WO2000067700A2 - Recombinant vaccine against botulinum neurotoxin - Google Patents
Recombinant vaccine against botulinum neurotoxin Download PDFInfo
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- WO2000067700A2 WO2000067700A2 PCT/US2000/012890 US0012890W WO0067700A2 WO 2000067700 A2 WO2000067700 A2 WO 2000067700A2 US 0012890 W US0012890 W US 0012890W WO 0067700 A2 WO0067700 A2 WO 0067700A2
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/33—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Clostridium (G)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/02—Bacterial antigens
- A61K39/08—Clostridium, e.g. Clostridium tetani
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Definitions
- This invention is directed to preparation and expression of synthetic genes encoding polypeptides containing protective epitopes of botulinum neurotoxin (BoNT).
- the invention is also directed to methods of vaccination against botulism using the expressed peptides.
- Tetanus neurotoxin (TeNT) is produced by Clostridium tetani while Clostridium botulinum produces seven different neurotoxins which are differentiated serologically by specific neutralization.
- the botulinum neurotoxins (BoNT) have been designated as serotypes A, B, Ci, D, E, F, and G.
- Botulinum neurotoxins (BoNT) are the most toxic substances known and are the causative agents of the disease botulism.
- BoNT exert their action by inhibiting the release of the neurotransmitter acetylcholine at the neuromuscular junction
- BoNT exert their action by inhibiting the release of the neurotransmitter acetylcholine at the neuromuscular junction
- Hibermann, E., et al., (1986) "Clostridial Neurotoxins: Handling and Action at the Cellular and Molecular Level," C r. Top. Microbiol. Immunol, 129:93-179; Schiavo, G., et al., (1992a), "Tetanus and Botulinum-B Neurotoxins Block Neurotransmitter Release by Proteolytic Cleavage of Synaptobrevin," Nature, 359:832-835; Simpson, L.L., (1986), "Molecular Pharmacology of Botulinum Toxin and Tetanus Toxin," Annu. Rev.
- Botulinum neurotoxins are translated as a single 150 kDa polypeptide chain and then posttranslationally nicked, forming a dichain consisting of a 100 kDa heavy chain and a 50 kDa light chain which remain linked by a disulfide bond (DasGupta, B.R., et al., (1972), "A Common Subunit Structure in Clostridium botulinum Type A, B, and E Toxins," Biophys. Res.
- the two chains can be separated; one chain has a Mr of -100 kDa and is referred to as the heavy chain while the other has a Mr -50 kDa and is termed the light chain.
- toxin is internalized into an endosome through receptor-mediated endocyctosis (Shone, C.C., et al., (1987), "A 50-kDa Fragment from the NH 2 -terminus of the Heavy Subunit of Clostridium botulinum Type A Neurotoxin Forms Channels in Lipid Vesicles, Euro. J. Biochem., 167:175-180).
- the amino terminal half of the heavy chain is believed to participate in the translocation mechanism of the light chain across the endosomal membrane (Simpson, 1986; Poulain, B., et al., (1991), "Heterologous Combinations of Heavy and Light Chains from Botulinum Neurotoxin A and Tetanus Toxin Inhibit Neurotransmitter Release in Aplysia," J. Biol.
- BoNT serotypes A, d, and E cleave SNAP-25 (synaptosomal-associated protein of M25,000), serotypes B, D, F, and G cleave VAMP/synaptobrevin (synaptic vesicle- associated membrane protein); and serotype Ci cleaves syntaxin.
- Inactivation of SNAP-25, VAMP, or syntaxin by BoNT leads to an inability of the nerve cells to release acetylcholine resulting in neuromuscular paralysis and possible death, if the condition remains untreated.
- Human botulism poisoning is generally caused by type A, B, E or rarely, by type F toxin.
- Type A and B are highly poisonous proteins which resist digestion by the enzymes of the gastrointestinal tract.
- Foodborne botulism poisoning is caused by the toxins present in contaminated food, but wound and infant botulism are caused by in vivo growth in closed wounds and the gastrointestinal tract respectively.
- the toxins primarily act by inhibiting the neurotransmitter acetylcholine at the neuromuscular junction, causing paralysis.
- Another means for botulism poisoning to occur is the deliberate introduction of the toxin(s) into the environment as might occur in biological warfare.
- toxin When the cause of botulism is produced by toxin rather than by in vivo infection the onset of neurologic symptoms is usually abrupt and occurs within 18 to 36 hours after ingestion. The most common immediate cause of death in respiratory failure due to diaphragmatic paralysis. Home canned foods are the most common sources of toxins. The most frequently implicated toxin is toxin A, which is responsible for more than 50% of morbidity resulting from botulinum toxin.
- botulinal toxin Because even small amounts of botulinal toxin can cause serious illness, persons such as laboratory workers who are exposed to toxin must learn to handle all samples that may contain toxin with extreme care. It is also suggested that such workers be protected from illness by vaccination against the toxins. Furthermore, persons exposed to conditions in which botulism toxins might be in the environment which might be inhaled or ingested, such as military personnel, need to be protected from the toxin.
- Microbiol, 26:2351-2356) available under Investigational New Drug (IND) status, is used to immunize specific populations of at-risk individuals, i.e., scientists and health care providers who handle BoNT and our armed forces who may be subjected to weaponized forms of the toxin
- serotypes A, B, and E are most associated with botulism outbreaks in humans, type F has also been diagnosed (Midura, T.F., et al., (1972), "Clostridium botulinum Type F: Isolation from Venison Jerky," Appl. Microbiol. ⁇ 24:165-167; Green, J., et al., (1983), "Human Botulism (Type F) - A Rare Type," Am. J.
- toxoid vaccines are available, there are numerous shortcomings with their current use and ease of production.
- C. botulinum is a spore- former, a dedicated facility is required to manufacture a toxin-based product.
- the requirement for a dedicated manufacturing facility is not trivial. It is extremely costly to renovate and upgrade an existing facility or to build a new one and then to maintain the facility in accordance with current Good Manufacturing Practices (cGMP) to manufacture one vaccine.
- cGMP Good Manufacturing Practices
- the yields of toxin production from C. botulinum are relatively low.
- the toxoiding process involves handling large quantities of toxin and thus is dangerous, and the added safety precautions increase the cost of manufacturing.
- the toxoid product for types A-E consists of a crude extract of clostridial proteins that may influence immunogenicity or reactivity of the vaccine, and the type F toxoid is only partially purified (IND 5077).
- the toxoiding process involves the use of formaldehyde, which inactivates the toxin, and residual levels of formaldehyde (not to exceed 0.02%) are part of the product formulation to prevent reactivation of the toxin, the vaccine is reactogenic.
- An additional component of the toxoid vaccines is the preservative thimerosal (0.01%), which also increases the reactogenicity of the product.
- a recombinant vaccine would eliminate the need for a dedicated manufacturing facility.
- many cGMP facilities are in existence and available that could manufacture a recombinant product.
- Production yields from a genetically engineered product is expected to be high.
