Abstract:
Methods for developing vaccines to protect from neurotoxins of  C. botulinum  have been developed. Truncated BoNT/A proteins of about 15-30 kDa in size produced immune responses that provided protection from neuronal damage by  botulinum  neurotoxins.

Description:
FIELD OF THE INVENTION 
     This invention relates to immunization against toxic effect of neurotoxins of  Clostridium botulinum.  Protective epitopes of the heavy chain of the neurotoxin of  C. botulinum  have been discovered. The invention also relates to preparation of protective immunotoxins of  C. botulinum.    
     BACKGROUND OF THE INVENTION 
       Botulinum  neurotoxin (BoNT) is one of the most potent toxins known to man. Ingestion or inhalation of toxin inhibits neurotransmitter release from synaptic vesicles, resulting in neuroparalysis and death. Seven serologically distinct forms of neurotoxin are produced by  Clostridium botulinum.  The toxin is synthesized as a 150 kDa precursor that is proteolytically nicked into two subunits. The light (L) chain, associated with the toxicity of BoNT, must be internalized in the cell in order to inhibit neurotransmitter release. It is linked by a disulfide bond to the heavy (H) chain, which mediates binding of the toxin to receptors located on the surface of the nerve cell. Although the heavy chain is required for BoNT to productively bind and enter the target cell, it is not toxic by itself. 
     The current pentavalent toxoid vaccine for botulism is composed of formalin-inactivated holotoxin. Although effective, this vaccine is difficult to manufacture. Furthermore, extensive treatment with formalin is required to inactivate the toxin. Prolonged treatment with formalin can affect the immunogenicity of protein antigens, and this may explain why certain lots of toxoid have been poorly immunogenic in the past. 
     There are several approaches that can be used to construct a new vaccine. One approach would be to express a non-toxigenic mutant of BoNT/A, as has already been done for other toxins. The advantage of this approach is that the immune response elicited by the modified protein would most closely approximate the response elicited by the native toxin, because almost all of the native protein structure would still be intact. However, high level expression of the C fragment of tetanus toxin (TeTx) could not be achieved in  E. coli  when the native clostridial gene sequence was used. Based on this information, expression of BoNT might be predicted to be difficult, as well. Another approach is to construct a synthetic peptide-based vaccine. The advantage of this approach is that large quantities of synthetic peptide can be easily manufactured for use in a vaccine. However, studies with MAbs have indicated that many of the neutralizing epitopes located on BoNT are conformationally sensitive. This suggests that a peptide-based vaccine may not necessarily be able to induce neutralizing antibody responses due to its lack of conformational epitopes. A genetically engineered vaccine for botulism would eliminate many problems, since it could be expressed in a recombinant host at high levels and would not require treatment with formalin before incorporation into a vaccine. 
     Recent developments have made the construction of a genetically engineered BoNT vaccine possible. The gene for BoNT serotype A (BoNT/A) has been cloned and sequenced (Binz, et al.,  J. Biol. Chem.  265:9153-9158.(1990), and the minimum length of the light chain needed to retain neurotoxicity has been defined (Kurazono, et al.,  J. Biol. Chem.  267:14721-14729 (1992)). While construction of such a vaccine is feasible, there has not been a systematic attempt to identify the domain(s) of BoNT/A that would be required to elicit protective immunity. Immunization with a fragment corresponding to the C-terminal half of the heavy chain (H C ) has been shown to stimulate protective immunity, but more definitive identification of sequences that elicit protective immune response had not previously been described. Monoclonal antibodies directed against either light chain or heavy chain determinants had been shown to provide some passive protection to mice against a lethal exposure to BoNT, indicating that protective epitopes may exist on either chain. However, many of these epitopes appear to be conformationally sensitive, which suggests that mapping their location by using synthetic peptides may be unproductive due to their lack of tertiary structure. 
     SUMMARY OF THE INVENTION 
     It is the purposes of this invention to provide methods for developing vaccines to protect from neurotoxins of  C. botulinum . The methods used to identify specific sequences consisted of amplifying and cloning overlapping segments of the BoNT/A gene. These segments are then expressed in suitable vectors such as  E. coli  to produce truncated BoNT/A proteins of about 15-30 kDa in size. The truncated proteins are purified by appropriate methods such as SDS-PAGE. The invention is exemplified using two particularly protective regions from the heavy chain of the type A  C. botulinum  toxin. The peptides giving rise to protective antibodies may be fused to other peptides that act as adjuvants to increase antigenicity. Such fusion proteins may be produced by recombinant technology using plasmids containing hybrid genes for expression of the desired fusion proteins. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     It is the purpose of this invention to identify and provide immunogenic polypeptides which give rise to protective antibodies against botulism. Compositions containing the subject polypeptides in pharmaceutically acceptable carriers are useful as vaccines and as diagnostic agents to identify protective antibodies. 
     The location of protective domains was identified, and those domains were produced by expressing fragments of BoNT/A in  E. coli  and then evaluating each for its protective efficacy. Using this approach, fragments of the BoNT/A gene were expressed that were of sufficient size to still possess some tertiary conformation, but that would greatly reduce the amount of the toxin utilized. By overlapping the regions of the BoNT/A protein being expressed, it was possible to minimize the possibility that a locally encoded epitope was accidently interrupted. The advantage of this approach is that the fragments were sufficiently small to be nontoxic. However, it is possible that not all protective determinants may have been encoded by these fragments. 
