# Patent

## Title
Subtype of Closteridium botulinum neurotoxin type A and uses thereof

## Abstract

A novel subtype of type A botulinum neurotoxin (BoNT/A) is disclosed in the application. Methods to purify the neurotoxin as well as uses thereof are also disclosed.

## Background
CROSS-REFERENCE TO RELATED APPLICATION
This application claims priority from U.S. provisional patent application Ser. No. 61/174,331 filed Apr. 30, 2009, which is incorporated by reference herein.


STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
This invention was made with United States government support awarded by the following agencies: NIH AI065359. The United States has certain rights in this invention.


BACKGROUND OF THE INVENTION
Neurotoxigenic strains of Clostridium butyricum and Clostridium baratii produce botulinum neurotoxins (BoNTs), which are the most potent neurotoxins known. BoNTs are characterized as Category A Select Agents and are considered potential bioterrorism threats (Arnon, S. S. et al. 2001, Jama 285:1059-1070).
BoNT is synthesized as a single chain polypeptide (molecular weight of 150 kDa) with relatively little toxic potency. It becomes toxic upon cleavage by trypsin or bacterial enzymes into a heavy chain (100 kDa) and a light chain (50 kDa). Three dimensional structure shows that BoNTs contain a receptor-binding domain located in the C-terminal region of the heavy chain, a catalytic domain (the light chain) with endopeptidase activity on neuronal substrates and a translocation domain located in the N-terminal region of the heavy chain.
BoNTs can be immunologically distinguished using homologous antitoxins into seven serotypes, designated A-G. Different serotypes of BoNTs have regions of homology, particularly in the residues defining the catalytic active site, in the translocation domain, and in the two cysteine residues forming the disulfide bond connecting the heavy chain and the light chain. The least degree of homology is in the carboxyl region of the heavy chain, which is involved in neurospecific binding. Indeed, among these serotype distinctions there is considerable genetic variation, as demonstrated by the recognition of at least 24 subtypes (Carter, A. T., et al., 2009, BMC Genomics 10:115; Dover, N., et al., 2009, J. Clin. Microbiol. 47:2349-2350; Hill, K. K. et al. 2007, J. Bacteriol 189:818-832; Smith, T. J. et al. 2005, Infect. Immun. 73:5450-5457). These subtypes have been distinguished based on their degree of genetic variation with subtypes having a minimum of 2.6% divergence on the amino acid level (Webb, R. P., et al., 2009, Vaccine 27:4490-4497). These subtypes can also be distinguished by mouse bioassay, as a new subtype is resistant to neutralization by antibodies raised against known subtypes.
Despite of the variations, different serotypes of BoNTs act through a similar mechanism: by inhibiting the release of acetylcholine, a neurotransmitter, from the presynaptic nerve terminal and thus causing local chemodenervation. The action of BoNTs involves a four step process: (1) high affinity, serotype specific binding by the heavy chains to receptors on presynaptic membrane of cholinergic nerve endings; (2) receptor mediated, energy dependent internalization of the complex; (3) translocation from the acidic endosome to the cytosol; and (4) enzymatic cleavage, by the light chain, of specific proteins that are critical for fusion of the presynaptic acetylcholine vesicle with the presynaptic membrane, thus preventing release of acetylcholine into the synapse.
BoNT/A is of particular importance and interest since it is the most significant threat in bioterrorism and has been increasingly used as a pharmaceutical modality (Aoki, K. R. 2003, Clin. Dermatol. 21:476-480; Delgado, M. R. 2003, J. Am. Acad. Orthop. Surg. 11:291-294). Thus far, four distinct subtypes of BoNT type A, i.e., BoNT/A1, BoNT/A2, BoNT/A3 and BoNT/A4, have been identified in this manner (Arndt, J. W. et al. 2006, J. Mol. Biol. 362:733-742; Smith, T. J. et al. 2007, PLoS ONE 2:e1271).
The success of BoNTs as a therapeutic derives from certain important attributes of the toxin: (a) exceptionally specific binding to the presynaptic membrane of cholinergic terminals; (b) extremely high potency; (c) remarkable specificity for catalytic cleavage of proteins involved in neurotransmitter trafficking and exocytosis; (d) minimal spread from the injection site; (e) limited and mild adverse effects, and (f) extraordinary long duration of action. Table 1 summarizes the clinical applications of BoNTs.
Although each injection of BoNT has a long duration of effective action, repeated injection is necessary at about three month intervals because although the affected nerve terminals are no longer capable of neurotransmitter exocytosis, newly formed sprouts do release acetylcholine and form a functional synapse. As a result, after about three months, the original terminal resumes exocytosis and the sprouts regress to return the neuromuscular junction to its original state.







