvs, BALB/c mice were immunized with Seam 3, an mAb raised against N-Pr MenB CP (9). ScFv libraries displayed on filamentous phages were prepared from spleen cells of immunized mice, and individual clones were selected using Seam 3-coated plates. A total of 48 clones were obtained and two of these clones, designated G1 and B5, were selected on the basis of strong ELISA signals. As these clones were obtained from the same library, they could share clonal ancestry. To exclude this possibility, fingerprint analysis was performed on PCR-amplified DNA fragments after BstNII digestion. The restriction pattern of the G1 fragment markedly differed from that of B5 (not shown), thus excluding shared ancestry. Nucleotide sequence analysis further confirmed that G1 and B5 were different scFvs.
Inhibition ELISA
Next, it was determined whether the G1 and B5 anti-idiotypic scFvs could mimic some of the antigenic properties of the nominal Ag, i.e., the N-Pr MenB CP. First, we looked at the ability of the scFvs to compete with the N-Pr MenB CP for binding to Seam 3. Fig. 1 shows the results obtained with an inhibition ELISA using N-Pr MenB CP as the coating Ag. As expected, soluble N-Pr MenB CP, used as a positive control, inhibited Seam 3 binding, with a 50% inhibitory concentration of 10 ng/ml (Fig. 1, left panel). In contrast, the negative control, consisting of group-specific polysaccharide from group B streptococci, did not produce any inhibition at doses up to 50 µg/ml. Both the G1 and B5 scFvs could block Seam 3 binding in a dose-dependent fashion, with 50% inhibitory concentrations of 7 and 9 µg/ml, respectively. In contrast, the irrelevant H6 scFv failed to inhibit Seam 3 binding at concentrations as high as 50 µg/ml.
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Induction of Ab responses
In additional experiments we investigated whether immunization with the G1 and B5 scFvs could induce the production of anti-N-Pr MenB CP Abs. Sera were collected from BALB/c mice at different times after immunization and tested for binding to N-Pr MenB CP-sensitized plates. Low Ab levels were detected in preimmune sera (Fig. 2, left panels). Immunization with both the G1 and B5 scFvs induced significant elevations of Abs reacting against the N-Pr MenB CP. In contrast, immunization with the irrelevant H6 scFv produced no variation in Ab titers. Both the G1- and B5-induced Ab responses were boostable and predominantly of the IgG class (Fig. 2). Isotype analysis indicated that IgG2a and, to a lesser extent, IgG1 were the predominant Abs produced (Fig. 2, right panels).
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In additional experiments it was of interest to ascertain whether the scFvs were also able to induce significant anti-N-Pr MenB CP responses when using alum as an adjuvant in place of CFA. To this end, alum-adsorbed G1 scFv-KLH conjugate was employed to immunize animals. Results were compared with those observed using an identical G1 scFvs dose in CFA. Under these conditions, both immunization treatments resulted in significant anti-N-Pr MenB CP IgG1 or IgG2a elevations, with no differences in Ab titers being observed between the two types of immunization (data not shown). These data indicated that CFA was not absolutely required for immunogenic activities of the scFvs.
Next, the ability of the G1 and B5 scFvs to induce anti-N-Pr MenB CP Ab responses was investigated in rabbits (Fig. 3). As previously observed in mice, a boostable anti-N-Pr MenB CP response, which was predominantly of the IgG type, was also observed in rabbits using the G1 scFv as an immunogen. The B5 scFv also induced elevations in IgG levels, although these were not statistically different from Ab levels in animals immunized with the irrelevant H6 scFv. This lack of statistical significance was probably secondary to the small number of animals examined. However, in view of the seemingly lower immunogenicity of the B5 scFv in rabbits, in further studies we focused on the G1 scFv only.
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The ability of priming with anti-idiotypes to modify subsequent responses to the mimicked Ags has been clearly demonstrated (12, 13). It was therefore of interest to determine whether the G1 scFv was capable of priming animals for increased anti-N-Pr MenB Ab responses upon encounter with the pathogen. To this end, animals were immunized twice with different scFvs before inoculation with whole killed group B meningococci in IFA. In animals primed with the irrelevant H6 scFv, inoculation with meningococci induced low grade elevations in anti-MenB CP IgM (Fig. 6, lower panel). In contrast, brisk elevations of anti-MenB CP Abs were observed in G1-primed animals (Fig. 6). These data indicated that the G1 scFv mimic of the MenB CP could effectively prime mice for increased Ab responses after exposure to whole bacteria.