     It was possible to express fragments of the BoNT/A gene at high levels in  E. coli  by using an inducible T7 expression system. It was not predictable that, in contrast to some of the problems encountered with expression of the C fragment of tetanus toxin (TeTx), this could be done for purposes of making a protective vaccine against botulism. Some difficulty encountered which was related to the fact that clostridial toxin is encoded by codons that are rarely used by  E. coli . Unexpectedly, this problem with the DNA sequence naturally encoded by Clostridium did not present the barrier that might have been expected. The possibility for expression of these proteins may be due to the size of the BoNT/A proteins encoded. The TeTx proteins being expressed in  E. coli  were two to three times larger than the BoNT/A proteins expressed as disclosed herein. The smaller size of the BoNT/A transcripts may have permitted  E. coli  to translate them more efficiently. However, the truncated BoNT/A proteins were expressed primarily in the form of insoluble inclusion bodies. Insertion of the BoNT/A gene fragments into the plasmid vector pMTD74 resulted in expression of a BoNT/A protein fused to the A2 peptide of cholera toxin (CtxA2) at its C-terminus. These fragments were fused to CtxA2 to associate noncovalently with the B subunit of cholera toxin (CtxB). Fusion of antigens to CtxB was shown to improve their immunogenicity when administered by mucosal routes of immunization (Dertzbaugh, et al.,  Infect. Immun.  61:48-55 (1993)). Hence, CtxB is used as a delivery system with these fragments of BoNT/A as part of a mucosally administered vaccine for botulism. 
     The ability of the BoNT/A fragments to induce an antibody response was affected by the antigen preparation used for immunization. Effective production of antibody to BoNT/A was inadequate when the crude lysates were used for immunization, even though they contained relatively large amounts of BoNT/A-specific protein. For this reason, immunization was performed again with highly enriched preparations of the BoNT/A proteins. Unlike the crude form of the antigen, the purified form was able to elicit BoNT/A-specific antibody whilst being well tolerated by the animals. The poor immunogenicity of the crude lysates may have been due to saturation of the antigen-presenting cells with other antigens present. It is possible that by purifying the BoNT/A proteins, other immunodominant antigens were removed which could have been competing for uptake and presentation to lymphocytes by the antigen-presenting cells. 
     Preparative SDS-PAGE was used to purify the BoNT/A fragments for several reasons. First, most of the BoNT/A protein present in the lysates were in the form of inclusion bodies that had to be solubilized before purification. SDS easily solubilized the BoNT/A proteins. Second, this method can be used to purify all of the fragments, regardless of their size or composition. Furthermore, the size range of the BoNT/A proteins permitted them to be separated from most of the other proteins present in the lysates. One potential disadvantage of using such a denaturing method is that the purified BoNT/A proteins may not have completely resumed their native conformation, resulting in the loss of some epitopes. The BoNT/A proteins should have been able to refold when the SDS was removed from the antigen preparations before immunization. 
     Hybrid gene fusion proteins may also be produced to increase protective immune response. For example, DNA sequences which encode desired antigenic polypeptides may be fused to DNA sequences which encode non-toxic peptides of other organisms such as cholera. U.S. Pat. No. 5,268,276 to Holmgren, et al., which is incorporated herein in its entirety by reference, discloses a means of producing an appropriate fusion gene to produce fusion proteins containing the immunogenic peptides of botulism. 
     Both BoNT peptides and fusion proteins containing BoNT amino acid sequences may be administered by mouth. Antigenic fusion proteins containing sequences of cholera subunits are useful for administration orally or to the mucosa (for example intranasally). The fusion proteins may be lyophilized and inhaled from a vial for administration. 
     Compositions containing the BoNT peptides in pharmaceutically acceptable carriers may also be administered parenterally. Preferred parenteral routes include intracutaneous or subcutaneous or intramuscular injection. Any of the compositions may contain, additionally, adjuvants such as alum or Freund&#39;s adjuvant. While the invention has been exemplified using the peptides of  C. botulinum  , serotype A, analogous polypeptides sequences of other serotypes can be made in the manner described herein. A cocktail of polypeptides from various serotypes may be administered to provide broad protection against toxins of  C. botulinum  serotypes. 
     MATERIALS AND METHODS 
     Construction of the BoNT/A gene fragments. The polymerase chain reaction (PCR) was used to amplify and clone overlapping fragments of the BoNT/A gene. Primers used to amplify each fragment are listed in Table 1. The primers were designed to include unique flanking restriction sites on the 5′ and 3′ ends of each amplified fragment in order to permit its insertion into the expression vector. Plasmids pCBA2, pCBA3, and pCBA4 encoding large overlapping regions of the BoNT/A gene and flanking DNA were used as template DNA (Thompson, et al.,  Eur. J. Biochem.  73-81 (1990)). Amplification was performed using Vent DNA polymerase (New England Biolabs, Beverly, Mass.). The reaction mixture was prepared according to the manufacturer&#39;s directions, and consisted of 100 ng/μl forward primer, 100 ng/μl reverse primer, and 10 ng/μl template. Each reaction was subjected to 25 cycles of amplification in a DNA thermocycler according to the following parameters: melting temperature, 94° C. for 1 min; annealing temperature, 45° C. for 1 min; extension temperature, 72° C. for 1 min. The amplified DNA was digested with the appropriate restriction enzymes and then was ligated into the expression vector pMTD74. 
     
