Additional Conjugation Methods and Immunogenicity of Bacillus anthraci
http://www.100md.com
《感染与免疫杂志》
Laboratory of Developmental and Molecular Immunity, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892
ABSTRACT
The capsule of Bacillus anthracis, composed of poly--D-glutamic acid (DPGA), is an essential virulence factor of B. anthracis. The capsule inhibits innate host defense through its antiphagocytic action. DPGA is a poor immunogen, but when covalently bound to a carrier protein, it elicits serum antibodies. To identify the optimal construct for clinical use, synthetic DPGAs of different lengths were bound to carrier proteins at different densities. The advantages of the synthetic over the natural polypeptide are the homogeneous chain length and end groups, allowing conjugates to be accurately characterized and standardized and their chemical compositions to be related to their immunogenicities. In the present study, we evaluated, in addition to methods reported by us, hydrazone, oxime, and thioether linkages between DPGA and several proteins, including bovine serum albumin, recombinant Pseudomonas aeruginosa exotoxin A, recombinant B. anthracis protective antigen (rPA), and tetanus toxoid (TT). The effects of the dosage and formulation on the immunogenicities of the conjugates were evaluated in mice. All conjugates were immunogenic. The optimal DPGA chain length of 10 to 15 amino acids and the density, an average of 15 mol DPGA per mol of protein, were confirmed. The thioether bond was the optimal linkage type, and TT and rPA were the best carriers. The optimal dosage was 1.2 to 2.5 μg of DPGA per mouse, and adsorption of the conjugates onto aluminum hydroxide significantly increased the antibody response to the protein with a lesser effect on anti-DPGA levels.
INTRODUCTION
Anthrax, a potentially lethal human disease, is a zoonotic infection that under natural conditions is contracted by humans directly or indirectly from animals. The causative organism, Bacillus anthracis, exists in vegetative or spore forms, the latter being the infecting agent. A veterinary vaccine based on a capsule-negative, toxin-positive strain (Sterne) is available and is routinely used throughout the world (25). In addition, anthrax vaccine adsorbed, containing the protective antigen, is licensed and used by veterinarians, animal by-product handlers, and the U.S. Army (2, 5). Anthrax is endemic in countries that do not immunize domesticated animals (9, 12, 24). Because of the ease of cultivating the organism and improved technology of spore preparation, B. anthracis is a potential bioterrorism agent. In 2001, B. anthracis spores were used successfully via the U.S. mail, though few people were affected. A major bioterrorism attack may be airborne, with a number of spores far exceeding a natural exposure, causing inhalation anthrax and affecting a large number of people, including children. These facts warrant devising an improved anthrax vaccine.
The addition of components other than those of anthrax toxin to improve vaccine-induced protection has been considered (22). The capsule, composed of poly--D-glutamic acid (DPGA), is an essential virulence factor and antiphagocytic, and antibodies to this polypeptide have been shown to be opsonophagocytic and protective in mice (3, 10, 22). DPGA by itself is a poor immunogen and does not induce booster responses, probably because of its simple homopolymeric structure, similar to those of capsular polysaccharides; it is a T-cell-independent antigen and of D-amino acid composition (7). These immunologic properties can be overcome by covalent binding of the T-cell-independent antigen to immunogenic proteins (22). Because of the success in inducing protective levels of antibodies in infants against systemic infection with capsulated pathogens, we developed conjugates of DPGA with several carrier proteins, including bovine serum albumin (BSA), recombinant B. anthracis protective antigen (rPA), and recombinant Pseudomonas aeruginosa exoprotein A (rEPA). Unlike DPGA alone, these conjugates were immunogenic in mice, with booster responses upon reinjection. Conjugate-induced antibodies were opsonophagocytic (22, 27). This study describes additional synthetic schemes in an attempt to develop the most immunogenic conjugates.
MATERIALS AND METHODS
Analyses. Amino acid analysis was done by gas-liquid chromatography-mass spectrometry (GLC-MS) after hydrolysis with 6 N HCl at 150°C for 1 h and derivatization to N-heptafluorobutyryl R-(–)-isobutyl esters and assay with a Hewlett-Packard apparatus (model HP 6890) with an HP-5 0.32- by 30-mm glass capillary column with temperature programming at 8°C/min from 125 to 250°C in the electron ionization (106-eV) mode (22). The number of peptide chains in the conjugates was calculated by the ratio between L- and D-glutamic acids. The protein concentration was measured by the method of Lowry et al. (16), free amino groups by Fields' assay (6), benzaldehyde groups by colorimetric reaction with 2-hydrazinopyridine (Solulink, San Diego, CA), hydrazide was measured as reported previously (23), and thiolation was measured by release of 2-pyridylthio groups (A343) (1). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis used 14% gels according to the manufacturer's instructions (Bio-Rad, Hercules, CA). Double immunodiffusion was performed in 1.0% agarose gels in phosphate-buffered saline (PBS) with rabbit anti-DPGA, rabbit anti-BSA (Sigma, St. Louis, MO), goat anti-exotoxin A (List Biological Laboratories, Inc.), anti-tetanus toxoid (obtained from W. Vann, FDA, Bethesda MD), and anti-B. anthracis protective antigen (obtained from S. Leppla, NIH/NIAID, Bethesda, MD). Aluminum hydroxide was used as Alhydrogel (Staten Serum Institut, Copenhagen, Denmark).