Passive immunoprotection
In additional experiments we looked at the ability of sera from scFv-immunized mice and rabbits to passively protect infant mice from lethality produced by group B meningococci. In these experiments we used a recently developed neonatal mouse model. Preliminary experiments showed that this model correlated well with the infant rat model in testing the protective activity of murine and human sera. Fig. 7 shows that sera from G1-scFv immunized animals moderately, but significantly, increased survival in pups inoculated with a 90 or 50% lethal dose of group B meningococci. These protective effects, however, were lower (p < 0.05) than those produced by passive immunization with sera from N-Pr Men B-TT-immunized mice (Fig. 7, upper panels).
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The ability of sera from scFv-immunized animals to kill N. meningitidis was examined in vitro using complement-dependent bactericidal or opsonophagocytosis assays. In these experiments we used selected serum samples obtained during the experiments shown in Fig. 2. Serum bactericidal activity (Fig. 8, 
) was expressed as the percent decrease in CFU observed in the presence of fresh complement, relative to heat-inactivated complement. Although sera obtained from N-Pr MenB-TT immunized mice showed significant bactericidal activity, no such activity was observed after immunization of mice or rabbits with the G1 scFv. However, significant increases in opsonizing activity (Fig. 8, 
) were detected in serum samples from G1-immunized animals compared with their respective preimmune controls.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the present study we have developed scFv mimics of a protective capsular MenB epitope that are devoid of cross-reactivity with host polysialic acid. These anti-idiotypic mimics not only compete with the nominal Ag (i.e., N-Pr MenB CP) for binding to specific mouse or rabbit Abs, but also induce, after immunization, N-Pr MenB CP-specific serum Abs. These scFv-induced Abs had the following characteristics: 1) lack of binding to human polysialic acid; 2) binding to a capsular epitope expressed on both the intact bacterial surface and the purified capsular polysaccharide; 3) opsonizing activity, resulting in increased bacterial killing by human PMN leukocytes; and 4) protective activity in a neonatal model of MenB disease. Moe et al. (12) and Shin et al. (10) used a strategy similar to the one described in this study to select peptide mimics of the MenB CP using MenB CP-specific Abs. However, the immunogenic properties of these peptide mimotopes were not described. It should be noted that none of these peptides has any sequence homology with the hypervariable regions of the anti-idiotypic scFvs described in this study.
Collectively, our data indicate that the anti-idiotypic strategy we used was effective in mimicking the immunogenic properties of a protective capsular epitope while avoiding the risks inherent to vaccination with the whole capsular Ag, which is known to contain host cross-reactive epitopes. In fact, both whole MenB cells and the purified capsular polysaccharide express human cross-reactive epitopes. Although N-Pr derivatization of the Ag can markedly increase immunogenicity after protein conjugation (7) (Fig. 2), cross-reactive epitopes are still present on N-Pr MenB CP, as shown in immunized mice by low, but detectable, levels of anti-polysialic acid serum IgGs (Fig. 5). These data are in agreement with previously described work (8, 9). A high frequency of polysialic acid-specific mAbs was observed in N-Pr MenB CP-immunized mice after fusion of spleen cells (9). Moreover, a pool of sera from N-Pr MenB TT-immunized mice showed weak, but specific, IgG binding to polysialic acid-rich neural cell adhesion molecule in embryonic brain extracts and on the surface of live cells derived from a mouse pituitary tumor (8). No binding to polysialic acid was detected, however, by other tests. Collectively, published work and the present study indicate that weak, but discernible, elevations of anti-mammalian polysialic acid Abs can be induced by N-Pr MenB TT immunization. Although the neuropathological potential of these Abs has not been shown, it may be difficult to exclude that after crossing the placenta, they can somehow damage the nervous system of the developing fetus.
In the present study the scFv-induced responses were boostable and predominantly of the IgG2a type, indicating that these anti-idiotypes were capable of producing a Th1-like response. This feature is of interest, because capsular polysaccharides are incapable of inducing Th1-dominant responses even after protein conjugation. In fact, although protein conjugation does confer T-dependent help, conjugate vaccines have limited ability to induce the Th1-associated murine IgG2a isotype, while inducing predominantly IgG1 and IgG3 isotypes (28). A Th1 response with the expression of IgG2a would be desirable because IgG2a and IgG3 isotypes have been reported to be particularly effective in conferring protection against encapsulated organisms (29). In addition, the scFv immunization primed the mice for a MenB-specific response that was rapidly activated upon encounter with whole MenB. These data suggest that scFv immunization generates a memory B cell population that can be expanded by encounter with MenB bacteria.