- There would be no need to treat the vaccine with formalin because the product would be non-toxic from the outset.
- Recombinant products would be purer, less reactogenic, and more fully characterized.
- the cost of a recombinant product would be expected to be much lower than a toxoid because there would be no expenditures required to support a dedicated facility, and the higher production yields would reduce the cost of the vaccine product.
- this invention provides a nucleic acid encoding the carboxy-terminal portion of the heavy chain (HC) of botulinum neurotoxin (BoNT), the BoNT being selected from the group consisting of BoNT serotype A, BoNT serotype B, BoNT serotype CI, BoNT serotype D, BoNT serotype E, BoNT serotype F, and BoNT serotype G, wherein said nucleic acid is expressable in a recombinant organism selected from Escherichia coli and Pichia pastoris.
- the nucleic acid comprises a nucleic acid sequence selected from SEQ ID No:l (serotype A), SEQ ID No:7 (serotype B), SEQ ID No:9 (serotype CI), SEQ ID No: 11 (serotype D), SEQ ID No: 13 (serotpye E), SEQ ID No: 15 (serotype F), and SEQ ID No: 17 (serotype G).
- the nucleic acid encodes an HC amino acid sequence of BoNT selected from SEQ ID No:2 (serotype A), SEQ LD No:8 (serotype B), SEQ ID No: 10 (serotype CI), SEQ JD No: 12 (serotype D), SEQ ID No: 14 (serotpye E), SEQ ID No: 16 (serotype F), and SEQ ID No: 18 (serotype G).
- this invention provides a nucleic acid encoding the amino-terminal portion of the heavy chain (HN) of botulinum neurotoxin (BoNT), the BoNT being selected from the group consisting of BoNT serotype B, BoNT serotype CI, BoNT serotype D, BoNT serotype E, BoNT serotype F, and BoNT serotype G, wherein said nucleic acid is expressable in a recombinant organism selected from Escherichia coli and Pichia pastoris.
- BoNT botulinum neurotoxin
- the nucleic acid comprises a nucleic acid sequence selected from SEQ ID No:21 (serotype B), SEQ ID No:23 (serotype CI), SEQ ID No:25 (serotype D), SEQ ID No:27 (serotpye E), SEQ ID No:29 (serotype F), and SEQ ID No:31 (serotype G).
- nucleic acid nucleic acid encodes an HN amino acid sequence of BoNT selected from SEQ ID No:22 (serotype B), SEQ ID No:24 (serotype CI), SEQ ID No:26 (serotype D), SEQ ID No:28 (serotpye E), SEQ ID No:30 (serotype F), and SEQ ID No:32 (serotype G).
- the nucleic acid of this invention is a synthetic nucleic acid.
- the sequence of the nucleic acid is designed by selecting at least a portion of the codons encoding HC from codons preferred for expression in a host organism, which may be selected from gram negative bacteria, yeast, and mammalian cell lines; preferably, the host organism is Escherichia coli or Pichia pastoris.
- the nucleic acid sequence encoding HC is designed by selecting codons encoding HC which codons provide HC sequence enriched in guanosine and cytosine residues.
- nucleic acid encoding HC or HN is expressed in a recombinant host organism with higher yield than a second nucleic acid fragment encoding the same HC sequence, said second nucleic acid fragment having the wild-type Clostridum botulinum sequence of HC.
- this invention provides anexpression vector comprising the nucleic acid of this invention, whereby HC and/or HN is expressed upon transfection of a host organism with the expression vector.
- Another embodiment of this invention provides a method of preparing a polypeptide comprising the carboxy-terminal portion of the heavy chain (HC) of botulinum neurotoxin (BoNT) or the amino-terminal portion of the heavy chain (HN) of botulinum neurotoxin (BoNT) selected from the group consisting of BoNT serotype A, BoNT serotype B, BoNT serotype C, BoNT serotype D, BoNT serotype E, BoNT serotype F, and BoNT serotype G, said method comprising culturing a recombinant host organism transfected with the expression vector of of this invention under conditions wherein HC or HN is expressed.
- the recombinant host organism is a eukaryote.
- the method of this invention further comprises recovering insoluble protein from the host organism, whereby a fraction enriched in HC or HN is obtained.
- the host organism is Pichia pastoris.
- this invention provides an immunogenic composition
- an immunogenic composition comprising the carboxy-terminal portion of the heavy chain (HC) of botulinum neurotoxin (BoNT) selected from the group consisting of BoNT serotype A, BoNT serotype B, BoNT serotype C, BoNT serotype D, BoNT serotype E, BoNT serotype F, and BoNT serotype G.
- the immunogenic composition is prepared by culturing a recombinant organism transfected with an expression vector encoding HC. More preferably, the immunogenic composition is prepared by a method wherein an insoluble protein fraction enriched in HC is recovered from said recombinant organism.
- this invention provides an immunogenic composition comprising the amino-terminal portion of the heavy chain (HN) of botulinum neurotoxin (BoNT) selected from the group consisting of BoNT serotype A, BoNT serotype B, BoNT serotype C, BoNT serotype D, BoNT serotype E, BoNT serotype F, and BoNT serotype G.
- the immunogenic composition comprising HN is prepared by culturing a recombinant organism transfected with an expression vector encoding HN. More preferably, the immunogenic composition is prepared from an insoluble protein fraction enriched in HN which is recovered from the recombinant organism.
- this invention provides an immunogenic composition
- a polypeptide comprising epitopes contained in the carboxy-terminal portion of the heavy chain (HC) of botulinum neurotoxin (BoNT) and/or the amino-terminal portion of the heavy chain (HN) of botulinum neurotoxin (BoNT) selected from the group consisting of BoNT serotype A, BoNT serotype B, BoNT serotype C, BoNT serotype D, BoNT serotype E, BoNT serotype F, and/or BoNT serotype G, said epitopes eliciting protective immunity toward the respective BoNT serotype.
- the immunogenic composition elicits an ELISA response to the respective BoNT serotype(s) in an animal which is detectable in serum from the animal even when the serum is diluted 100-fold.
- Figure 1 shows the sequence for a synthetic gene encoding the He fragment of BoNT serotype A and the encoded amino acids sequence.
- Figure 2 shows the sequence for a synthetic gene encoding the He fragment of BoNT serotype A and the encoded amino acids sequence.
- Figure 3 shows the sequence for a synthetic gene encoding the He fragment of BoNT serotype A and the encoded amino acids sequence.
- Figure 4 shows the sequence for a synthetic gene encoding the He fragment of BoNT serotype B and the encoded amino acids sequence.
- Figure 5 shows the sequence for a synthetic gene encoding the He fragment of BoNT serotype C and the encoded amino acids sequence.
- Figure 6 shows the sequence for a synthetic gene encoding the He fragment of BoNT serotype D and the encoded amino acids sequence.
- Figure 7 shows the sequence for a synthetic gene encoding the He fragment of BoNT serotype E and the encoded amino acids sequence.
- Figure 8 shows the sequence for a synthetic gene encoding the He fragment of BoNT serotype E and the encoded amino acids sequence.