TABLE 1





Clinical applications of botulinum toxin















Dystonia


Blepharospasm and lid apraxia


Oromandibular-facial-lingual dystonia


Cervical dystonia (torticollis)


Laryngeal dystonia (spasmodic dysphonia)


Limb dystonia


Task specific dystonia (eg, writer's or other occupational cramps)


Other focal/segmental dystonias (primary, secondary)


Other involuntary movements


Hemifacial spasm


Limb, head, voice, chin tremor


Palatal myoclonus


Motor and phonic tics (including coprolalia)


Nystagmus and oscillopsia


Myokymia


Inappropriate muscle contractions


Spasticity (stroke, cerebral palsy, head injury, multiple sclerosis)


Painful rigidity


Strabismus


Bruxism and temporo-mandibular joint syndrome


Stuttering


Chronic tension (muscle contraction) headaches


Lumbosacral strain and back spasms


Radiculopathy with secondary muscle spasm


Myofascial pain syndromes


Achalasia (lower oesophageal sphincter spasm)


Spasm of the inferior constrictor of the pharynx


Spasm of the sphincter of Oddi


Spastic bladder, detrusor sphincter dyssynergia


Anismus


Vaginismus


Other applications


Protective ptosis


Hyperlachrymation


Drooling (sialorrhoea)


Hyperhidrosis


Gustatory sweating


Anal fissure


Constipation


Obesity (distal stomach)


Cosmetic (wrinkles, brow furrows, frown lines, “crow's feet”, platysma 


lines, facial asymmetry)