MALDI-TOF. Mass spectra were obtained with an OmniFlex matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) instrument (Bruker Daltonics) operated in the linear mode. Samples for analysis were desalted, and 1 μl was mixed with 20 μl of sinapinic acid matrix made in 30% CH3CN and 0.1% trifluoroacetic acid. Then, 1 μl of the mixture was dried on the sample stage and placed in the mass spectrometer.
Antigens. BSA (66.5 kDa; Sigma, St. Louis, MO) was dialyzed against pyrogen-free water, sterile filtered, and freeze-dried. rPA (83 kDa) from B. anthracis and rEPA (67 kDa) from P. aeruginosa were prepared and characterized (8, 21). Tetanus toxoid (TT) (150 kDa) was obtained from Merieux, Lyon, France.
DPGA was purified from the culture supernatant of B. anthracis strain A34 toxin-negative by cetavlon precipitation, acidification to pH 1.5, precipitation with ethanol, and passage through a 2.5- by 100-cm Sephacryl S-1000 column in 0.2 M NaCl (26). Its structure was confirmed by 1H nuclear magnetic resonance and 13C nuclear magnetic resonance, and its enantiomeric composition was determined by GLC-MS spectroscopy. DPGA peptides were synthesized by the method of Merrifield (AnaSpec, San Jose, CA). Peptides were divided into groups depending on the types of linkages through which they were bound to proteins: (i) thioether linkage, NAc-DPGA10-Gly3-L-Cys-CONH2 (DPGA10-Cys) or NBrAc-Gly3-DPGA10-COOH (Br-DPGA10); (ii) hydrazone linkage, 4-formylbenzoyl-Gly3-DPGA10-COOH (CHO-DPGA10), NAc-DPGA10-Gly3-CO-NH-NH-CO-(CH2)4-CO-NH-NH2 (DPGA10-AH), or NAc-DPGA15-CO-NH-NH-CO-(CH2)4-CO-NH-NH2 (DPGA15-AH), where AH is adipic acid hydrazide; and (iii) oxime linkage, 4-formylbenzoyl-Gly3-DPGA10-COOH (CHO-PGA10).
Conjugation. (i) Thioether linkage. First, protein was bromoacylated using succinimidyl 3-(bromoacetamido)propionate (SBAP) (Pierce, Rockford, IL) and reacted with peptides equipped with a terminal cysteine residue as reported previously (22) (protein/S-Cys-Gly3-DPGA10-NAc, protein/S-Cys-DPGA10-NAc, or protein/S-Cys-DPGA15-NAc).
Second, protein was derivatized with an N-hydroxysuccinimide ester of 3-(2-pyridyldithio)propionic acid (SPDP) (Pierce, Rockford, IL) and reacted with bromoacylated peptide (22) (protein-S/Gly3-DPGA10-COOH).
Third, sulfhydryl groups were introduced into the protein using 2-iminothiolane (ITL) (ScienceLab, Houston, TX). Protein (20 mg) was derivatized with 2.8 mg ITL in 1.5 ml of buffer A (PBS, 0.1% glycerol, 0.005 M EDTA, pH 7.4) for 60 min. Next, a solution of Br-DPGA10 (35 mg) in 300 μl of 1 M K2HPO4 was added while the pH was maintained at 7.4 with the addition of 0.2 N NaOH at room temperature. After 90 min, the reaction mixture was passed through a Sepharose CL-6B column (1 by 120 cm) and eluted with 0.2 M NaCl. Fractions reacting with anti-protein and anti-DPGA were pooled (protein-ITL/Gly3-DPGA10-COOH).
(ii) Hydrazone linkage. First, protein was derivatized with succinimidyl 4-formylbenzoate (SFB) (Solulink, San Diego, CA). To a solution of protein (30 mg) in 1.2 ml buffer A, SFB (7.5 mg) in 100 μl dimethyl sulfoxide was added and reacted for 2 h at pH 7.4. The product 4-formylbenzoyl-protein was passed through a Sephadex G-50 column (1 by 100 cm) in 0.2 M NaCl. Protein-containing fractions were pooled and assayed for the presence of benzaldehyde, antigenicity, and protein concentration. To 4-formylbenzoyl-protein (20 mg) in 1.25 ml buffer A, a solution of 15 mg of AH-DPGA10 or AH-DPGA15 in 200 μl 1 M K2HPO4 was added. The pH of the reaction mixture was adjusted to 7.4, and the mixture was incubated overnight at room temperature and then passed through a Sepharose CL-6B column as described above. Fractions reacting with anti-protein and anti-DPGA were pooled and assayed (protein-SFB/AH-Gly3-DPGA10-NAc or protein-SFB/AH-DPGA15-NAc).
Second, derivatization of protein with adipic acid dihydrazide using a water-soluble carbodiimide was done as reported previously (23). The incorporation of hydrazide residues was 2 to 5% per protein. To protein-AH (20 mg) in 1.3 ml buffer A, a solution of 20 mg of CHO-DPGA10 in 200 μl 1 M K2HPO4 was added. The pH was adjusted to 7.4, and the solution was incubated overnight at room temperature and then passed through a Sepharose CL-6B column as described above. Fractions reacting with anti-protein and anti-DPGA were pooled and assayed (protein-AH/SFB-Gly3-DPGA15-COOH).
