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1 ,3 Department of Bacteriology1 and Department of Immunology3 , School of Medical Sciences, Tarbiat Modares University, Tehran, Iran
2 Iranian Blood Transfusion Organization, Research Center, Tehran, Iran
4 Academic Center for Education, Culture, and Research, Tehran, Iran
Correspondence
Nasim Kashef
na_kashef{at}yahoo.com
Received 24 April 2006
Accepted 13 July 2006
Abbreviations: ADH, adipic acid dihydrazide; CF, cystic fibrosis; EDAC, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide; i.p., intraperitoneal(ly); KLH, keyhole-limpet haemocyanin; MEP, mucoid exopolysaccharide; TT, tetanus toxoid.
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INTRODUCTION |
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 TOP INTRODUCTION METHODSRESULTS AND DISCUSSIONREFERENCES |
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Immunization with alginate antigen gives rise to antibodies that have opsonic activity and lead to clearance of mucoid P. aeruginosa from the respiratory tract in mice and rats (Pier et al., 1983, 1990, 1994).
One of the most effective modern technologies applied to active vaccination has been the conjugation of surface carbohydrate capsular antigens to carrier proteins to increase their immunogenicity, particularly in young children (Lakshman & Finn, 2002; Makela, 2003; Pelton et al., 2003). This converts polysaccharide from a T-cell-independent to a T-cell-dependent antigen, and elicits a higher and boostable immune response in animals (Sood et al., 1996). The applicability of this technology to the alginate of P. aeruginosa has been investigated (Pier, 2005; Cryz et al., 1991; Theilacker et al., 2003). Cryz et al. (1991) used detoxified exotoxin A as a carrier protein, whereas Theilacker et al. (2003) used keyhole-limpet haemocyanin (KLH).
In the present study, we describe the synthesis and characterization of alginate–tetanus toxoid (TT) conjugate using the native non-depolymerized polymer of alginate.
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METHODS |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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Chemical analysis of alginate. The purified antigen was analysed for uronic acid content by the carbazole-borate assay (Knutson & Jeanes, 1986) with sodium alginate as the standard, for protein by the Bradford assay (Bradford, 1976) with BSA as the standard, for nucleic acid by A260, and for LPS (endotoxin) by the Limulus amoebocyte lysate assay with Escherichia coli endotoxin as the standard.
Protein. TT was obtained from Razi Vaccine and Serum Research Institute of Iran. TT preparations were concentrated by ultrafiltration with a molecular-size cutoff of 100 000 Da. Chromatography was performed with a Sephacryl S-200 (Pharmacia XK 16) column. The concentrated TT preparations were applied to the column, which was equilibrated with 0.2 M NaCl (flow rate 30 ml h–1). Optical densities of eluted fractions were measured by spectrophotometry at 280 nm. The peak corresponding to a molecular mass of 150 000 Da was pooled and concentrated. The final material was passed through a 0.45 µm pore-size filter and stored at 4 °C.
Derivatization and conjugation of alginate. The alginate was derivatized as follows. Alginate (10 mg) and adipic acid dihydrazide (ADH) (0.5 M final concentration) were dissolved in 5 ml 0.05 M PBS buffer, pH 7.4, and the pH was adjusted to 5.0 by adding 0.3 M HCl. After stirring at 4 °C for 4 h, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDAC) was added (0.2 M final concentration), and the reaction mixture was stirred at 4 °C for 18 h while the pH was maintained between 4.9 and 5.1. The reaction mixture was dialysed exhaustively against distilled H2O at 4 °C.
A total of 10 mg TT was added to the adipic hydrazide (AH) derivative of alginate, and coupling was done with 0.1 M EDAC for 1 h at room temperature and 24 h at 4 °C. The reaction mixture was passed through a Sepharose CL-4B column with PBS, pH 7.4, used as running buffer (flow rate 30 ml h–1). Void-volume fractions that assayed positive for both protein and uronic acid were designated polysaccharide–protein conjugate and were pooled, passed through a 0.45 µm pore-size filter and stored at 4 °C.
Chemical analysis of alginate–TT conjugate. The amounts of protein and uronic acid present in the conjugate were quantified by the Folin–Lowry assay with BSA as the standard (Lowry et al., 1951), and the carbazole-borate assay with sodium alginate as the standard, respectively.
Pyrogenicity determination. New Zealand White rabbits (2–2.5 kg each), three in each group, were used. Alginate–TT antigen was administered intravenously at 1 ml per kg rabbit body weight. Rectal temperatures were recorded at 15 min intervals for 3 h after challenge.
Toxicity test. The lethal effect of alginate–TT conjugate was evaluated in five female mice (weight 22 g) and two guinea pigs (weight 250 g). One human dose (10 µg ml–1) of conjugate was administered intraperitoneally (i.p.). Animals were observed for 5 days post-challenge.
Stability test. Alginate–TT conjugate was placed at 37 °C for 1 week, and rerun over the same size-exclusion chromatography column. The profiles of the protein and polysaccharide were determined to check stability.