       
         
               
             
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 PCR primers 
               
             
          
           
               
                 n.t. a   
                 Direction c   
                 Sequence 
               
               
                   
               
             
          
           
               
                 367-741 
                 F: 
                 5′-ATATGGAATTCGTTAATAAACAATTTAATTATAAAGATCC-3′ 
                 Seq. #1 
               
               
                 L 4-128   b   
                 R: 
                 5′-AGTATCGTCGACTTTTAATTCTGTATCTATTGTACTTCCACC-3′ 
                 Seq. #2 
               
               
                 732-1170 
                 F: 
                 5′-GATACAGAATTCAAAGTTATTGATACTAATAG-3′ 
                 Seq. #3 
               
               
                 L l26-271   b   
                 R: 
                 5′-CTTTGCGTCGACTCCCCCAAATGTTCTAAGTTCC-3′ 
                 Seq. #4 
               
               
                 1126-1750 
                 F: 
                 5′-GGGTTAGAATTCAGCTTTGAGGAACTTAGAACATTTGGG-3′ 
                 Seq. #5 
               
               
                 L 257-465   b   
                 R: 
                 5′-AGGACTGTCGACCAAGTCCCAATTATTAACTTTGATTGATAAATC3′ 
                 Seq. #6 
               
               
                 720-2340 
                 F: 
                 5′-TTAAATGAATTCTCAATCAAAGTTAATAATTGGGAC-3′ 
                 Seq. #7 
               
               
                 H 455-661   b   
                 R: 
                 5′-CTCTGGGTCGACTTCTAACAGAATAACAGCTCC-3′ 
                 Seq. #8 
               
               
                 2150-2780 
                 F: 
                 5′-GAAGTAAGAGCTCTGGATAAAATTGCGGATATAAC-3′ 
                 Seq. #9 
               
               
                 H 630-808   b   
                 R: 
                 5′-TAACCGGTCGACACCATAAGGGATCATAGAG-3′ 
                 Seq. #11 
               
               
                 2695-3175 
                 F: 
                 5′-GCTATGATTAATATAAATAAATTTTTGAATCAATGC-3′ 
                 Seq. #10 
               
               
                 H 780-939   b   
                 R: 
                 5′-AGTACTAAGCTTTTCATACATACTATTATATACAATAGC-3′ 
                 Seq. #12 
               
               
                 3100-3530 
                 F: 
                 5′-AAAAATAGAGCTCAATTATTTAATTTAGAAAGTAG-3′ 
                 Seq. #13 
               
               
                 H 915-1059   b   
                 R: 
                 5′-ACCATCGTCGACAAACATTATATTATTACTAGC-3′ 
                 Seq. #14 
               
               
                 3301-3726 
                 F: 
                 5′-TATGGTGAATTCATCTGGACTTTACAGGATACTCAGG-3′ 
                 Seq. #15 
               
               
                 H 982-1123   b   
                 R: 
                 5′-ATTTACGTCGACATATTTATTTGGATC-3′ 
                 Seq. #16 
               
               
                 3590-4020 
                 F: 
                 5′-GATAAGGAATTCAATGAAAAAGAAATCAAAG-3′ 
                 Seq. #17 
               
               
                 H 1078-1220   b   
                 R: 
                 5′-CTTCATGTCGACTACTTGACTTAGATTTCC-3′ 
                 Seq. #18 
               
               
                 3806-4223 
                 F: 
                 5′-AACATTGAATTCAATTCAAGTTTGTATAGGGGG-3′ 
                 Seq. #19 
               
               
                 H 1150-1289   b   
                 R: 
                 5′-TCCATCGTCGACAGGAATAAATTCCCATGAGCTACC-3′ 
                 Seq. #20 
               
               
                   
               
               
                   a Nucleotide sequence  number designation based on EMBL/Genbank ™ accession file X52066.  
               
               
                   b Amino acid residue number of the light (L) chain and the heavy (H) chain.  
               
               
                   c F, forward primer; R, reverse primer.  
               
             
          
         
       
     
     Bacterial strains and plasmids. Plasmids constructed are listed in Table 2. All plasmids were transformed by the CaCl 2 -heat shock method (See Morrison, D. A.,  J. Bacteriol.  132:349-351 (1977)) into  E. coli  strain HMS174(DE3) (Campell, et al.,  Proc. Natl. Acad. Sci., U.S.A.,  75:2276-2280 (1978)). Bacterial strains were grown at 37° C. in M-9 medium in accord with the methods of Miller (Miller, J. H.,  Experiments in Molecular Genetics,  Cold Spring Harbor Laboratory, Cold Spring Harbor (1972)) supplemented with 10 g of Casamino Acids (Difco Laboratories, Ann Arbor, Mich.) per liter, 20 μg of leucine per ml, 20 μg of proline per ml, 2 μg of thiamine per ml, 50 μg of ampicillin per ml, and 25 μg of rifampicin per ml. Plasmid pMTD74 was used to express the BoNT/A fragments in  E. coli  . It was derived from the T7 translation vector pET-8c (Studier, et al.,  Methods in enzymology,  Academic Press, New York (1990)) and has a multiple cloning site encoding unique restriction sites. This plasmid was used to express fragments of the BoNT/A gene in  E. coli  . It encodes a gene for ampicillin (Ap) resistance and a ColE1 origin of replication (ORI). Transcription is initiated from the T7 promoter and is terminated by the Tφ transcriptional terminator (t.t.). Proper in-frame insertion of DNA within the multiple cloning site (MCS) results in expression of protein fused to the A2 peptide of cholera toxin (CtxA2). (Lockman, et al.,  J. Biol. Chem.  258:13722-13726 (1983)). The MCS contains several unique restriction sites, and is shown in more detail below the plasmid map. Translation is initiated by the Shine-Delgarno (S.D.) sequence located upstream of the initiator methionine encoded by the NcoI site. Transformation of this plasmid into the lysogenic  E. coli  strain HMS174(DE3) permits inducible expression of protein from the T7 promoter. T7 RNA polymerase is required for initiation of transcription from the T7 promoter, and this protein is inducibly expressed in HMS174(DE3) by using isopropyl-beta-D-thiogalactopyranoside (IPTG). 
     