(iii) Oxime linkage. BSA was first bromoacylated with SBAP and then reacted with O-(3-thiolpropyl)hydroxylamine, a heterobifunctional aminooxy-thiol linker (11). To 20 mg of aminooxylated protein (protein-ONH2) in 1.3 ml buffer A, a solution of 20 mg of CHO-PGA in 200 μl 1 M K2HPO4 was added. The pH was adjusted to 7.4, and the mixture was incubated overnight at room temperature and then passed through a Sepharose CL-6B column as described above. Fractions reacting with anti-protein and anti-DPGA were pooled and assayed (protein-ONH2/SFB-Gly3-DPGA10-COOH).
Immunization. All animal experiments were approved by the National Institute of Child Health and Human Development (NICHD) Animal Care and Use Committee. Five- to 6-week-old female NIH general-purpose mice were immunized subcutaneously three times at 2-week intervals with 2.5 μg DPGA as a conjugate in 0.1 ml PBS. Groups of 10 were exsanguinated 7 days after the second or third injection (23). Controls received PBS.
Antibodies. Serum immunoglobulin G (IgG) antibodies were measured by enzyme-linked immunosorbent assay (ELISA) (23). Nunc Maxisorb plates were coated with B. anthracis DPGA, 20 μg/ml PBS, or 4 μg protein/ml PBS (determined by checkerboard titration). The plates were blocked with 0.5% BSA (or with 0.5% HSA for assay of BSA conjugates) in PBS for 2 h at room temperature. An MRX Dynatech reader was used. Antibody levels were calculated relative to standard sera: for DPGA, a hyperimmune murine serum (22); for PA, a monoclonal antibody containing 4.7 mg antibody/ml (15); for BSA and rEPA, a pool of highest-titer sera obtained from mice immunized three times and assigned a value of 100 EU. The results were computed with an ELISA data-processing program provided by the Biostatistics and Information Management Branch, CDC (19). IgG levels are expressed as geometric means (GM).
Statistics. The Bonferroni multiple-comparison test was used for different groups of mice.
RESULTS
Characterization of conjugates. The conjugation methods used in the study for binding DPGA to protein carriers are illustrated in Fig. 1. The purity of the conjugates and the absence of free protein were confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by MALDI-TOF spectroscopy. The molar ratio of DPGA to protein in conjugates, calculated by GLC-MS analysis, was based on the ratio of D-glutamic acid to L-glutamic acid in the conjugate and by the increase in molecular mass, measured by MALDI-TOF, of the conjugate compared to that of the protein derivatized with the appropriate linker.
Antibodies to DPGA. Conjugates varied in DPGA chain length, density on the protein, carrier proteins, and types of linkage between DPGA and the protein. Additional variables were the formulation and the dosage injected into mice. All conjugates reacted with anti-DPGA and anti-protein sera with a line of identity. All conjugates were highly immunogenic in mice; most immune sera precipitated B. anthracis DPGA by double immunodiffusion (results not shown).
To identify the most immunogenic conjugates, we compared the immunogenicities of new constructs with those reported previously (22). Our previous results showed that protein conjugates of 10-mers of DPGA were more immunogenic than the conjugates of 5- or 20-mers (22). Here, we have used 10-mers with three glycine residues (Gly3-DPGA10) at the end linked to protein as before and 10-mers (DPGA10) and 15-mers (DPGA15) without glycine to avoid the generation of antibodies to the glycine linker. There were no statistical differences in the anti-DPGA levels between the conjugates of the three peptides after three injections (Table 1). For example, the two conjugates TT/Cys-DPGA10 and TT/Cys-DPGA15, having the same average density of DPGA on the protein (16 chains), gave similar responses, irrespective of the peptide chain length (after the second injection, the TT/Cys-DPGA10 GM was 6,868 and the TT/Cys-DPGA15 GM was 6,547; after the third injection, the TT/Cys-DPGA10 GM was 6,667 and the TT/Cys-DPGA15 GM was 7,688). Conjugate TT/Cys-Gly3-DPGA10, with an average density of 11 DPGA chains per protein, induced a lower level of antibodies after the second injection (GM = 421; P < 0.001), but after the third injection, the GM antibody level was similar to that of the conjugate without the glycine linker (GM = 7,762).
Table 1 summarizes the influence of the DPGA chain density on the protein on the conjugate's immunogenicity. The best responses were achieved with an average of 10 to 15 chains per protein. The antibody level induced by TT/Cys-DPGA15 with 16 chains per protein (GM = 7,688) was significantly higher than that induced by TT-CHO/AH-DPGA15 with 37 chains per protein (GM = 2,322; P < 0.001). Similarly, conjugate rPA-CHO/AH-DPGA15 with 11 chains (GM = 5,225) induced a higher level of antibodies than the conjugate with an average of 5 chains per protein (GM = 315; P < 0.001) or rPA-CHO/AH-Gly3-DPGA10 with 29 chains per protein (GM = 1,256; P < 0.001). There were no statistical differences between rPA-ITL/Br-Gly3-DPGA10 with either 8 or 18 chains per protein and rPA/Cys-Gly3-DPGA10 with 15 chains per protein.