Immunization of mice. Female BALB/c mice, 6–8 weeks old, were injected i.p. in groups of five on days 0, 14 and 28 with either purified alginate or alginate–TT conjugate suspended in PBS. Blood was obtained from the orbital sinus on days 14, 28 and 42. All immunizations were done without adjuvant. Alginate-specific IgG was determined by ELISA.
ELISA. ELISAs were performed as follows. Microtitre plates were coated with alginate derived from P. aeruginosa strain 8821M (6 µg ml–1 in PBS, pH 7.4), and kept overnight at 4 °C. Between incubation steps, plates were washed three times with PBS containing 0.05 % Tween 20 (PBS-Tw). Individual mouse sera were diluted in the blocking buffer (1/50) and assayed in triplicate. Incubation was performed for 1.5 h at 37 °C. Bound antibodies were allowed to react with a horseradish peroxidase-conjugated goat anti-mouse IgG diluted 1 : 8000 as secondary antibody, for 1.5 h at 37 °C. o-Phenylenediamine dihydrochloride (Sigma; 0.4 mg ml–1 in 0.2 M Na2HPO4, 0.1 M citric acid, pH 5) and 10 µl H2O2 was used as substrate. After 15 min incubation in the dark, the reaction was stopped by the addition of 0.05 ml H2SO4 (20 %), and A492 was measured.
Opsonophagocytosis assay. Opsonophagocytic killing was determined by using 100 µl heat-inactivated mouse serum diluted 1 : 10, 100 µl mouse macrophages at 1x107 ml–1, 100 µl 4 % fresh infant rabbit serum as a complement source, and 100 µl mucoid P. aeruginosa 8821M at 1x107 ml–1. These components were mixed in sterile microfuge tubes. Control tubes, from which antibody, complement or macrophages were omitted, and 100 µl RPMI medium/fetal calf serum was substituted, were run with each assay. For all assays involving mouse sera, pooled serum from members of the respective immunization groups was used. The tubes were held at 37 °C for 90 min with gentle shaking and a 10 µl sample was removed, diluted in saline, and plated for bacterial counts. The plates were incubated overnight at 37 °C, and mucoid colonies were counted. The percentage kill was calculated as follows:
Percentage kill=[1–(c.f.u. of immune serum at 90 min/c.f.u. of preimmune serum at 90 min)]x100.
Active protection. Mice were divided into three groups, A–C, each containing six mice. Groups A and B were immunized i.p. three times (on days 0, 7 and 14) with 4 µg MEP–TT and MEP (in 0.1 ml PBS, pH 7.4), respectively. Group C contained six unimmunized control mice. Two weeks after the last immunization, mice were challenged i.p. with 3x108 c.f.u. (4x LD50) of the heterologous strain of mucoid P. aeruginosa suspended in sterile PBS, pH 7.4; the inoculum was given in a volume of 1 ml. Mice were observed for 7 days, and mortality was recorded.
Statistical analysis.
The statistical analysis was performed using SPSS version 11.5 (SPSS). Differences in the mean ELISA absorbance and the mean percentage of opsonic killing were compared by analysis of variance (ANOVA) by using a post hoc multiple comparison (Bonferroni correction) test. The chi square test was used to analyse the survival data from the protection experiment. P
0.05 was considered significant.
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RESULTS AND DISCUSSION |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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On a Sephacryl S-400 column, sodium alginate eluted at a lower volume than purified alginate, indicating a somewhat larger molecular size. Chemical analysis showed that the purified antigen contained 91.6 % (w/w) uronic acid, <7.6 % protein, <0.0061 % LPS, and 0.7 % nucleic acid.
Only the large-size polymer fractions of alginate were collected, because it has been shown that only the highest-molecular-size polymers of alginate are able to induce opsonic antibodies in mice with pre-existing levels of non-opsonic antibodies (Pedersen & Kharazmi, 1990). Non-opsonic antibodies are frequently seen in healthy individuals as well as in most patients with CF, even before the onset of detectable infection (Pedersen & Kharazmi, 1990).
Efficiency of coupling reaction
The conjugation of capsular polysaccharide (CP) or other bacterial polysaccharide-based vaccines to a carrier protein is a well-established approach to increase the immunogenicity of the former (Fattom et al., 1995). Because of the very large molecular mass of alginate, conjugating it to carrier proteins to produce immunogenic vaccines has proven difficult. Cryz et al. (1991) constructed a conjugate vaccine of depolymerized alginate with exotoxin A of P. aeruginosa. This approach, however, has some potential disadvantages, as conformational epitopes of bacterial polysaccharides are often stabilized by polymer length, which can be destroyed by depolymerization (Watson et al., 1992). Another alginate conjugate vaccine was synthesized and evaluated by Theilacker et al. (2003), who bound thiolated alginate to KLH by using succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) as a linker. Using this technology, they were able to construct a water-soluble conjugate of native, large-molecular-weight alginate. A potential disadvantage of the chosen carrier protein and conjugation chemistry is that neither KLH nor the cross-linker SMCC have yet been used in bacterial polysaccharide conjugate vaccines injected into humans.