       
         
               
             
               
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 Bacterial strains and plasmids 
               
             
          
           
               
                 
                   E. Coli 
                 
                   
                   
                 Refer- 
               
               
                 Strain 
                 Plasmid 
                 Comments 
                 ences 
               
               
                   
               
               
                   
                 pMTD74 
                   
                   
               
               
                 HMS174 
                   
                 F −   hsdR (r K   −  m K   + ) 
                 Campbell* 
               
               
                   
                   
                 
                   recA 
                   rif 
                   
                     R 
                   
                 
               
               
                 HMS174(DE3) 
                   
                 T7 expression host 
                 Studier** 
               
               
                 MTD103 
                 pMTD89 
                 BoNT/A L 4-128   
               
               
                 MTD143 
                 pMTD143 
                 BoNT/A H 455-661   
               
               
                 MTD145 
                 pMTD130 
                 BoNT/A H 780-939   
               
               
                 MTD150 
                 pMTD74 
                 HMS174(DE3) host 
               
               
                   
                   
                 for background 
               
               
                   
                   
                 control 
               
               
                 MTD164 
                 pMTD162 
                 BoNT/A H 982-1123   
               
               
                 MTD165 
                 pMTD163 
                 BoNT/A H 1150-1289   
               
               
                 MTD191 
                 pMTD186 
                 BoNT/A L 126-271   
               
               
                 MTD193 
                 PMTD188 
                 BoNT/A H 1078-1220   
               
               
                 MTD196 
                 pMTD187 
                 BoNT/A L 257-465   
               
               
                 MTD203 
                 PMTD195 
                 BoNT/A H 915-1059   
               
               
                 MTD210 
                 pMTD148 
                 BoNT/A H 630-808   
               
               
                   
               
               
                 *Campbell, et al.,  Proc. Natl. Acad. Sci., U.S.A.  75:2276-2280 (1994)  
               
               
                 **Studier, et al.,  Methods in Enzymology , Academic Press, New York (1990)  
               
             
          
         
       