- Figure 9 shows the sequence for a synthetic gene encoding the He fragment of BoNT serotype F and the encoded amino acids sequence.
- Figure 10 shows the sequence for a synthetic gene encoding the He fragment of BoNT serotype G and the encoded amino acids sequence.
- Figure 11 shows the sequence for a synthetic gene encoding the H N fragment of BoNT serotype A and the encoded amino acids sequence.
- Figure 12 shows the sequence for a synthetic gene encoding the H N fragment of BoNT serotype B and the encoded amino acids sequence.
- Figure 13 shows the sequence for a synthetic gene encoding the H N fragment of BoNT serotype C and the encoded amino acids sequence.
- Figure 14 shows the sequence for a synthetic gene encoding the H N fragment of BoNT serotype D and the encoded amino acids sequence.
- Figure 15 shows the sequence for a synthetic gene encoding the H N fragment of BoNT serotype E and the encoded amino acids sequence.
- Figure 16 shows the sequence for a synthetic gene encoding the H N fragment of BoNT serotype F and the encoded amino acids sequence.
- Figure 17 shows the sequence for a synthetic gene encoding the H N fragment of BoNT serotype G and the encoded amino acids sequence.
- Figure 18 shows the sequence for a synthetic gene encoding the He fragment of BoNT serotype F and the encoded amino acids sequence.
- Figure 19 shows (A) AT base content of a putative fragment C region in native C. botulinum DNA. (B) Reduction at AT content after the first design (rBoNTF(Hc)l) of the synthetic gene. (C) AT content of the final gene design (rBoNTF(Hc)2) used to express recombinant rBoNTF(Hc) in P. pastoris.
- Figure 20 shows (A) SDS-PAGE and (B) Western blot of samples at various steps along the rBoNTF(Hc) purification. Lanes from both figures are identical except lane 1, where SDS-PAGE shows No vex mark 12 wide-range molecular weight markers and Western blot shows Novex See Blue prestained molecular weight markers. Lane 2 is the cell lysate, lane 3 is the cell extract, lane 4 is the cell extract after dialysis, lane 5 is pool of rBoNTF(Hc) positive fractions after Mono S column chromatography, and lane 6 is pool of rBoNTF(Hc)-positive fractions after hydrophobic interaction chromatography. Figure 21 shows purification of rBoNTF(Hc) by sequential chromatography.
- Figure 22 shows CD spectra of purified soluble ( — ) and resolubilized (-) rBoNTF(Hc) at 30 ⁇ g/ml (0.62 ⁇ M) in 10 M sodium phosphate, pH 7.0 in a 1-cm path length cell. Spectra were the average of four accumulations, scanned from 260 to 200 nm at a scan rate of 10 nm/min with a 2-s response and a 1-nm bandwidth. The temperature was maintained at 20°C using a Peltier thermocontrol device.
- the present inventors have determined that animals, including primates, may be protected from the effects of botulinum neurotoxin (BoNT) by immunization with fragments of the botulinum neurotoxin protein expressed by recombinant organisms.
- BoNT botulinum neurotoxin
- peptides comprising protective epitopes from the receptor binding domain and/or the translocation domain, found in the carboxy terminal and the amino terminal portions of the heavy chain of the BoNT protein, respectively, are expressed by recombinant organisms transfected with expression vectors encoding the peptides for each serotype of BoNT. Immunization with these recombinantly produced peptides will elicit antibodies capable of protecting animals against intoxication with the BoNT of the respective serotype.
- This invention provides a genetically engineered vaccine for protection against botulism.
- the vaccine comprises fragments of the A and B toxins known as "C fragments" (He domain). It is now possible to produce the He fragments of the A and B toxins in E. coli using gene segment constructs of the HC fragment or an HC polypeptide fused to E. coli maltose binding protein. It has been found that the fusion product provides excellent protection against the native toxin challenge.
- the invention provides plasmids and recombinant proteins for use as vaccines to provide protection against toxins of Clostridium botulinum.
- codons found in clostridial DNA are very unique both in terms of base composition (i.e., very high A+T base composition) and in the use of codons not normally found in E. coli or yeast.
- Table 1 is a chart depicting codon usage in Pichia pastoris. This table was generated by listing the codons found in a number of highly expressed genes in P. pastoris. The codon data was obtained by sequencing the genes and then listing which codons were found in the genes.
- the amino acid residues can be encoded for by multiple codons.
- synthetic genes using P. pastoris codon usage it is preferred to use only those codons that are found in the naturally occurring genes in P. pastoris, and it should be attempted to keep them in the same ratio found in the genes of the natural organism.
- the clostridial gene has an overall A+T richness of greater than 70% and A+T regions that have spikes of A+T of 95% or higher, they have to be lowered for expression in expression systems like yeast. (Preferably, the overall A+T richness is lowered below 60% and A+T in spikes is also lowered to 60% or below).
- the amino acid sequence encoded by such synthetic genes will preferably be the sequence of one of the BoNT serotype proteins, or a fragment thereof which contains protective epitopes. Suitable fragments include the He fragments of BoNT serotypes A, B Ci, D, E, F, and G, and the H N fragments of BoNT serotypes A, B, Ci, D, E, F, and G. Such alternative gene sequences are within the contemplation of this invention.
- proteins containing protective epitopes from both the N-terminal and the C-terminal domains of the respective serotype BoNT proteins may be prepared by fusing a sequence encoding the translocation domain (HN) to the sequence of the HC region. This may be accomplished by removing the restriction enzyme site of the 3' end of the translocation domain gene as well as the termination codon, and also removing the initiation codon, restriction enzyme site and any other nucleotides on the 5' end of the gene that are not part of the botulinum toxin gene. Then a common restriction enzyme site not found in either synthetic gene may be inserted on the 3' end of the H gene and the 5' end of the He gene, and this common restriction site may be used to fuse the two genes together.
- HN translocation domain
- the nontoxin fragment is very safe, will not require formalin treatment, and has been shown to produce significant immunity against the fully toxic parent molecule.
- the recombinantly-produced botulinum neurotoxin (rBoNT) protein fragments are completely nontoxic and, is thus, very safe.
- the fermentation of the host cell harboring the rBoNT gene e.g., Escherichia coli or Pichia pastoris
- the synthetic gene can be placed in high expression systems and used to make much larger quantities of the fragment than toxin produced by the parent organism, Clostridium botulinum.
- Clostridium botulinum the parent organism
- Synthetic genes as described herein may be transfected into suitable host organisms to create recombinant production organisms, and cultures of these recombinant organisms can then be used to produce immunogenic peptide fragments capable of conferring protective immunity against BoNT of the respective serotypes. Exemplary techniques for transfection and production of BoNT fragments are shown in the Examples. Alternative techniques are well documented in the literature (See, e.g., Maniatis, Fritsch & Sambrook, "Molecular Cloning: A Laboratory Manual” (1982); “DNA Cloning: A Practical Approach,” Volumes I and II (D.N. Glover, ed., 1985); “Oligonucleotide Synthesis” (M.J.