A comparison of different linkage methods, including those tested previously (22), is presented in Table 2. The best results were obtained by thioether-linked conjugates created between thiolated protein with SPDP and bromoacylated DPGA or, conversely, bromoacylated protein and SH-DPGA. These antibody levels were significantly higher over thioether-linked conjugates created using 2-iminothiolane and over hydrazone-linked conjugates (rPA-SH/Br-Gly3-DPGA10 [GM = 7,206] versus rPA-ITL/Br-Gly3-DPGA10 [GM = 3,726; P < 0.05]; rPA-SH/Br-Gly3-DPGA10 versus rPA-CHO/AH-DPGA15 [GM = 3,004; P < 0.01]; rPA-SH/Br-Gly3-DPGA10 versus rPA-AH/CHO-Gly3-DPGA10 [GM = 2,478; P < 0.001]; rPA-Br/Cys-Gly3-DPGA10 [GM = 5,822] versus rPA-CHO/AH-DPGA15 [GM = 3,004; P < 0.05]; and rPA-Br/Cys-Gly3-DPGA10 versus rPA-AH/CHO-Gly3-DPGA10 [GM = 2,478; P < 0.05]). There were no statistically significant differences between the best thioether-linked conjugate (GM = 7,206) and the aminooxy-linked conjugate (GM = 4,257), but the higher level of antibodies and the ease with which the thioether bond is formed (one less step is required) make the first the better choice.
The effects of the carrier proteins on immunogenicity are summarized in Table 3. Among all carriers, rPA and TT conjugates produced the best responses to DPGA (rPA and TT versus BSA [P < 0.001]; rPA and TT versus rEPA [P < 0.01]; and rEPA versus BSA [no statistical difference]). Formulation of the vaccine with aluminum hydroxide significantly increased the antibody response to the carrier protein with minimal significant effect upon the response to DPGA (Table 4). In only one case (TT/Cys-DPGA15) did the aluminum hydroxide significantly (P < 0.01) increase the response to DPGA, rendering it the best conjugate, inducing 14,950 EU. The optimal dose of conjugated DPGA was between 1.2 and 2.5 μg per mouse (Table 5).
Antibody to the protein carrier. The antibody levels were calculated in ELISA units relative to a standard for each protein, arbitrarily assigned a value of 100 EU for BSA and rEPA and μg/ml for rPA. Therefore, the comparison is possible only within each type of carrier. The responses to protein varied among the conjugates, since the dosage was always based on the amount of DPGA in the conjugate. Overall, the highest levels were induced by conjugates prepared by thioether linkage between bromoacylated protein and peptide containing terminal cysteine formulated with aluminum hydroxide. The antibody response was directly related to the dosage of the carrier protein.
DISCUSSION
The pathogenicity of B. anthracis requires two essential virulence factors: the tripartite toxin and the DPGA capsule. The licensed vaccine against anthrax (Anthrax Vaccine Adsorbed) contains the protective antigen, the binding component of the toxin complex (14). The protective effects of the anticapsular antibodies were investigated recently (3, 10, 22).
We have described the preparation of conjugates of synthetic DPGA peptides with several carrier proteins bound through thioether linkages (22). Peptides of different lengths were used, at an average of 5 to 32 mol of peptide per mol of protein. Unlike DPGA alone, these conjugates were immunogenic in 5- to 6-week old general-purpose mice when injected at a dosage and schedule relevant for use in humans, and the antibodies induced opsonophagocytic killing of B. anthracis. This finding was confirmed by others (27).
The conjugation techniques, including the types of chemical linkages between the hapten and the protein, as well as the peptide/sugar chain length and the density on the protein, influence the serum antibody response to both components (4, 13, 17, 18, 20). Therefore, we studied several constructs for human use by employing additional methods and formulations of the DPGA conjugates. Previously, we created thioether bonds between the protein and the DPGA by introduction of a bromoacyl group (SBAP) or a thiol group (SPDP) into the protein. The activated protein was then bound to a peptide with a thiol or bromoacetyl group at its N or C terminus, respectively. In the present study, we tested (i) thioether bonds generated by derivatization of the protein with thiol groups using 2-iminothiolane, followed by binding to bromoacetyl-peptide; (ii) hydrazone bonds generated by derivatizing the proteins with adipic acid dihydrazide, followed by reaction with benzaldehyde-derivatized peptides or, alternatively, a benzaldehyde group was introduced into the protein using SFB and the formylated protein bound to a hydrazide-derivatized peptide; (iii) oxime bonds created by derivatization of the protein with SBAP, which was then coupled to an aminoxy-thiol linker; the aminooxylated protein was then bound to 4-formylbenzoyl-DPGA. The immunogenicities of these conjugates in mice were similar to those previously prepared: the optimal density was 15 mol DPGA per mol protein with a peptide chain length of 10 or 15 amino acids. Tetanus toxoid and rPA were better carriers than BSA or rEPA. The most successful and reproducible linkage was formed by introduction of bromoacetyl groups onto the lysine residues of the protein, followed by conjugation with DPGA equipped with a terminal cysteine residue. A dosage of 1.2 to 2.5 μg of conjugated DPGA per mouse gave the best DPGA response. Aluminum hydroxide should be included in the formulation if a high response to rPA or to another carrier protein is needed.
The choice of an anthrax conjugate to be further studied can be evaluated by (i) its immunogenicity, (ii) its yield, and (iii) the ease of preparation. Based on our studies, a highly immunogenic and simple-to-prepare investigational vaccine, inducing antibody to either one DPGA only (TT/Cys-DPGA15) or two anthrax virulence factors, DPGA and PA (rPA/Cys-Gly3-DPGA10), can be prepared and considered for clinical testing.
ACKNOWLEDGMENTS
This research was supported by the Intramural Research Program of the NIH, NICHD.
We thank Vince Pozsgay for synthesizing the oxime linker and Chunyan Gou for technical assistance.
FOOTNOTES
Corresponding author. Mailing address: National Institutes of Health, NICHD/LDMI, 9000 Rockville Pike, Bldg. 6, Rm. 1A05, Bethesda, MD 20892. Phone: (301) 496-6141. Fax: (301) 480-3442. E-mail: kielbj@mail.nih.gov.