     
     Analysis of protein expression. Transformants were screened for protein expression by immunoblotting analysis. Individual colonies were grown at 37° C. in 2 ml of M-9 medium to an optical density of 0.8-1.0 at 660 nm. Expression was induced by adding IPTG to a concentration of 0.25 mM. After induction, cultures were incubated at 37° C. for 2 h before harvesting. The cells were pelleted in a microfuge tube and resuspended in 100 μl of sample loading buffer containing 1% sodium dodecyl sulfate (SDS) and 0.2 M 2-mercaptoethanol. The samples were boiled for 5 min and then separated by discontinuous SDS-polyacrylamide gel electrophoresis (PAGE) (Lammeli, U. K., Nature 227:680-685 (1970)). The proteins were transferred to nitrocellulose sheets using a semi-dry electroblotter (Integrated Separation Systems, Hyde Park, Mass.) and then stained for the presence of BoNT/A-specific protein using horse antiserum to BoNT/A. Strain MTD150 was prepared as described above and used as a background control. Purified BoNT/A (Sigma, St. Louis, Mo.) was included in each gel as a positive control. 
     Cell fractionation. Bacterial strains encoding the truncated BoNT/A proteins were grown in M-9 medium and were induced to express protein as described above. Cells were pelleted by centrifugation at 3,000×g for 10 min. The pellet was suspended in lysis buffer containing 1 mg/ml lysozyme, 50 mM Tris, 50 mM EDTA, and 20% sucrose (pH 8.0) and was incubated at 37° C. for 30 min. To ensure complete lysis, the cell suspension was subjected to two cycles of rapid freeze-thaw. MgSO 4  was added to the lysate to a concentration of 20 mM, DNase (Sigma) and RNase (Sigma) were added to a concentration of 0.01 mg/ml each, and then the lysate was incubated at 37° C. for 30 min. The lysate was clarified by centrifugation at 3,000×g for 10 min. The clarified lysate was centrifuged at 20,000×g for 30 min at 4° C. and the resulting pellet was dissolved in sample loading buffer. The sample was boiled for 5 min and stored at −20° C. before use. 
     Purification of BoNT/A proteins. The truncated BoNT/A proteins were purified by preparative SDS-PAGE with a Model 491 Prep Cell (Bio-Rad, Richmond, Calif.). The percentage of acrylamide used in the resolving gel was adjusted to maximize the separation of the protein of interest. Separation was typically performed at 12 watts constant power with a 37-mm diameter tube gel. The length of the stacking and resolving gels were 2 cm and 10 cm, respectively. The eluate was collected at a flow rate of 0.75 ml/min as 4-ml fractions. Aliquots of the fractions were separated by analytical SDS-PAGE and stained with Coomassie blue to visualize total protein. In some cases, a duplicate gel was transferred to nitrocellulose and analyzed for immunoreactivity to polyclonal horse antiserum to BoNT/A. Fractions containing truncated BoNT/A protein were pooled and concentrated by ultrafiltration (Amicon, Danvers, Mass.). The concentrated protein was passed through a column containing Extracti-Gel™ D resin (Pierce, Rockford, Ill.) to remove any remaining SDS. The protein was subjected to extensive diafiltration in buffer containing 120 mM NaCl, 2.7 mM KCl, 10 mM phosphate buffer (pH 7.4), 20% glycerol (v/v), and 5 mM EDTA. Each protein preparation was examined by Coomassie staining and immunoblotting analysis for its composition and for the presence of BoNT/A-specific protein . Protein concentrations were determined by the BCA assay (Pierce). The protein preparations were aliquoted and stored at −70° C. before use. 
     Immunization and challenge. The protocol used in this study was approved by the USAMRIID Institutional Animal Care and Use Committee. Female CB6F1 mice (Jackson Laboratory, Bar Harbor, Me.), 4-6 weeks old, were provided food and water ad libitum. The mice were immunized with 10 μg of BoNT/A-specific protein suspended in adjuvant emulsion (Ribi Immunochem, Hamilton, Mont.). some mice were immunized with saline emulsified in adjuvant for use as negative controls. For comparison, some mice were immunized with pentavalent toxoid. The mice were immunized i.p. four times at 2-week intervals. One week after the last immunization, the mice were bled and the serum was analyzed by immunoblot for the presence of chain-specific antibody. Two weeks after the last immunization, each mouse was challenged i.p. with 2 lethal doses of BoNT/A (2 MIPLD 99 ). Four days after challenge, the mice were scored for survivors. 
     Immunoblotting analysis. BoNT/A was separated by SDS-PAGE on a 10% gel and then transferred to nitrocellulose using a semi-dry electroblotter. The nitrocellulose was blocked and loaded into a Multi-Screen immunoblotting apparatus (Bio-Rad). Pooled serum from each group of immunized mice was diluted and then incubated in separate wells of the apparatus. The blot was developed using an alkaline phosphatase-conjugated goat antibody to mouse IgG (Kirkegaard &amp; Perry Labs, Gaithersburg, Md.) to identify the presence of BoNT/A-specific antibody. 
     Construction and expression of BoNT/A gene fragments. The BoNT/A gene was subcloned into overlapping fragments ranging in size from ˜300-600 base pairs by using PCR. The primers encoded flanking restriction sites that permitted convenient insertion into the expression vector used, and allowed transcriptional and translational read-through of the amplified fragments to occur (Table 1). Plasmid vector pMTD74 was used to express the amplified BoNT/A gene fragments in  E. coli  . Insertion of the PCR-amplified fragments into the expression vector pMTD74 resulted in translational fusion to the A2 peptide of cholera toxin (CtxA2) (8). The fragments were fused to CtxA2 to provide a vaccine for administration mucosally. The presence of BoNT/A-specific protein was determined by immunoblotting analysis, using polyclonal horse antiserum to BoNT/A, and by comparison of the predicted size of the truncated protein to its actual size. Fusion to CtxA2 increased the predicted size of the truncated BoNT/A proteins expressed by an additional 5.4 kDa, but it did not appear to affect their ability to be produced. By expressing overlapping segments of the toxin, all potential linear epitopes were encoded. BoNT/A is post translationally cleaved into the light (L) and heavy (H) chains which are joined together by a disulfide bond. The position of each fragment within BoNT/A is indicated by the chain it was derived from (L or H), followed by the amino acid residues of BoNT/A encoded.). The T7 promoter expressed these proteins at high levels in  E. coli  . The BoNT/A-specific proteins were expressed primarily in the form of inclusion bodies that could be isolated by differential centrifugation upon lysis of the cells. 
     Purification of BoNT/A proteins. While the crude lysates containing the BoNT/A proteins were initially used for immunization of mice, it was found preferable to use at least partially purified materials to provide improved tolerance and to effectively produce strong, specific antibody response. For these reasons, the lysates containing the truncated BoNT/A proteins were subjected to purification by preparative SDS-PAGE and then used for immunization of mice. Preparative SDS-PAGE provided a convenient method of both solubilizing and separating the BoNT/A proteins from the majority of other contaminants present in the lysates. Although the BoNT/A proteins were not always purified to homogeneity, they were highly enriched. Furthermore, the BoNT/A proteins remained soluble after the SDS was removed, which facilitated the administration of these proteins to mice. 
     Immunogenicity of BoNT/A proteins. Mice were immunized i.p. with the truncated BoNT/A proteins emulsified in Ribi™ adjuvant. The mice were immunized at 2-week intervals, and one week after the last immunization, their serum was analyzed for the presence of antibody to BoNT/A. Since BoNT/A can be separated by SDS-PAGE into a 50 kDa light chain and 100 kDa heavy chain, immunoblotting analysis was used to evaluate whether the antibody elicited by each truncated protein reacted with the appropriate chain. Optimal antibody responses were observed in mice after the fourth dose. All of the truncated proteins were able to elicit an antibody response except H 1078-1220 . Although this fragment was non-immunogenic, it was highly antigenic when reacted with polyclonal horse antiserum to BoNT/A. Unlike the crude lysates used for immunization previously, the purified proteins were well-tolerated and could be repeatedly administered to the mice. In addition, the purified proteins were able to elicit an BoNT/A-specific antibody response in mice. This difference in the immunogenicity of the crude lysates cannot be accounted for by the lack of BoNT/A-specific protein, since the lysates used for immunization were known to contain appreciable quantities of truncated protein. 
     Protective efficacy of BoNT/A proteins. Two weeks after the final immunization, each mouse was challenged i.p. with 2 lethal doses of BoNT/A (2 MIPLD 99 ). This dose was chosen for initial screening to observe any potential ability of the proteins to elicit protective immunity. As shown in Table 3, only two proteins protected the majority of animals from death. Both of these fragments were derived from the heavy chain and encoded amino acid residues H 455-661  and H 1150-1289 . 
     H 455-661  of serotype A neurotoxin is the sequence 
     H 3 N-IKVNN WDLFF SPSED NFTND LNKGE EITSD TNIEA AEENI SLDLI QQYYL TFNFD NEPEN ISIEN LSSDI IGQLE LMPNI ERFPN GKKYE LDKYT MFHYL RAQEF EHGKS RIALT NSVNE ALLNP SRVYT FFSSD YVKKV NKATE AAMFL GWVEQ LVYDF TDETS EVSTT DKIAD ITIII PYIGP ALNIG NMLYK DDFVG ALIFS GA-COOH Seq. #21 
     and H 1150-1289  of serotype A neurotoxin is the sequence 
     H 3 N-LNSSL YRGTK FIIKK YASGN KDNIV RNNDR VYINV VVKNK EYRLA TNASQ AGVEK ILSAL EIPDV GNLSQ VVVMK SKNDQ GITNK CKMNL QDNNG NDIGF IGFHQ FNNIA KLVAS NWYNR QIERS SRTLG CSWEF IPVDD-COOH Seq. #22. 
     Although some of the other truncated proteins appeared to provide partial protection at the challenge dose initially used, none were as definitive as H 455-661  and H 1150-1289 . Rechallenge of the survivors with 2 MIPLD 99  of BoNT/A resulted in the death of all mice except those immunized with the two protective fragments. To confirm these results, separate groups of mice were immunized with fragments H 455-661  and H 1150-1289  as before and then challenged with 10 MIPLD 50 . The survival rate for mice immunized with H 455-661  and H 1150-1289  at this challenge dose was determined to be 87.5% and 60.0%, respectively. 
     