- the synthetic gene for BoNT serotype B fragment He has been inserted into the yeast expression vector pHIL-D4, and integrated into the chromosome of Pichia pastoris strain GS115.
- the expressed product (see amino acid sequence in Figure 4B) had the expected molecular weight as shown by denaturing polyacrylamide gel electrophoresis (PAGE) and Western blot analysis using antibodies directed against botulinum neurotoxin serotype B.
- the synthetic gene for BoNT serotype C fragment H c has been inserted into the yeast expression vector pHIL-D4, and integrated into the chromosome of Pichia pastoris strain GS115.
- the expressed product (see amino acid sequence in Figure 5B) had the expected molecular weight as shown by denaturing polyacrylamide gel electrophoresis (PAGE) and Western blot analysis using antibodies directed against botulinum neurotoxin serotype C.
- the expressed recombinant BoNTC (He) elicited high antibody titers as judged by the Enzyme Linked Immunosorbent Assay (ELISA) and, more importantly, these circulating serum titers protected mice from challenges with active toxin.
- the synthetic gene for BoNT serotype D fragment He has been inserted into the yeast expression vector pHIL-D4, and integrated into the chromosome of Pichia pastoris strain GS115.
- the expressed product (see amino acid sequence in Figure 6B) had the expected molecular weight as shown by denaturing polyacrylamide gel electrophoresis (PAGE) and Western blot analysis using antibodies directed against botulinum neurotoxin serotype D.
- PAGE denaturing polyacrylamide gel electrophoresis
- ELISA Enzyme Linked Immunosorbent Assay
- the synthetic gene for BoNT serotype E fragment He has been inserted into the yeast expression vectors pHILD2, pHILD3, and pPIC9K (see Figures 7B).
- a modified form of the synthetic gene in which an internal EcoRI site was removed and the gene was enlarged was inserted into the yeast vector pHJL-D4, and integrated into the chromosome of Pichia pastoris strain GS115.
- the expressed product (see amino acid sequence in Figure 8) had the expected molecular weight as shown by denaturing polyacrylamide gel electrophoresis (PAGE) and Western blot analysis using antibodies directed against botulinum neurotoxin serotype E.
- BoNT serotype F fragment H c (see Figure 9A) has been inserted into the yeast expression vector pHIL-D4, and integrated into the chromosome of Pichia pastoris strain GS115.
- the initial step in the development of the rBoNTF(Hc) vaccine candidate was to design a gene which could satisfactorily be expressed in a pichia host.
- a synthetic gene encoding rBoNTF(H ) was constructed to lower the inherent AT richness of the native clostridial gene and to remove any potentially rare codons.
- Clostridial genes having an AT content in excess of 65% or having an average AT content but containing AT-rich tracts usually contain multiple terminators/polyadenylation signals, which can result in premature termination of transcripts when expression is attempted in yeast (Romanos, M.A., et al., (1995), "Expression of Cloned Genes in Yeast," DNA Cloning 2: Expression Systems,” (Glover D., et al., Eds.), Oxford Univ. Press, London).
- the synthetic gene in this study required two successive rounds of alterations before the yeast could properly produce full-length antigen.
- the expressed product (see amino acid sequence in Figure 9B) had the expected molecular weight as shown by denaturing polyacrylamide gel electrophoresis (PAGE) and Western blot analysis using antibodies directed against botulinum neurotoxin serotype F.
- the synthetic gene for BoNT serotype G fragment He has been inserted into the yeast expression vector pHJX,-D4, and integrated into the chromosome of Pichia pastoris strain GS115.
- the expressed product (see amino acid sequence in Figure 10B) had the expected molecular weight as shown by denaturing polyacrylamide gel electrophoresis (PAGE) and Western blot analysis using antibodies directed against botulinum neurotoxin serotype G.
- the expressed recombinant BoNTG (He) elicited high antibody titers as judged by the Enzyme Linked Immunosorbent Assay (ELISA) and, more importantly, these circulating serum titers protected mice from challenges with active toxin.
- ELISA Enzyme Linked Immunosorbent Assay
- CHAPS zwiterionic detergent
- the subsequent task is to separate that antigen from the myriad of pichia host proteins, lipids, and other impurities that exist in the extracted medium.
- the chemical separations it is critical that polynucleic acids be efficiently removed. Nucleotides will either bind to the C-fragment (serotypes A, E, and F due to their elevated pis) or will bind to the anion-exchange chromatography resin (as is used in the first purification step of the C ⁇ process). With either case, the chromatography is rendered futile.
- the C-fragment product will either fail to bind to the chromatography media or it will elute over an unacceptably large sodium chloride concentration range.
- Pichia cells possess an abundant amount of DNA.
- Polyethyleneimine (PEI) is a polycationic agent that readily precipitates nucleotides. When pichia cell extracts are treated with PEI, the nucleic acids are efficiently precipitated and removed by centrifugation without significant loss of product. More importantly, the chromatographic separation of C-fragments from pichia proteins are dramatically improved.
- the soluble portion of the cell lysate may typically be purified in two conventional chromatographic steps.
- the ultimate objective of this work is to obtain FDA licensure of rBoNT as a safe and effective vaccine.
- a preferred separation employs a cation-exchange step followed by hydrophobic interaction chromatography (HIC). These two steps complement each other as they provide separations based on electrostatic and hydrophobic interactions.
- the cation- exchange step was particularly efficient in increasing the purity of rBoNTF(Hc), as the antigen was estimated to be purified greater than 52-fold.
- Precipitate that results when the cation-exchange pool is treated with ammonium sulfate contains mostly pichia proteins and very little rBoNT product.
- the HIC step removes most or all of the remaining impurities.
- the yield of soluble rBoNTF(Hc) from the total recombinant yeast cell lysate was estimated to be greater than 28% with a purity greater than 98%.
- Use of similar purification steps for rBoNTA(H c ) produced greater than 95% pure material.
- rBoNTF(Hc) product (30-40%) was identified in the insoluble portion of the cell lysate. Also, the antigen was 35% of the total protein present in the pellet; in effect it was more pure than the soluble rBoNTF(Hc) was after the ion-exchange step. This suggests an alternative process whereby insoluble rBoNT product produced in yeast may be resolubilized and purified to homogeneity. The resolubilization may be performed by resuspending the pellet in urea and subsequently removing the urea by dialyses in nondenaturing buffer.
- a single chromatographic step using cation-exchange chemistry may be sufficient to purify the resolubilized antigen, in some cases to greater than 98%.
- the yield of resolubilized rBoNTF(Hc) product from the total cell lysate was estimated to be >19%.
- the overall bench scale yield of purified soluble and resolubilized rBoNTF(Hc) was estimated to be greater than 47% or 240 mg/Kg of the cell paste.
- a similar procedure would be suitable for purification of rBoNTA(H ) and other rBoNT fragment peptides from yeast.
- mice were inoculated with rBoNTF(Hc) and subsequently challenged with a high level of rBoNTF toxin
- the purified soluble rBoNTF(Hc) completely protected mice receiving three inoculations of 0.2 ⁇ g from challenge with 1000 mouse i.p.