However, some limitations of the anti-Id strategy used in this study need to be pointed out. First, the protective activities of the sera from scFv-immunized animals were only moderate in degree and considerably lower that those produced by the Seam-3 mAb, used as a template to develop the scFv mimics. Second, the scFv-induced sera had no significant bactericidal activity, whereas Seam 3 is highly bactericidal. The reasons underlying these phenomena are not clear, but are probably related to insufficient concentration and/or affinity of the Abs induced by the scFv immunization. Different approaches are being used to overcome these problems, all taking advantage of the ease with which the Ab fragments used can be manipulated by molecular techniques. Higher Ab responses could conceivably be obtained by expressing the scFvs as fusion proteins containing T cell epitopes or adjuvants, such as IL-12 or other cytokines. Indeed, IL-12 treatment was shown to dramatically enhance Ab responses and, in particular, IgG2a responses to both unconjugated and protein-conjugated capsular polysaccharide vaccines (28). New DNA immunization strategies are also likely to result in enhanced anti-scFv responses due to increased in vivo Ag persistence and the inclusion of DNA immunostimulatory sequences in the plasmid used for vaccination. It was recently shown that DNA vaccination with mimotope-encoded plasmid induced strong IgG2a anti-carbohydrate responses (30).
An additional strategy to increase the size and affinity of Ab responses is the development of further refined, second-generation mimics that would more closely resemble, from a structural standpoint, the natural Ag. Recent advances in structural chemistry involving x-ray and nuclear magnetic resonance spectroscopy may soon allow the creation of more "faithful" molecular mimics by design (31). Finally, the scFv described in this study may be used as fusion proteins in conjunction with other meningococcal Ags. Recently, promising vaccine candidates have been identified using whole genome sequencing of group B N. meningitidis (32). As all group B strains are sensitive to anti-capsular Abs, the inclusion of a capsule-based immunogen in such vaccines may help prevent bacterial escape due to the emergence of mutant strains in vaccinated populations.
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Footnotes |
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2 Abbreviations used in this paper; CP, capsular polysaccharide; KLH, keyhole limpet hemocyanin; MenB, serogroup B meningococcal; N-Ac, N-acetyl; N-Pr, N-propionyl; PMN, polymorphonuclear leukocyte; scFv, single-chain Ab fragment; TT, tetanus toxoid.
Received for publication March 17, 2003. Accepted for publication December 5, 2003.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2–8 N-acetyl neuraminic acid, the capsular polysaccharide of Neisseria meningitidis serogroup B. J. Infect. Dis. 172:1279.[Medline]
26 and 21 polyfructan reactive clones in BALB/c mice treated at birth with minute amounts of anti-A48 idiotype antibodies. J. Exp. Med. 158:1129.This article has been cited by other articles:
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  C. Lo Passo, A. Romeo, I. Pernice, P. Donato, A. Midiri, G. Mancuso, M. Arigo, C. Biondo, R. Galbo, S. Papasergi, et al. Peptide Mimics of the Group B Meningococcal Capsule Induce Bactericidal and Protective Antibodies after Immunization J. Immunol., April 1, 2007; 178(7): 4417 - 4423. [Abstract] [Full Text] [PDF]
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 C. Beninati, A. Midiri, G. Mancuso, C. Biondo, M. Arigo, E. Gerace, S. Papasergi, M. Gambuzza, M. Boretti, W. Magliani, et al. Antiidiotypic DNA vaccination induces serum bactericidal activity and protection against group B meningococci J. Exp. Med., January 23, 2006; 203(1): 111 - 118. [Abstract] [Full Text] [PDF]
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 I. H. Park, J. H. Youn, I.-H. Choi, M. H. Nahm, S. J. Kim, and J.-S. Shin Anti-Idiotypic Antibody as a Potential Candidate Vaccine for Neisseria meningitidis Serogroup B Infect. Immun., October 1, 2005; 73(10): 6399 - 6406. [Abstract] [Full Text] [PDF]
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-chain- and µ-chain-specific anti-mouse Ig (left panels) or anti-mouse IgG1, IgG2a, IgG2b, or IgG3 (right panels) HRP-conjugated Abs. Data represent the mean ± SD of five determinations, each conducted in duplicate on a different animal. *, Significantly different from anti-H6 scFv sera; 
, significantly different from anti-G1 or anti-B5 scFv serum.