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ABSTRACT
The capsule of Bacillus anthracis, composed of poly--D-glutamic acid (DPGA), is an essential virulence factor of B. anthracis. The capsule inhibits innate host defense through its antiphagocytic action. DPGA is a poor immunogen, but when covalently bound to a carrier protein, it elicits serum antibodies. To identify the optimal construct for clinical use, synthetic DPGAs of different lengths were bound to carrier proteins at different densities. The advantages of the synthetic over the natural polypeptide are the homogeneous chain length and end groups, allowing conjugates to be accurately characterized and standardized and their chemical compositions to be related to their immunogenicities. In the present study, we evaluated, in addition to methods reported by us, hydrazone, oxime, and thioether linkages between DPGA and several proteins, including bovine serum albumin, recombinant Pseudomonas aeruginosa exotoxin A, recombinant B. anthracis protective antigen (rPA), and tetanus toxoid (TT). The effects of the dosage and formulation on the immunogenicities of the conjugates were evaluated in mice. All conjugates were immunogenic. The optimal DPGA chain length of 10 to 15 amino acids and the density, an average of 15 mol DPGA per mol of protein, were confirmed. The thioether bond was the optimal linkage type, and TT and rPA were the best carriers. The optimal dosage was 1.2 to 2.5 μg of DPGA per mouse, and adsorption of the conjugates onto aluminum hydroxide significantly increased the antibody response to the protein with a lesser effect on anti-DPGA levels.
INTRODUCTION
Anthrax, a potentially lethal human disease, is a zoonotic infection that under natural conditions is contracted by humans directly or indirectly from animals. The causative organism, Bacillus anthracis, exists in vegetative or spore forms, the latter being the infecting agent. A veterinary vaccine based on a capsule-negative, toxin-positive strain (Sterne) is available and is routinely used throughout the world (25). In addition, anthrax vaccine adsorbed, containing the protective antigen, is licensed and used by veterinarians, animal by-product handlers, and the U.S. Army (2, 5). Anthrax is endemic in countries that do not immunize domesticated animals (9, 12, 24). Because of the ease of cultivating the organism and improved technology of spore preparation, B. anthracis is a potential bioterrorism agent. In 2001, B. anthracis spores were used successfully via the U.S. mail, though few people were affected. A major bioterrorism attack may be airborne, with a number of spores far exceeding a natural exposure, causing inhalation anthrax and affecting a large number of people, including children. These facts warrant devising an improved anthrax vaccine.
The addition of components other than those of anthrax toxin to improve vaccine-induced protection has been considered (22). The capsule, composed of poly--D-glutamic acid (DPGA), is an essential virulence factor and antiphagocytic, and antibodies to this polypeptide have been shown to be opsonophagocytic and protective in mice (3, 10, 22). DPGA by itself is a poor immunogen and does not induce booster responses, probably because of its simple homopolymeric structure, similar to those of capsular polysaccharides; it is a T-cell-independent antigen and of D-amino acid composition (7). These immunologic properties can be overcome by covalent binding of the T-cell-independent antigen to immunogenic proteins (22). Because of the success in inducing protective levels of antibodies in infants against systemic infection with capsulated pathogens, we developed conjugates of DPGA with several carrier proteins, including bovine serum albumin (BSA), recombinant B. anthracis protective antigen (rPA), and recombinant Pseudomonas aeruginosa exoprotein A (rEPA). Unlike DPGA alone, these conjugates were immunogenic in mice, with booster responses upon reinjection. Conjugate-induced antibodies were opsonophagocytic (22, 27). This study describes additional synthetic schemes in an attempt to develop the most immunogenic conjugates.
MATERIALS AND METHODS
Analyses. Amino acid analysis was done by gas-liquid chromatography-mass spectrometry (GLC-MS) after hydrolysis with 6 N HCl at 150°C for 1 h and derivatization to N-heptafluorobutyryl R-(–)-isobutyl esters and assay with a Hewlett-Packard apparatus (model HP 6890) with an HP-5 0.32- by 30-mm glass capillary column with temperature programming at 8°C/min from 125 to 250°C in the electron ionization (106-eV) mode (22). The number of peptide chains in the conjugates was calculated by the ratio between L- and D-glutamic acids. The protein concentration was measured by the method of Lowry et al. (16), free amino groups by Fields' assay (6), benzaldehyde groups by colorimetric reaction with 2-hydrazinopyridine (Solulink, San Diego, CA), hydrazide was measured as reported previously (23), and thiolation was measured by release of 2-pyridylthio groups (A343) (1). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis used 14% gels according to the manufacturer's instructions (Bio-Rad, Hercules, CA). Double immunodiffusion was performed in 1.0% agarose gels in phosphate-buffered saline (PBS) with rabbit anti-DPGA, rabbit anti-BSA (Sigma, St. Louis, MO), goat anti-exotoxin A (List Biological Laboratories, Inc.), anti-tetanus toxoid (obtained from W. Vann, FDA, Bethesda MD), and anti-B. anthracis protective antigen (obtained from S. Leppla, NIH/NIAID, Bethesda, MD). Aluminum hydroxide was used as Alhydrogel (Staten Serum Institut, Copenhagen, Denmark).
MALDI-TOF. Mass spectra were obtained with an OmniFlex matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) instrument (Bruker Daltonics) operated in the linear mode. Samples for analysis were desalted, and 1 μl was mixed with 20 μl of sinapinic acid matrix made in 30% CH3CN and 0.1% trifluoroacetic acid. Then, 1 μl of the mixture was dried on the sample stage and placed in the mass spectrometer.