- the ELISA is performed by coating a microtiter plate with toxin or fragment C itself, then sera from the vaccinated mice is added at various dilutions (i.e., sera diluted 1/100, 1/400, 1/1600, 1/6400, etc.). Since fragment C is sufficient to elicit protection in animals, preferably assays for neutralizing antibody titer in sera from animals vacinated with fragment C are performed using microtiter plates coated with fragment C. Antibody in the sera will bind to the toxin or the fragment C, and the bound antibody may be detected by a secondary antibody (e.g., anti-mouse IgG) that is coupled to horse-radish peroxidase or alkaline phosphatase.
- a secondary antibody e.g., anti-mouse IgG
- the secondary IgG will bind to the anti-BoNT antibody that was raised to the fragment C vaccine.
- a substrate for the peroxidase or phosphatase enzymes is added to the wells.
- the substrate will give off a color once the enzyme has cleaved the substrate, and the intensity of the color measured (e.g., at 405 nm. Typically, a reading of 0.2 is used as the base.
- a susceptible host may be immunized using the appropriate peptide vaccine formulated in adjuvant to increase the immune response.
- adjuvants include but are not limited to Freund's (complete and incomplete), mineral gels, such as aluminum hydroxide, surface active substances such as keyhole limpet hemocyanin, lysolecithin, pluronic polyols, polyanions, peptides, BCG (Bacille Calmette-Guerin), oil emulsions and dinotrophenols. Immunization can be carried out with additional various presentation and cross-linking permutations.
- such permutations include rBoNT peptides cross-linked to KLH as a carrier, any rBoNT peptide cross-linked to any other rBoNT protein as carrier, rBoNT peptides cross-linked to themselves, and these combinations presented by the various adjuvants listed above. It will become evident that such permutations are available in regard to other peptides and self-assembled peptides disclosed throughout this specification. It will also be known to one of ordinary skill in the art that use of the term
- susceptible host includes any such mammalian host susceptible to intoxication by BoNT. It will be further evident that any such susceptible host is a candidate for treatment to promote protection from BoNT utilizing the peptide vaccines and associated methods desc ⁇ bed in this specification.
- a synthetic gene encoding a putative fragment C region of botulinum neurotoxin serotype F was designed and constructed for expression in Escherichia coli (Holley et al, submitted to Vaccine)
- the recombinant BoNTF(H c ,) ⁇ , gene was expressed in E. coli as a fusion protein with maltose-bmdmg protein (MBP) with yields of 1 mg/L culture (See Figure 18)
- rBoNTF(Hc) 2 A second synthetic gene, rBoNTF(Hc) 2 , was subsequently designed to facilitate expression in P. pastoris. Redesigning the gene was intended to lower specific regions of the rBoNTF(Hc) ⁇ gene in which spikes of AT-rich tracts still remained. Previous work had shown that rare codons (Makoff, A.J., et al., (1989), "Expression of Tetanus Toxin Fragment C in E.
- Transformants expressing selectable markers were isolated and tested for their ability to express rBoNTF(H c ). Unlike the rBoNTF(H c ) ⁇ gene, rBoNTF(Hc) 2 was expressed after induction with methanol and yielded the expected molecular weight of approximately 50000 daltons as judged by SDS/PAG ⁇ and Western blot analysis ( Figure 20). The deduced molecular mass of the encoded polypeptide was 50,250 daltons.
- a stock seed culture of P pastoris was grown in shake-flasks containing 0.5 L of YNB medium (13.4 g/L yeast nitrogen base without amino acids, 20 g/L glycerol, 0.4 mg/L biotin, in 100 mM sodium phosphate, pH 6.0) Cultures were grown at 30°C until an A 600 of 20 absorbance units was achieved, and then used to inoculate a 5-L BioFlo 3000 fermentor (New Brunswick Scientific, Edison, NJ, U.S.A.) containing 2.5 L basal-salt medium plus PTM 4 trace mineral salts and 4% glycerol Dissolved oxygen was maintained at 40% and the pH was maintained at 5.0 with 30% ammonium hydroxide After the initial glycerol was consumed, 50% (w/v) glycerol was added at a rate of 20 g/L/h for 1 h then decreased linearly to 0 g/L/h over 3 h The medium was enriched with 1.5 g methanol/L of medium M
- YNB medium (13.4 g/L yeast nitrogen base without amino acids, 20 g/L glycerol, 0.4 mg/L biotin, in 100 mM sodium phosphate, pH 6.0) Cultures were grown at 30°C until an A 6 oo of 20 absorbance units was achieved, and then used to inoculate a 5-L BioFlo 3000 fermentor (New Brunswick Scientific, Edison, NJ, U.S.A.) containing 2.5 L basal-salt medium plus PTM t trace mineral salts and 4% glycerol .
- Dissolved oxygen was maintained at 40% and the pH was maintained at 5.0 with 30% ammonium hydroxide After the initial glycerol was consumed, 50% (w/v) glycerol was added at a rate of 20 g/L/h for 1 h then decreased linearly to 0 g/IJh over 3 h .
- the medium was enriched with 1.5 g methanol/L of medium Methanol feed was started at 4 g/IJh and linearly increased to 9 g/IJh over 10 h
- the methanol feed rate was adjusted by using the dissolved oxygen-spike method (Chiruvolu, 1997) After 10 h of methanol induction, the cells were harvested by centrifugation at 6000 x g for 10 min at 4 ° C with a Beckman JA-10 rotor (Beckman Instruments, Palo Alto, CA, U.S.A.) and then stored at -20°C. Cell disruption and sample preparation
- the resulting extract was noticeably turbid due to the presence of lipids and significant quantities of nucleic acids As rBoNTF(Hc) possessed a calculated isoelectric point of 9.1 and presumably interacted strongly with DNA, DNase was added to the cell extract in order to digest the polynucleotides and facilitate purification.
- the extract was treated with DNase (100 units/ml, Aldrich) and ZnCl 2 (2 mM, Aldrich) at room temperature for 30 min and then dialyzed extensively with 10 kDa molecular weight cut off (MWCO) Slide-A-Lyzer dialysis cassettes (Pierce) in 50 mM Na 2 HPO 4 /2 mM Na 2 EDTA/l mM PMSF, pH 6.8 at 4°C A precipitate developed during dialysis that was separated by centrifugation at 15000 g for 15 min at 4°C with a Sorval SS-34 rotor The clarified extract contained 7.8 mg/ml of total protein and was used as starting material for the FPLC purification of soluble rBoNTF(Hc) while the pellet was used as starting material for the resolubilized rBoNTF(Hc) purification.
- Example 4 Conventional Purification of rBoNTF(Hc) from P.