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 3 
               
             
             
               
                   
               
               
                 Immunogenicity and protective efficacy of the truncated 
               
               
                 BoNT/A proteins 
               
             
          
           
               
                   
                 Protein 
                   
                   
                   
               
               
                   
                 Segment   a     
                 Immuno- 
                 Number of 
                   
               
               
                   
                 Survival 
                 Blot   b     
                 Survivors   c     
                 % 
               
               
                   
                   
               
             
          
           
               
                   
                 L 4-128   
                 + 
                  1/10 
                 10.0 
               
               
                   
                 L 126-271   
                 + 
                 0/8 
                 0.0 
               
               
                   
                 L 257-465   
                 + 
                 0/9 
                 0.0 
               
               
                   
                 H 455-661   
                 + 
                 7/9 
                 77.8 
               
               
                   
                 H 630-808   
                 + 
                 0/5 
                 0.0 
               
               
                   
                 H 780-939   
                 + 
                 2/7 
                 28.6 
               
               
                   
                 H 915-1059   
                 + 
                 0/8 
                 0.0 
               
               
                   
                 H 982-1123   
                 + 
                 1/9 
                 11.1 
               
               
                   
                 H 1078-1220   
                 − 
                 0/5 
                 0.0 
               
               
                   
                 H 1150-1289   
                 + 
                 6/8 
                 75.0 
               
               
                   
                   
               
               
                   
                     a   Amino acid residue number of the light (L) chain and the heavy (H) chain.  
               
               
                   
                     b   CB6F1 mice were immunized i.p. with four doses of each protein at 2-week intervals. One week after the last dose, the mice were bled and the serum was analyzed by immunoblot for the presence of antibody specific for BoNT/A.  
               
               
                   
                     c   Number of survivors/total number 4 days after challenge with 2 MIPLD 99  of BoNT/A.  
               
             
          
         
       