We used TT as the carrier protein because in practice, only a handful of proteins of bacterial origin, such as TT and diphtheria toxoid, have been used for the preparation of the conjugate vaccines that have been licensed for human use or are currently under development (Sood et al., 1996). The TT molecule (Mr 150 000) is a more effective carrier protein than diphtheria toxoid (Mr 58 000), perhaps owing to its size (Watson et al., 1992). However, its use as a universal carrier may prove to be undesirable, as its frequent use may overload the immune system with large doses of this protein in combined vaccines, and therefore result in a higher frequency of adverse reactions due to pre-existing antibodies in targeted populations (Peters et al., 1991; Barington et al., 1994). Overall, diversifying our carrier-protein pool may prove to be crucial for the development of human conjugate vaccines.
Our initial attempts to conjugate native, non-depolymerized alginate to TT were not successful. We found that ADH coupling via EDAC had to be repeated once or twice to obtain sufficient ADH bound to the alginate, or alternatively that the reaction time of ADH, alginate and EDAC needed to be increased.
In preliminary studies, the concentration of the reactants during the coupling reaction, and the reaction time, influenced the yield of conjugate. The coupling reactions were not effective at ADH and EDAC concentrations lower than 0.5 and 0.2 M, respectively, and optimal results were obtained with concentrations of TT and alginate of 10 mg ml–1. There was a progressive increase in the efficiency of the coupling reaction with time (data not shown).
Characterization of conjugate
Gel filtration of the alginate–TT conjugate on a Sepharose CL-4B column yielded a large peak at the void volume consisting of both protein and polysaccharide, one protein peak at higher elution volumes, and two minor peaks positive for uronic acid (Fig. 1
). Since native alginate elutes from this resin at higher volumes, only fractions eluting at and just past the void volume were presumed to be free of non-conjugated polysaccharide, and these represented alginate–TT conjugate containing 74.6 % protein and 25.4 % uronate. There were no overt signs of toxicity or pyrogenicity after i.p. or intravenous administration (respectively) of the conjugate vaccine to animals. The elution profile of the conjugate did not alter on storage at 37 °C.
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). The first immunization with alginate alone elicited low levels of IgG antibody, but this did not increase significantly after second and third immunizations (P=0.214 and P=0.133, respectively). The first immunization with conjugate also induced low levels of IgG. No significant rise in alginate-specific IgG was observed 14 days after the second vaccine dose (P=0.963), but a third immunization boosted antibody levels significantly (P<0.0001). As shown in Table 1, immunization with the alginate–TT conjugate also induced IgG against the carrier protein. The second immunization with conjugate induced high levels of TT-specific IgG compared to the first immunization (P=0.007), but no significant rise was observed 14 days after the third immunization (P=0.10). Native alginate therefore acted as a typical T-cell-independent antigen, whereas a T-cell-dependent response was observed with the conjugate.
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. Mice immunized with conjugate not only elicited IgG against the two components of the conjugate, but also induced significant levels of IgG antibodies to the complete vaccine. A considerable rise in alginate–TT-specific IgG was observed after the second and third vaccine dose (P<0.0001).
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. In the opsonophagocytic killing assay, antisera from mice immunized with the conjugate mediated phagocytic killing (86.3 %) against the vaccine strain on day 42, but antiserum from mice primed with native alginate was less efficient in mediating phagocytic killing (68.5 %). There was no phagocytic killing when antibody, complement or macrophages were omitted.
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Active protection
Three immunizations of mice with 4.0 µg conjugate per dose showed significant protection (P<0.01) against intreperitoneal challenge with 4x LD50 of wild-type mucoid P. aeruginosa. This challenge dose killed 6/6 of mice that were uninoculated, 1/6 of mice immunized with conjugate and 3/6 of mice immunized with alginate alone. There was no significant difference in the rates of survival of mice immunized with alginate and those of uninoculated mice (P=0.54).
A key feature of any vaccine is its ability to protect against infection with strains of the organism heterologous to the one from which the vaccine was derived. For alginate, with its considerable variation in the ratio of mannuronic to guluronic acid and degree of O-acetylation, it is crucial to establish that vaccination with a single preparation induces antibodies reactive against heterologous mucoid P. aeruginosa strains (Sherbrock-Cox et al., 1984). Our data suggest that immunization of mice with the alginate–TT conjugate results in an increased LD50 after heterologous-type challenge, and IgG induced by the conjugate was cross-reactive to the heterologous mucoid strain. In contrast to our results, in another study, cross-reactive IgG was virtually absent after immunization of rats with an alginate–exotoxin A conjugate (Johansen et al., 1994, 1995). These data suggest that extensive depolymerization and/or de-O-acetylation may lead to the loss of epitopes shared between heterologous strains.
In conclusion, conjugation of alginate to TT utilizing ADH coupling via EDAC yielded an alginate-based conjugate rich in protein and uronate, which was non-toxic, non-pyrogenic, and elicited high titres of alginate-specific IgG. Antisera raised against the conjugate had high opsonic activity against the vaccine strain. The alginate conjugate was also able to protect mice against a lethal dose of mucoid P. aeruginosa. These data indicate that an alginate-based vaccine has significant potential to protect against chronic infection with mucoid strains of P. aeruginosa in the CF host.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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