Antigens. BSA (66.5 kDa; Sigma, St. Louis, MO) was dialyzed against pyrogen-free water, sterile filtered, and freeze-dried. rPA (83 kDa) from B. anthracis and rEPA (67 kDa) from P. aeruginosa were prepared and characterized (8, 21). Tetanus toxoid (TT) (150 kDa) was obtained from Merieux, Lyon, France.
DPGA was purified from the culture supernatant of B. anthracis strain A34 toxin-negative by cetavlon precipitation, acidification to pH 1.5, precipitation with ethanol, and passage through a 2.5- by 100-cm Sephacryl S-1000 column in 0.2 M NaCl (26). Its structure was confirmed by 1H nuclear magnetic resonance and 13C nuclear magnetic resonance, and its enantiomeric composition was determined by GLC-MS spectroscopy. DPGA peptides were synthesized by the method of Merrifield (AnaSpec, San Jose, CA). Peptides were divided into groups depending on the types of linkages through which they were bound to proteins: (i) thioether linkage, NAc-DPGA10-Gly3-L-Cys-CONH2 (DPGA10-Cys) or NBrAc-Gly3-DPGA10-COOH (Br-DPGA10); (ii) hydrazone linkage, 4-formylbenzoyl-Gly3-DPGA10-COOH (CHO-DPGA10), NAc-DPGA10-Gly3-CO-NH-NH-CO-(CH2)4-CO-NH-NH2 (DPGA10-AH), or NAc-DPGA15-CO-NH-NH-CO-(CH2)4-CO-NH-NH2 (DPGA15-AH), where AH is adipic acid hydrazide; and (iii) oxime linkage, 4-formylbenzoyl-Gly3-DPGA10-COOH (CHO-PGA10).
Conjugation. (i) Thioether linkage. First, protein was bromoacylated using succinimidyl 3-(bromoacetamido)propionate (SBAP) (Pierce, Rockford, IL) and reacted with peptides equipped with a terminal cysteine residue as reported previously (22) (protein/S-Cys-Gly3-DPGA10-NAc, protein/S-Cys-DPGA10-NAc, or protein/S-Cys-DPGA15-NAc).
Second, protein was derivatized with an N-hydroxysuccinimide ester of 3-(2-pyridyldithio)propionic acid (SPDP) (Pierce, Rockford, IL) and reacted with bromoacylated peptide (22) (protein-S/Gly3-DPGA10-COOH).
Third, sulfhydryl groups were introduced into the protein using 2-iminothiolane (ITL) (ScienceLab, Houston, TX). Protein (20 mg) was derivatized with 2.8 mg ITL in 1.5 ml of buffer A (PBS, 0.1% glycerol, 0.005 M EDTA, pH 7.4) for 60 min. Next, a solution of Br-DPGA10 (35 mg) in 300 μl of 1 M K2HPO4 was added while the pH was maintained at 7.4 with the addition of 0.2 N NaOH at room temperature. After 90 min, the reaction mixture was passed through a Sepharose CL-6B column (1 by 120 cm) and eluted with 0.2 M NaCl. Fractions reacting with anti-protein and anti-DPGA were pooled (protein-ITL/Gly3-DPGA10-COOH).
(ii) Hydrazone linkage. First, protein was derivatized with succinimidyl 4-formylbenzoate (SFB) (Solulink, San Diego, CA). To a solution of protein (30 mg) in 1.2 ml buffer A, SFB (7.5 mg) in 100 μl dimethyl sulfoxide was added and reacted for 2 h at pH 7.4. The product 4-formylbenzoyl-protein was passed through a Sephadex G-50 column (1 by 100 cm) in 0.2 M NaCl. Protein-containing fractions were pooled and assayed for the presence of benzaldehyde, antigenicity, and protein concentration. To 4-formylbenzoyl-protein (20 mg) in 1.25 ml buffer A, a solution of 15 mg of AH-DPGA10 or AH-DPGA15 in 200 μl 1 M K2HPO4 was added. The pH of the reaction mixture was adjusted to 7.4, and the mixture was incubated overnight at room temperature and then passed through a Sepharose CL-6B column as described above. Fractions reacting with anti-protein and anti-DPGA were pooled and assayed (protein-SFB/AH-Gly3-DPGA10-NAc or protein-SFB/AH-DPGA15-NAc).
Second, derivatization of protein with adipic acid dihydrazide using a water-soluble carbodiimide was done as reported previously (23). The incorporation of hydrazide residues was 2 to 5% per protein. To protein-AH (20 mg) in 1.3 ml buffer A, a solution of 20 mg of CHO-DPGA10 in 200 μl 1 M K2HPO4 was added. The pH was adjusted to 7.4, and the solution was incubated overnight at room temperature and then passed through a Sepharose CL-6B column as described above. Fractions reacting with anti-protein and anti-DPGA were pooled and assayed (protein-AH/SFB-Gly3-DPGA15-COOH).
(iii) Oxime linkage. BSA was first bromoacylated with SBAP and then reacted with O-(3-thiolpropyl)hydroxylamine, a heterobifunctional aminooxy-thiol linker (11). To 20 mg of aminooxylated protein (protein-ONH2) in 1.3 ml buffer A, a solution of 20 mg of CHO-PGA in 200 μl 1 M K2HPO4 was added. The pH was adjusted to 7.4, and the mixture was incubated overnight at room temperature and then passed through a Sepharose CL-6B column as described above. Fractions reacting with anti-protein and anti-DPGA were pooled and assayed (protein-ONH2/SFB-Gly3-DPGA10-COOH).