     
     Immunoblotting analysis was used to detect the presence of BoNT/A-specific antibody in the immunized mice for several reasons. First, the sensitivity of this method maximized the probability of detecting the presence of any fragment-specific antibody, regardless of whether it was directed towards a linear or a conformational epitope. Second, by separating BoNT/A into its heavy and light chains, this procedure also permitted the chain specificity of the antibody to be confirmed. By this method, all fragments were able to elicit an antibody response, except H 1078-1220 . 
     Although most of the BoNT/A fragments were able to elicit antibody, only two were clearly able to confer protective immunity (Table 3). The protective efficacy of H 455-661  and H 1150-1289  correlates well with the potential functional role of these domains. The N-terminal half of the heavy chain (H N ) of BoNT/A, from which H 455-661  was derived, has been shown to be important in productive binding and internalization of the toxin to the cell. The C-terminal half of the heavy chain (H C ), from which H 1150-1289  was derived, has been associated with the initial binding of the toxin to the cell. If these functions are encoded by either fragment, then antibody specific to these domains would be predicted to interfere with the binding and/or internalization of BoNT/A. This, in turn, would prevent intoxication of the cell. The location of these protective domains on the extreme N- and C-terminal ends of the heavy chain suggest that important functional roles may also be encoded by these fragments. We are currently exploring this possibility. 
     The light chain fragment L 126-271 , did not confer protection even though it elicited an antibody response (Table 3). This fragment encodes a highly conserved histidine-rich motif characteristic of zinc-dependent metalloproteases, such as BoNT/A. Although unproven, antibody directed to this region may block the enzymatic activity of BoNT/A. The inability of L 126-271  to protect suggests that the antibody elicited by this fragment may not have been directed towards epitopes involved in the enzymatic activity of the light chain. 
     Studies with MAbs suggest that many of the antibody determinants of BoNT/A may be conformationally sensitive, and there is evidence to suggest that BoNT/A is an oligomeric protein. If BoNT/A is indeed oligomeric, then it is possible that some epitopes are formed by the interaction of adjoining subunits. Alternatively, linear-distant parts of the toxin molecule may come together when folded to form epitopes, as appears to be the case for the light chain. Comparison of the amino acid sequence of these fragments with the amino acid sequence of similar regions from the other serotypes did not show any significant homology. A cocktail of recombinant proteins containing amino acid sequences from analogous domains other serotypes (H 455-661  and H 1150-1289 ) should be prepared using the methods of the invention to provide immune protection against more than one serotype of organism. 
     The entire domains of H 455-661  and/or H 1150-1289  need not be used to provide a vaccine. However, at least 100 amino acids from one of the domains of any serotype should be used to provide sufficient antigenicity and immunoprotection. 
     
       
         
           
             22 
           
           
             
               40 base pairs 
               nucleic acid 
               unknown 
               linear 
             
             
               DNA (genomic) 
             
             NO 
             NO 
             
               C. botulinum 
             
             1
ATATGGAATT CGTTAATAAA CAATTTAATT ATAAAGATCC                           40 
           
           
             
               42 base pairs 
               nucleic acid 
               unknown 
               linear 
             
             
               DNA (genomic) 
             
             NO 
             NO 
             
               C. botulinum 
             
             2
AGTATCGTCG ACTTTTAATT CTGTATCTAT TGTACTTCCA CC                        42 
           
           
             
               32 base pairs 
               nucleic acid 
               unknown 
               linear 
             
             
               DNA (genomic) 
             
             NO 
             NO 
             
               C. botulinum 
             
             3
GATACAGAAT TCAAAGTTAT TGATACTAAT AG                                   32 
           
           
             
               34 base pairs 
               nucleic acid 
               unknown 
               linear 
             
             
               DNA (genomic) 
             
             NO 
             NO 
             
               C. botulinum 
             
             4
CTTTGCGTCG ACTCCCCCAA ATGTTCTAAG TTCC                                 34 
           
           
             
               39 base pairs 
               nucleic acid 
               unknown 
               linear 
             
             
               DNA (genomic) 
             
             NO 
             NO 
             
               C. botulinum 
             
             5
GGGTTAGAAT TCAGCTTTGA GGAACTTAGA ACATTTGGG                            39 
           
           
             
               45 base pairs 
               nucleic acid 
               unknown 
               linear 
             
             
               DNA (genomic) 
             
             NO 
             NO 
             
               C. botulinum 
             
             6
AGGACTGTCG ACCAAGTCCC AATTATTAAC TTTGATTGAT AAATC                     45 
           
           
             
               36 base pairs 
               nucleic acid 
               unknown 
               linear 
             
             
               DNA (genomic) 
             
             NO 
             NO 
             
               C. botulinum 
             
             7
TTAAATGAAT TCTCAATCAA AGTTAATAAT TGGGAC                               36 
           
           
             
               33 base pairs 
               nucleic acid 
               unknown 
               linear 
             
             
               DNA (genomic) 
             
             NO 
             NO 
             
               C. botulinum 
             
             8
CTCTGGGTCG ACTTCTAACA GAATAACAGC TCC                                  33 
           
           
             
               35 base pairs 
               nucleic acid 
               unknown 
               linear 
             
             
               DNA (genomic) 
             
             NO 
             NO 
             
               C. botulinum 
             
             9
GAAGTAAGAG CTCTGGATAA AATTGCGGAT ATAAC                                35 
           
           
             
               36 base pairs 
               nucleic acid 
               unknown 
               linear 
             
             
               DNA (genomic) 
             
             NO 
             NO 
             
               C. botulinum 
             
             10
GCTATGATTA ATATAAATAA ATTTTTGAAT CAATGC                               36 
           
           
             
               31 base pairs 
               nucleic acid 
               unknown 
               linear 
             
             
               DNA (genomic) 
             
             NO 
             NO 
             
               C. botulinum 
             
             11
TAACCGGTCG ACACCATAAG GGATCATAGA G                                    31 
           
           
             
               39 base pairs 
               nucleic acid 
               unknown 
               linear 
             
             
               DNA (genomic) 
             
             NO 
             NO 
             
               C. botulinum 
             
             12
AGTACTAAGC TTTTCATACA TACTATTATA TACAATAGC                            39 
           
           
             
               35 base pairs 
               nucleic acid 
               unknown 
               linear 
             
             
               DNA (genomic) 
             
             NO 
             NO 
             
               C. botulinum 
             
             13
AAAAATAGAG CTCAATTATT TAATTTAGAA AGTAG                                35 
           
           
             
               33 base pairs 
               nucleic acid 
               unknown 
               linear 
             