- the rBoNTF(Hc) protein was pu ⁇ fied to homogeneity using an FPLC system and two chromatographic steps. First, the mate ⁇ al was subjected to cation exchange chromatography ( Figure 21 A) FPLC purification of soluble rBoNTF(H c )
- Soluble rBoNTF(Hc) was pu ⁇ fied by using a Pharmacia model 500 FPLC system (Pharmacia, Uppsala, Sweden) with programmed elution and A 280 monito ⁇ ng
- the starting mate ⁇ al was loaded onto a Pharmacia HR 10/10 Mono S cation-exchange column equilibrated with 50 mM Na 2 HP0J2 mM Na EDTA/l mM PMSF, pH 6.8 (buffer A) at a flow rate of 2 ml/min (150 cm h)
- the column was washed with 16 ml (2 bed volumes) of buffer A Flow through and wash were collected separately and stored for subsequent analysis
- Protein was eluted from the column with a linear gradient from 0 to 300 mM NaCl over 80 ml (10 bed volumes), then a linear gradient from 300 to 1000 mM NaCl over 20 ml (2.5 bed volumes), and then an isocratic gradient at 1000 mM over 10
- HIC was used as a second chromatographic step ( Figure 21B) and separated proteins based on their differences in surface hydrophobicity. It was determined that neopentyl chemistry provided the approp ⁇ ate hydrophobic interaction with rBoNTF(Hc). The supernatant was loaded onto a Pharmacia alkyl superose 10/10 hydrophobic interaction chromatography (HIC) column equilibrated with 1.5 M (NH ) 2 SO 4 /50 mM Na 2 HPOJ2 mM Na 2 EDTA/25 mM NaCl, pH 7.5 (buffer B) at a flow rate of 1 ml/min (75 cm/h).
- HIC Pharmacia alkyl superose 10/10 hydrophobic interaction chromatography
- Total protein concentration was determined by Pierce BCA , T"M V1 assay rBoNTF(H c ) was identified by Western blot analysis and purity was estimated by analysis of individual lanes of SDS/PAGE by pixel densitometry using NIH imaging software.
- CD of purified soluble and resolubilized rBoNTF(Hc) Purified soluble and resolubilized rBoNTF(Hc) were subjected to CD spectroscopy in a Jasco 600 spectropolarimeter (Japan Spectroscopy company, Tokyo, Japan) Experiments were performed at a concentration of 30 ⁇ g/ml (0.62 ⁇ M) in a 1 cm path length cell in 10 mM Na 2 HPO , pH 7.0 Spectra were obtained as an average of four accumulations, scanned from 260-200 nm, at a scan rate of 10 nm/min, with a 2 sec response, and a 1 nm band width The temperature was maintained at 20°C with a Peltier thermocontrol device.
- the cell lysate pellet was resuspended into 20 ml of 3 M urea/50 mM Na 2 HPO 4 , pH 7.0 and extracted 15 h at 4°C on a Labquake rotator .
- the cellular components not solubilized by the denaturing buffer were removed by centrifugation with a Sorval SS-34 rotor at 15000 g for 10 min at 4°C
- the supernatant was dialyzed extensively using 10 kDa MWCO Pierce Slide-A-Lyzer dialysis cassettes in buffer A .
- a slight precipitate formed during the dialysis which was removed by centrifugation as described above Western blot analysis showed that rBoNTF(H c ) was present only in the supernatant, which was estimated to be about 35% pure by
- the rBoNTF(Hc) was greater than 98% pure as judged by SDS-PAGE.
- the total yield of purified resolubilized rBoNTF(Hc) was 100 mg/kg of cell paste.
- the conformation of purified resolubilized antigen showed significant ⁇ -sheet as determined by CD spectral analysis ( Figure 22). However, the overall fold appeared slightly different than that shown by rBoNTF(Hc) purified from the cell lysate supernatant. The primary difference was the lack of a positive peak at 233 nm, indicating differences in ⁇ -sheet. content.
- mice were inoculated with either one, two, or three doses of purified rBoNTF(H ) from the soluble fraction of lysate at doses ranging from 0.008 to 5 ⁇ g per mouse.
- mice Crl:CD-l, ICR mice (Charles River, NC, U.S.A.) weighing 16-22 g on receipt, were injected intramuscularly (i.m.) with purified rBoNTF(H c ) Mice were challenged intraperitoneally (i.p.) 21 days after their last rBoNTF(Hc) injection with BoNTF toxin complex (Langeland strain) diluted in .0.2% (w/v) gelatin/0.4% (w/v) Na 2 HPO 4 , pH 6.2, in 100 ⁇ l total volume per mouse. Groups of five naive mice were also used as toxin controls Mice were observed daily and deaths were recorded five days post challenge . All animal manipulations were in accordance with applicable regulations in AAALAC-accredited facilities.
- the efficacy of the purified soluble rBoNTF(H c ) was determined by inoculating groups of five female mice with one, two, or three doses of 0.008, 0.04, 0.2, 1.0, or 5.0 ⁇ g rBoNTF(H c ) (diluted in 100 ⁇ l of 0.2% (v/v) Alhydrogel (Superfos Biosector, Kvistgaard, Denmark) in 0.9% (w/v) saline) per mouse at 14 day intervals . Two days before challenge, mice were bled retroorbitally and serum was collected for ELISA testing Mice were challenged with 1000 mouse i.p LD 5 o of BoNTF toxin complex.
- mice All of the mice, including five na ⁇ ve controls, were challenged with 1000 mouse ip LD 50 of BoNTF toxin. The controls all died within 2-4 h. A dose response was observed from groups of mice receiving different numbers of inoculations (Table 3). A single inoculation of 5 ⁇ g protected four of five mice, while a dose of 0.2 ⁇ g or below protected one or no mice. Two and three inoculations protected four of five and five of five mice at doses of 0.2 and 0.04 ⁇ g, respectively. At all dose levels studied, the number of surviving mice increases with the number of inoculations. Serum antibody titers for each individual mouse were determined by ELISA, followed by calculation of the geometric mean titers for each group in the study.
- Botulinum neurotoxin serotype F (Langeland strain, Food Research Institute, University of Wisconsin, Madison, Wl, U.S.A.) was used as the coating antigen and the positive control for each assay was a mouse IgG monoclonal antibody, 7F8.G2.H3 (Brown, D.R., et al., (1997), "Identification and Characterization of a Neutralizing Monoclonal Antibody against Botulinum Neurotoxin, Serotype F, Following Vaccination with Active Toxin," Hybridoma, 16:447-456).
- the logistic regression model was used to test associations of geometric mean ELISA titers and individual titers with survival by using SAS, version 6.10. Geometric mean titers correlated well with protection (Table 3). The three groups with no survivors had geometric means titers below the detection limit of the assay (1.4). Similarly, the four groups that showed complete protection had geometric means titers of 2.8 or greater. Individual mouse antibody titers correlated extremely well with protection (Table 4). Only 7 out of 38 mice survived whose titers were below 100. On the other hand, 34 out of 34 survived whose titers were 100 or greater.
- mice in the study could be classified as a "nonresponder.”
- the mouse, receiving two injections at the highest dose level had an antibody titer below the detection limit and did not survive the BoNTF challenge.
- the rest of the mice in that particular group had titers of 1600 or greater.