Immunization. All animal experiments were approved by the National Institute of Child Health and Human Development (NICHD) Animal Care and Use Committee. Five- to 6-week-old female NIH general-purpose mice were immunized subcutaneously three times at 2-week intervals with 2.5 μg DPGA as a conjugate in 0.1 ml PBS. Groups of 10 were exsanguinated 7 days after the second or third injection (23). Controls received PBS.
Antibodies. Serum immunoglobulin G (IgG) antibodies were measured by enzyme-linked immunosorbent assay (ELISA) (23). Nunc Maxisorb plates were coated with B. anthracis DPGA, 20 μg/ml PBS, or 4 μg protein/ml PBS (determined by checkerboard titration). The plates were blocked with 0.5% BSA (or with 0.5% HSA for assay of BSA conjugates) in PBS for 2 h at room temperature. An MRX Dynatech reader was used. Antibody levels were calculated relative to standard sera: for DPGA, a hyperimmune murine serum (22); for PA, a monoclonal antibody containing 4.7 mg antibody/ml (15); for BSA and rEPA, a pool of highest-titer sera obtained from mice immunized three times and assigned a value of 100 EU. The results were computed with an ELISA data-processing program provided by the Biostatistics and Information Management Branch, CDC (19). IgG levels are expressed as geometric means (GM).
Statistics. The Bonferroni multiple-comparison test was used for different groups of mice.
RESULTS
Characterization of conjugates. The conjugation methods used in the study for binding DPGA to protein carriers are illustrated in Fig. 1. The purity of the conjugates and the absence of free protein were confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by MALDI-TOF spectroscopy. The molar ratio of DPGA to protein in conjugates, calculated by GLC-MS analysis, was based on the ratio of D-glutamic acid to L-glutamic acid in the conjugate and by the increase in molecular mass, measured by MALDI-TOF, of the conjugate compared to that of the protein derivatized with the appropriate linker.
Antibodies to DPGA. Conjugates varied in DPGA chain length, density on the protein, carrier proteins, and types of linkage between DPGA and the protein. Additional variables were the formulation and the dosage injected into mice. All conjugates reacted with anti-DPGA and anti-protein sera with a line of identity. All conjugates were highly immunogenic in mice; most immune sera precipitated B. anthracis DPGA by double immunodiffusion (results not shown).
To identify the most immunogenic conjugates, we compared the immunogenicities of new constructs with those reported previously (22). Our previous results showed that protein conjugates of 10-mers of DPGA were more immunogenic than the conjugates of 5- or 20-mers (22). Here, we have used 10-mers with three glycine residues (Gly3-DPGA10) at the end linked to protein as before and 10-mers (DPGA10) and 15-mers (DPGA15) without glycine to avoid the generation of antibodies to the glycine linker. There were no statistical differences in the anti-DPGA levels between the conjugates of the three peptides after three injections (Table 1). For example, the two conjugates TT/Cys-DPGA10 and TT/Cys-DPGA15, having the same average density of DPGA on the protein (16 chains), gave similar responses, irrespective of the peptide chain length (after the second injection, the TT/Cys-DPGA10 GM was 6,868 and the TT/Cys-DPGA15 GM was 6,547; after the third injection, the TT/Cys-DPGA10 GM was 6,667 and the TT/Cys-DPGA15 GM was 7,688). Conjugate TT/Cys-Gly3-DPGA10, with an average density of 11 DPGA chains per protein, induced a lower level of antibodies after the second injection (GM = 421; P < 0.001), but after the third injection, the GM antibody level was similar to that of the conjugate without the glycine linker (GM = 7,762).
Table 1 summarizes the influence of the DPGA chain density on the protein on the conjugate's immunogenicity. The best responses were achieved with an average of 10 to 15 chains per protein. The antibody level induced by TT/Cys-DPGA15 with 16 chains per protein (GM = 7,688) was significantly higher than that induced by TT-CHO/AH-DPGA15 with 37 chains per protein (GM = 2,322; P < 0.001). Similarly, conjugate rPA-CHO/AH-DPGA15 with 11 chains (GM = 5,225) induced a higher level of antibodies than the conjugate with an average of 5 chains per protein (GM = 315; P < 0.001) or rPA-CHO/AH-Gly3-DPGA10 with 29 chains per protein (GM = 1,256; P < 0.001). There were no statistical differences between rPA-ITL/Br-Gly3-DPGA10 with either 8 or 18 chains per protein and rPA/Cys-Gly3-DPGA10 with 15 chains per protein.
A comparison of different linkage methods, including those tested previously (22), is presented in Table 2. The best results were obtained by thioether-linked conjugates created between thiolated protein with SPDP and bromoacylated DPGA or, conversely, bromoacylated protein and SH-DPGA. These antibody levels were significantly higher over thioether-linked conjugates created using 2-iminothiolane and over hydrazone-linked conjugates (rPA-SH/Br-Gly3-DPGA10 [GM = 7,206] versus rPA-ITL/Br-Gly3-DPGA10 [GM = 3,726; P < 0.05]; rPA-SH/Br-Gly3-DPGA10 versus rPA-CHO/AH-DPGA15 [GM = 3,004; P < 0.01]; rPA-SH/Br-Gly3-DPGA10 versus rPA-AH/CHO-Gly3-DPGA10 [GM = 2,478; P < 0.001]; rPA-Br/Cys-Gly3-DPGA10 [GM = 5,822] versus rPA-CHO/AH-DPGA15 [GM = 3,004; P < 0.05]; and rPA-Br/Cys-Gly3-DPGA10 versus rPA-AH/CHO-Gly3-DPGA10 [GM = 2,478; P < 0.05]). There were no statistically significant differences between the best thioether-linked conjugate (GM = 7,206) and the aminooxy-linked conjugate (GM = 4,257), but the higher level of antibodies and the ease with which the thioether bond is formed (one less step is required) make the first the better choice.