             
               DNA (genomic) 
             
             NO 
             NO 
             
               C. botulinum 
             
             14
ACCATCGTCG ACAAACATTA TATTATTACT AGC                                  33 
           
           
             
               37 base pairs 
               nucleic acid 
               unknown 
               linear 
             
             
               DNA (genomic) 
             
             NO 
             NO 
             
               C. botulinum 
             
             15
TATGGTGAAT TCATCTGGAC TTTACAGGAT ACTCAGG                              37 
           
           
             
               27 base pairs 
               nucleic acid 
               unknown 
               linear 
             
             
               DNA (genomic) 
             
             NO 
             NO 
             
               C. botulinum 
             
             16
ATTTACGTCG ACATATTTAT TTGGATC                                         27 
           
           
             
               31 base pairs 
               nucleic acid 
               unknown 
               linear 
             
             
               DNA (genomic) 
             
             NO 
             NO 
             
               C. botulinum 
             
             17
GATAAGGAAT TCAATGAAAA AGAAATCAAA G                                    31 
           
           
             
               30 base pairs 
               nucleic acid 
               unknown 
               linear 
             
             
               DNA (genomic) 
             
             NO 
             NO 
             
               C. botulinum 
             
             18
CTTCATGTCG ACTACTTGAC TTAGATTTCC                                      30 
           
           
             
               33 base pairs 
               nucleic acid 
               unknown 
               linear 
             
             
               DNA (genomic) 
             
             NO 
             NO 
             
               C. botulinum 
             
             19
AACATTGAAT TCAATTCAAG TTTGTATAGG GGG                                  33 
           
           
             
               36 base pairs 
               nucleic acid 
               unknown 
               linear 
             
             
               DNA (genomic) 
             
             NO 
             NO 
             
               C. botulinum 
             
             20
TCCATCGTCG ACAGGAATAA ATTCCCATGA GCTACC                               36 
           
           
             
               207 amino acids 
               amino acid 
               unknown 
               unknown 
             
             
               peptide 
             
             NO 
             NO 
             N-terminal 
             
               C. botulinum 
             
             21
Ile Lys Val Asn Asn Trp Asp Leu Phe Phe Ser Pro Ser Glu Asp Asn
1               5                   10                  15
Phe Thr Asn Asp Leu Asn Lys Gly Glu Glu Ile Thr Ser Asp Thr Asn
            20                  25                  30
Ile Glu Ala Ala Glu Glu Asn Ile Ser Leu Asp Leu Ile Gln Gln Tyr
        35                  40                  45
Tyr Leu Thr Phe Asn Phe Asp Asn Glu Pro Glu Asn Ile Ser Ile Glu
    50                  55                  60
Asn Leu Ser Ser Asp Ile Ile Gly Gln Leu Glu Leu Met Pro Asn Ile
65                  70                  75                  80
Glu Arg Phe Pro Asn Gly Lys Lys Tyr Glu Leu Asp Lys Tyr Thr Met
                85                  90                  95
Phe His Tyr Leu Arg Ala Gln Glu Phe Glu His Gly Lys Ser Arg Ile
            100                 105                 110
Ala Leu Thr Asn Ser Val Asn Glu Ala Leu Leu Asn Pro Ser Arg Val
        115                 120                 125
Tyr Thr Phe Phe Ser Ser Asp Tyr Val Lys Lys Val Asn Lys Ala Thr
    130                 135                 140
Glu Ala Ala Met Phe Leu Gly Trp Val Glu Gln Leu Val Tyr Asp Phe
145                 150                 155                 160
Thr Asp Glu Thr Ser Glu Val Ser Thr Thr Asp Lys Ile Ala Asp Ile
                165                 170                 175
Thr Ile Ile Ile Pro Tyr Ile Gly Pro Ala Leu Asn Ile Gly Asn Met
            180                 185                 190
Leu Tyr Lys Asp Asp Phe Val Gly Ala Leu Ile Phe Ser Gly Ala
        195                 200                 205 
           
           
             
               140 amino acids 
               amino acid 
               unknown 
               unknown 
             
             
               peptide 
             
             NO 
             NO 
             N-terminal 
             
               C. botulinum 
             
             22
Leu Asn Ser Ser Leu Tyr Arg Gly Thr Lys Phe Ile Ile Lys Lys Tyr
1               5                   10                  15
Ala Ser Gly Asn Lys Asp Asn Ile Val Arg Asn Asn Asp Arg Val Tyr
            20                  25                  30
Ile Asn Val Val Val Lys Asn Lys Glu Tyr Arg Leu Ala Thr Asn Ala
        35                  40                  45
Ser Gln Ala Gly Val Glu Lys Ile Leu Ser Ala Leu Glu Ile Pro Asp
    50                  55                  60
Val Gly Asn Leu Ser Gln Val Val Val Met Lys Ser Lys Asn Asp Gln
65                  70                  75                  80
Gly Ile Thr Asn Lys Cys Lys Met Asn Leu Gln Asp Asn Asn Gly Asn
                85                  90                  95
Asp Ile Gly Phe Ile Gly Phe His Gln Phe Asn Asn Ile Ala Lys Leu
            100                 105                 110
Val Ala Ser Asn Trp Tyr Asn Arg Gln Ile Glu Arg Ser Ser Arg Thr
        115                 120                 125
Leu Gly Cys Ser Trp Glu Phe Ile Pro Val Asp Asp
    130                 135                 140