- Serum was bled from each mouse individually Titer is reciprocal of the highest dilution having an OD 05 greater than 0.2 AU after correcting for background .
- Mice were challenged with 1000 i.p LDso BoNTF toxin 21 days after last inoculation.
- mice were not measured . Two mice did not offer enough serum and one mouse was not challenged.
- the resolubilized antigen was also evaluated for immunogencity and protective efficacy by its ability to protect mice from a BoNTF toxin challenge.
- Groups of 10 male mice each received three inoculations of either 1 ⁇ g or 5 ⁇ g of rBoNTF(Hc) (diluted in 100 ⁇ l 0.2% (v/v) Alhydrogel in 0.9% (w/v) saline) per mouse at 14 day intervals . Two days before challenge, mice were bled retroorbitally and serum was collected for ELISA testing.
- mice inoculated with 1 ⁇ g doses were challenged with 5000 mouse ip LD 50 of BoNTF toxin. Ten of ten mice survived the challenge. Because 100% protection was observed with the group inoculated with 1 ⁇ g doses, the group that received three doses of 5 ⁇ g were subjected to a challenged level two orders of magnitude greater in order to test the limits of the antigen. Therefore, the 5 ⁇ g dose group was challenged with 500,000 mouse ip LD 5 o of BoNTF toxin. None of the mice survived the challenge; however, a significant delay in time to death was observed (24-48 h). All the control mice succumbed within 2-4 h after challenge.
- GIBCO BRL Restriction endonucleases and DNA modifying enzymes were obtained from GIBCO BRL (Gaithersburg, Maryland).
- Polymerase chain reaction (PCR) reagents were purchased from Perkin-Elmer Cetus (Norwalk, CT). SDS PAGE precast gels and running buffers were acquired from Amersham (Arlington Heights, Illinois). All oligonucleotides were synthesized by Macromolectular Resources (Ft. Collins, Colorado).
- ELISA reagents were obtained in house or from Sigma (St. Louis, Missouri) or Kirkegard and Perry Laboratories (Gaithersburg, Maryland). The Escherichia coli host was K12DH5a, purchased as competent cells from GIBCO BRL.
- Oligonucleotide primers incorporating appropriate terminal restriction enzyme sites were used to PCR amplify the He region of the C. botulinum clone pCBA3.
- Gel-purified insert DNA and vector DNA were cleaved with the appropriate restriction enzymes, purified on low melting point agarose, and ligated overnight at room temperature.
- Competent DH5a host cells were transformed according to suppliers recommendations and plated on LB plates with 100 ug/ml ampicillin. Protein electrophoresis was run on precast 11-20% SDS PAGE at the manufacturer's recommended parameters.
- ELISA plates were incubated with capture antibody (horse an ti -botulinum A polyclonal serum) overnight, then blocked with skim milk prior to application of various dilutions of test material, signal antibody (rabbit antibotulinum A polyclonal serum), signal HRP conjugated anti-(rabbit IgG) and ABTS substrate solution. Plates were read on an automated reader at 405 nm.
- the C fragment protein sequence was reverse translated using E. coli optimal codon usage. The gene was then altered in many places to insert restriction sites, start codon, stop codon. Other changes were also effected to make the molecule more appropriate for use in the vector. Throughout, the fidelity of the protein sequence generated therefrom was maintained.
- This gene has been synthesized using a large number of oligomers of approximately 60-65 bases corresponding to the sequences of the + and - strands.
- the oligomers had overlaps of 7 bases.
- the oligomers were allowed to anneal and were ligated to form 5 subunits of 250-300 base pairs each. Each subunit had been designed to have restriction sites at their termini which allowed them to be assembled in the right order to form the complete gene. On confirmation there was shown that the correct gene had 7 deletion errors. These errors were repaired using in vitro mutagenesis and the repair sites sequenced to confirm.
- the C fragment for botulism toxin serotype B of Whelan was studied and the portion of the protein having the sequence
- the synthetic gene for expression in E. coli was produced in the manner described for synthesis of the gene for the C fragment of the A strand, namely, using a large number of oligomers of approximately 60-65 bases corresponding to the sequences of the _+ and - strands with overlaps of 7 bases.
- the oligomers were allowed to anneal and were ligated to form subunits of 250-300 base pairs each. Each subunit had been designed to have restriction sites at their termini which allowed them to be assembled in the right order to form the complete gene, the synthetic gene for encoding the c fragment of the B toxin was as follows:
- BoNTA(Hc) peptide was produced recombinantly in yeast.
- the first step in the purification process for BoNTA(Hc) was a Streamline expanded bed chromatography column.
- the product was eluted by a sodium chloride step gradient.
- Product eluted from the expanded bed chromatography column was estimated to be 10% pure with a total protein concentration of 0.92 mg/ml.
- After dialyzing the salt away, the material was loaded onto a mono S cation exchange column for further purification.
- Western blot and ELISA data indicated that BoNTA(H c ) eluted from the column at 110 mM sodium chloride.
- the Mono S pool was subjected to HIC as a final purification step and thus, the material was adjusted to 1.5 M ammonium sulfate.
- the Mono S product was loaded onto a HIC column and eluted with a gradient of decreasing ammonium sulfate.
- Product eluted at 1.04 M ammonium sulfate and BoNTA(Hc) immunologically positive fractions were combined and dialyzed to remove ammonium sulfate. Only a 50 kDa BoNTA(Hc) band was detected by SDS-PAGE and Western blot analysis and was judged to be greater than 95% pure after the final step.
- Example 11 rBoNTB(Hc) purification and protective effect Recombinant BoNTC(Hc) peptide was produced recombinantly in yeast.
- the first separation technique employed for the purification process for BoNTB(Hc) was Streamline chromatography (Pharmacia), which is a single pass expanded bed adsorption operation where proteins can be recovered from crude feed stock or cell lysate without prior clarification. Significant clean-up was accomplished in this step as the MES buffer system prohibited binding of a large percentage of unwanted proteins to the SP resin.
- Protein was loaded onto the column at a concentration of 123 mg/mL-resin, using 20 mM MES buffer, pH 5.7 with 10 mM NaCl. The product pool was eluted in a single step. Under the conditions investigated, on average 3.9% of the total protein loaded was recovered in the elution peak, and the product pool was approximately 70% BoNTB(Hc) fragment based on SDS-PAGE.
- the second chromatography step in the process utilizes Poros HS, another strong cation exchange resin.
- the buffer system was similar to that used for Streamline SP, however enhanced selectivity of Poros HS enriched the product peak to about 85% purity.
- the product peak eluted during the gradient at approximately 130 mM NaCl. Strongly bound proteins were eluted with 1 M NaCl.
- the final chromatography step utilized a Poros PI column. Analysis of the PI fractions by SDS-PAGE and IEF revealed that the product band, a single band at 50 kD on SDS-PAGE, was present in the pH 8.0 fraction. Analysis of purified BoNTB(H c ) fragment by 2-D electrophoresis resulted in one major spot and two minor, faint spots from the Pi-peak 1 fraction. Peak 2 contained several spots at two different molecular weights corresponding to 50 kD and 47 kD. Presumably these spots represent different isoforms. IEF banding patterns detected in the first dimension are in agreement with those seen in Phast IEF for the two peaks. The protective efficacy of this material was determined by potency assay of 1 dose followed by challenge with 1000 LD50 of BoNTB(Hc). The results are shown in the following Table 7.