The effects of the carrier proteins on immunogenicity are summarized in Table 3. Among all carriers, rPA and TT conjugates produced the best responses to DPGA (rPA and TT versus BSA [P < 0.001]; rPA and TT versus rEPA [P < 0.01]; and rEPA versus BSA [no statistical difference]). Formulation of the vaccine with aluminum hydroxide significantly increased the antibody response to the carrier protein with minimal significant effect upon the response to DPGA (Table 4). In only one case (TT/Cys-DPGA15) did the aluminum hydroxide significantly (P < 0.01) increase the response to DPGA, rendering it the best conjugate, inducing 14,950 EU. The optimal dose of conjugated DPGA was between 1.2 and 2.5 μg per mouse (Table 5).
Antibody to the protein carrier. The antibody levels were calculated in ELISA units relative to a standard for each protein, arbitrarily assigned a value of 100 EU for BSA and rEPA and μg/ml for rPA. Therefore, the comparison is possible only within each type of carrier. The responses to protein varied among the conjugates, since the dosage was always based on the amount of DPGA in the conjugate. Overall, the highest levels were induced by conjugates prepared by thioether linkage between bromoacylated protein and peptide containing terminal cysteine formulated with aluminum hydroxide. The antibody response was directly related to the dosage of the carrier protein.
DISCUSSION
The pathogenicity of B. anthracis requires two essential virulence factors: the tripartite toxin and the DPGA capsule. The licensed vaccine against anthrax (Anthrax Vaccine Adsorbed) contains the protective antigen, the binding component of the toxin complex (14). The protective effects of the anticapsular antibodies were investigated recently (3, 10, 22).
We have described the preparation of conjugates of synthetic DPGA peptides with several carrier proteins bound through thioether linkages (22). Peptides of different lengths were used, at an average of 5 to 32 mol of peptide per mol of protein. Unlike DPGA alone, these conjugates were immunogenic in 5- to 6-week old general-purpose mice when injected at a dosage and schedule relevant for use in humans, and the antibodies induced opsonophagocytic killing of B. anthracis. This finding was confirmed by others (27).
The conjugation techniques, including the types of chemical linkages between the hapten and the protein, as well as the peptide/sugar chain length and the density on the protein, influence the serum antibody response to both components (4, 13, 17, 18, 20). Therefore, we studied several constructs for human use by employing additional methods and formulations of the DPGA conjugates. Previously, we created thioether bonds between the protein and the DPGA by introduction of a bromoacyl group (SBAP) or a thiol group (SPDP) into the protein. The activated protein was then bound to a peptide with a thiol or bromoacetyl group at its N or C terminus, respectively. In the present study, we tested (i) thioether bonds generated by derivatization of the protein with thiol groups using 2-iminothiolane, followed by binding to bromoacetyl-peptide; (ii) hydrazone bonds generated by derivatizing the proteins with adipic acid dihydrazide, followed by reaction with benzaldehyde-derivatized peptides or, alternatively, a benzaldehyde group was introduced into the protein using SFB and the formylated protein bound to a hydrazide-derivatized peptide; (iii) oxime bonds created by derivatization of the protein with SBAP, which was then coupled to an aminoxy-thiol linker; the aminooxylated protein was then bound to 4-formylbenzoyl-DPGA. The immunogenicities of these conjugates in mice were similar to those previously prepared: the optimal density was 15 mol DPGA per mol protein with a peptide chain length of 10 or 15 amino acids. Tetanus toxoid and rPA were better carriers than BSA or rEPA. The most successful and reproducible linkage was formed by introduction of bromoacetyl groups onto the lysine residues of the protein, followed by conjugation with DPGA equipped with a terminal cysteine residue. A dosage of 1.2 to 2.5 μg of conjugated DPGA per mouse gave the best DPGA response. Aluminum hydroxide should be included in the formulation if a high response to rPA or to another carrier protein is needed.
The choice of an anthrax conjugate to be further studied can be evaluated by (i) its immunogenicity, (ii) its yield, and (iii) the ease of preparation. Based on our studies, a highly immunogenic and simple-to-prepare investigational vaccine, inducing antibody to either one DPGA only (TT/Cys-DPGA15) or two anthrax virulence factors, DPGA and PA (rPA/Cys-Gly3-DPGA10), can be prepared and considered for clinical testing.
ACKNOWLEDGMENTS
This research was supported by the Intramural Research Program of the NIH, NICHD.
We thank Vince Pozsgay for synthesizing the oxime linker and Chunyan Gou for technical assistance.
FOOTNOTES
Corresponding author. Mailing address: National Institutes of Health, NICHD/LDMI, 9000 Rockville Pike, Bldg. 6, Rm. 1A05, Bethesda, MD 20892. Phone: (301) 496-6141. Fax: (301) 480-3442. E-mail: kielbj@mail.nih.gov.
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