- BoNTC(Hc) peptide was produced recombinantly in yeast.
- the initial chromatography step used for the purification process for BoNTC ⁇ Hc) was a Mono Q anion-exchange column.
- the column was equilibrated with 50 mM sodium phosphate, 0.2% (W/V) CHAPS, 2 mM EDTA, pH 7.0.
- the CHAPS was incorporated into the column buffers to allow product to elute from the column over a narrower sodium chloride concentration.
- Fractions positive for BoNTC ⁇ Hc) by Western analysis were pooled and adjusted to 1 M ammonium sulfate. A moderate precipitate formed which was removed by passing the material through a 0.2 ⁇ filtration unit.
- the clarified Mono Q product pool was subjected to hydrophobic interaction chromatography using a Pharmacia alkyl superose column. This final step removed the remainder of the impurities liberating BoNTC ⁇ (Hc) product which was estimated to be greater than 98% pure as judged by SDS/PAGE. Protective effect of this purified material was measured by immunizing mice with 1 dose followed by challenge with 1000 LD50 of BoNTC ⁇ (H c ). The results are shown in Table 8 below.
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AU50035/00A AU783450B2 (en) | 1999-05-12 | 2000-05-12 | Recombinant vaccine against botulinum neurotoxin |
EP00932296A EP1180999A4 (en) | 1999-05-12 | 2000-05-12 | Recombinant vaccine against botulinum neurotoxin |
CA002371279A CA2371279A1 (en) | 1999-05-12 | 2000-05-12 | Recombinant vaccine against botulinum neurotoxin |
JP2000616731A JP2004512004A (en) | 1999-05-12 | 2000-05-12 | Recombinant vaccine against botulinum neurotoxin |
US09/611,419 US7214787B1 (en) | 1993-09-21 | 2000-07-06 | Recombinant vaccine against botulinum neurotoxin |
US09/910,186 US7081529B2 (en) | 1993-09-21 | 2001-07-20 | Recombinant vaccine against botulinum neurotoxin |
US11/437,212 US7786285B2 (en) | 1993-09-21 | 2006-05-19 | Recombinant vaccine against botulinum neurotoxin |
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US13386899P | 1999-05-12 | 1999-05-12 | |
US13386799P | 1999-05-12 | 1999-05-12 | |
US13386599P | 1999-05-12 | 1999-05-12 | |
US60/133,873 | 1999-05-12 | ||
US60/133,867 | 1999-05-12 | ||
US60/133,865 | 1999-05-12 | ||
US60/133,869 | 1999-05-12 | ||
US60/133,868 | 1999-05-12 | ||
US60/133,866 | 1999-05-12 | ||
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JP (1) | JP2004512004A (en) |
AU (1) | AU783450B2 (en) |
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Cited By (11)
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---|---|---|---|---|
WO2003012117A1 (en) * | 2001-07-28 | 2003-02-13 | The Secretary Of State For Defence | Dna vaccine |
US7037680B2 (en) * | 1993-09-21 | 2006-05-02 | The United States Of America As Represented By The Secretary Of The Army | Recombinant light chains of botulinum neurotoxins and light chain fusion proteins for use in research and clinical therapy |
FR2889066A1 (en) * | 2005-07-28 | 2007-02-02 | Centre Nat Rech Scient | METHOD OF GENETIC IMMUNIZATION BY ELECTROTRANSFER AGAINST TOXIN AND ANTISERUM THAT CAN BE OBTAINED BY THIS PROCESS |
US7227010B2 (en) * | 1993-09-21 | 2007-06-05 | United States Of America As Represented By The Secretary Of The Army | Recombinant light chains of botulinum neurotoxins and light chain fusion proteins for use in research and clinical therapy |
JP2008508886A (en) * | 2004-08-04 | 2008-03-27 | アラーガン、インコーポレイテッド | Optimization of expression of active botulinum toxin type A |
US7825233B2 (en) | 2004-06-30 | 2010-11-02 | Allergan, Inc. | Optimizing expression of active Botulinum Toxin type E |
WO2015004461A1 (en) * | 2013-07-09 | 2015-01-15 | Syntaxin Limited | Cationic neurotoxins |
WO2016110662A1 (en) * | 2015-01-09 | 2016-07-14 | Ipsen Bioinnovation Limited | Cationic neurotoxins |
JP2018515542A (en) * | 2015-05-15 | 2018-06-14 | ボード オブ リージェンツ オブ ザ ユニバーシティ オブ ネブラスカ | Genetically engineered Clostridium botulinum toxin for molecular delivery to selected cells |
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JP2009106163A (en) * | 2007-10-26 | 2009-05-21 | Kyushu Univ | Nucleic acid sequence, vector, transformant, production method, and nucleic acid sequence primer |
PT2491384E (en) | 2009-10-21 | 2014-02-21 | Merz Pharma Gmbh & Co Kgaa | System for determining unprocessed and partially processed neurotoxin type a |
GB201407525D0 (en) * | 2014-04-29 | 2014-06-11 | Syntaxin Ltd | Manufacture of recombinant clostridium botulinum neurotoxins |
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GB9511909D0 (en) * | 1995-06-12 | 1995-08-09 | Microbiological Res Authority | Vaccine |
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2000
- 2000-05-12 WO PCT/US2000/012890 patent/WO2000067700A2/en active IP Right Grant
- 2000-05-12 CA CA002371279A patent/CA2371279A1/en not_active Abandoned
- 2000-05-12 AU AU50035/00A patent/AU783450B2/en not_active Ceased
- 2000-05-12 JP JP2000616731A patent/JP2004512004A/en active Pending
- 2000-05-12 EP EP00932296A patent/EP1180999A4/en not_active Ceased
Non-Patent Citations (4)
Title |
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CAMPBELL K. ET AL.: 'Gene probes for identification of the botulinal neurotoxin gene and specific identification of neurotoxin types B, E and F, J' J. CLIN. MICROBIOLOGY vol. 31, no. 9, September 1993, pages 2255 - 2262, XP002934084 * |
LI L.: 'In vitro translation of type A clostridium botulinum neurotoxin heavy chain and analysis of its binding to rat synaptosomes' J. PROTEIN CHEMISTRY vol. 18, no. 1, January 1999, pages 89 - 95, XP002934086 * |
POTTER K. ET AL.: 'Production and purification of the heavy-chain fragment C of botulinum neurotoxin, serotype B, expressed in the methylotrophic yeast pichia pastoris' PROTEIN EXPRESSION AND PURIFICATION vol. 13, no. 3, 1998, pages 357 - 365, XP002934085 * |
See also references of EP1180999A2 * |
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AU5003500A (en) | 2000-11-21 |
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