Tennis elbow and other sports injuries









A growing impediment in BoNT administration is the development in patients of antibodies that react with and neutralize the toxin, thereby eliminating the effectiveness of the toxin for medicinal and cosmetic purposes. As a result, some patients become unresponsive to subsequent repeated treatments. Studies have shown that the heavy chain of the protein, used for substrate binding, is the primary portion against which humans develop antibodies against BoNT/A1, the predominat form of botulinum toxin used clinically.
The observation that BoNTs are chimeric molecules comprised of distinct protein domains suggests that designed chimeric neurotoxin could be constructed with enhanced or distinct therapeutic utility.
Needed in the art are novel subtypes of BoNTs which are not easily neutralized by existing antibodies in patients and thus possess distinct therapeutic utility, as well as novel subtypes of BoNTs which cause no or less development of antibodies in patients.
SUMMARY OF THE INVENTION
We disclose here the identification, purficiation and characterization of a novel isoform of Clostridium botulinum type A botulinum neurotoxin (BoNT/A), BoNAT/A5, in Clostridium botulinum strain A661222.
In one embodiment, the present invention is an isolated nucleotide encoding BoNT/A5. Preferably, the nucleotide sequence is SEQ ID NO:1.
In another embodiment, the present invention is a substantially purified BoNT/A5 protein. Preferably, the protein is encoded by the nucleotide comprising SEQ ID NO:1. More preferably, the protein is at least 90% pure. Most preferably, the protein is at least 95% pure.
In another embodiment, the present invention is a method of purifying BoNT/A5 complex comprising the steps of inoculating appropriate C. botulinum strain culture capable of producing BoNT/A5 complex, subjecting the C. botulinum culture to acid precipitation, and isolating BoNT/A5 complex by chromatography. Preferably, the chromatography is at pH5.5 using a DEAE-Sephadex A-50 column. Preferably, the method further comprises the step of separation of a 150 kDa toxin from complexing proteins by anion exchange chromatography and a final purification of the 150 kDa toxin by cation exchange chromatography.
In yet another embodiment, the present invention is a BoNT/A5 complex purified by the method described above.
In another embodiment, the present invention is a method of obtaining an antibody specific to BoNT/A5. In one embodiment, the method comprises the steps of obtaining purified BoNT/A5 either in its complete form or its separate domain parts, inactivating the purified BoNT/A5, administering the inactivated BoNT/A5 to a mammalian host in a standard process and obtaining an antibody. Preferably, the inactivation of the purified BoNT/A5 is via heating the purified BoNT/A5 or via formaldehyde treatment.
In one preferred embodiment, the antibody is a monoclonal antibody. Preferably, the monoclonal antibody is obtained via the method described above, wherein the monoclonal antibody is obtained via generating hybridomas from the fusion of spleen cells, which are from the mammalian host administered with the inactivated BoNT/A5, with myeloma cells from the same mammalian species and harvesting monoclonal antibodies from the hybridomas. Preferably, the mammalian host is a mouse.
In another preferred embodiment, the antibody is a polyclonal antibody. Preferably, the polyclonal antibody is obtained via the method described above, wherein the administration of inactivated BoNT/A5 to the mammalian host is followed by booster injections to increase antibody yield against inherent epitopes, collecting serum from the mammalian host, and purifying antibody specific to BoNT/A5 by affinity chromatography. Preferably, the method further comprises the step of testing the purified antibody for specificity to BoNT/A5.
In one embodiment, the present invention is an antibody obtained by the method described above.
In another embodiment, the present invention is a method of treating a patient in need of botulinum toxin therapy comprising the step of supplying substantially purified BoNT/A5 complex or neurotoxin to the patient.
In yet another embodiment, the present invention is a method of improving the medicinal use of BoNTs, comprising the step of substituting BoNT/A5 for BoNT/A1 for patients refractive to treatment with BoNT/A1.
Other embodiments, features and advantages of the present invention will become apparent on review of the specification, claims and drawings.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 A-B show graphical representations of degree of relatedness among the amino acid sequences of the BoNT/A1-5 subtypes. Panel A shows comparison of the entire BoNT among all 5 C. botulinum type A subtypes. Panel B shows comparison of the heavy chain of toxins of all 5 C. botulinum type A subtypes. A1 is from the ATCC 3502 strain, A2 is from the Kyoto F strain, A3 from the CDC/A3 strain, A4 from the 657Ba strain and the BoNT/A5 sequence was derived from the strain listed in the figure.
FIG. 2 shows 3D model of the new BoNT/A5 subtype. The varied residues are shown as sticks with black color. Overall structure is displayed as ribbon diagram with a grey Cα.
FIG. 3 is ribbon diagrams of the important epitopes area. (A) The varied residues are shown as sticks with black color. (B) This view localizes the area in the overall structure of the protein.
FIG. 4 shows close-up view of sequence variability between H1064 of BoNT/A1 (A), modeled R1064 of BoNT/A2 (B) and Q1064 of BoNT/A5 (C) in complex with CR1 (VL in dark grey and positioned at the upper part of each panel, and VH in light grey and positioned at the lower left part of each panel). BoNT/A1, BoNT/A2 and BoNT/A5 are showed at the lower right part of each panel.
FIG. 5 is a representation of BoNT/A5 LC in complex with SNAP-25 (arrows). The three mutated amino acids in BoNT/A5 versus BoNT/A1 are shown as sticks with black color. SNAP-25 is the stick pointed out by the arrow in panel A and the Cα pointed out by the arrow in panel B.
FIG. 6 is a view of the BoNT/A5 active site in complex with the N-Ac-CRATKML (black sticks pointed out by the block arrow) and the two closer mutated amino acids (A27 and D171) are shown as sticks with black color.
FIG. 7 shows BoNT/A1 in complex with GT1b (PDB: 2VU9). GT1b is represented as sticks. BoNT/A1 is represented as sticks with Cα. Leu 1278 and Ala 1259 that correspond to Phe 1278 and Asp 1259 in BoNT/A5 are marked out.
FIG. 8 shows coomassie blue stained SDS-PAGE gel of purified BoNT/A5 under reducing and non-reducing condition. NR: non-reducing, R: reducing, M: Marker, HC: heavy chain, LC: light chain.
FIG. 9 shows nucleotide sequence encoding BoNAT/A5 from Clostridium Botulinum strain A661222.
FIG. 10 shows amino acid sequence of BoNT/A5 from Clostridium Botulinum strain A661222.
FIG. 11 shows genomic DNA sequence of BoNT/A5 neurotoxin cluster with HA genes from Clostridium Botulinum strain A661222.
FIG. 12 shows nucleotide sequence encoding HA70 from Clostridium Botulinum strain A661222.
FIG. 13 shows amino acid sequence of HA70 from Clostridium Botulinum strain A661222.
FIG. 14 shows nucleotide sequence encoding HA17 from Clostridium Botulinum strain A661222.
FIG. 15 shows amino acid sequence of HA17 from Clostridium Botulinum strain A661222.
FIG. 16 shows nucleotide sequence encoding HA33 from Clostridium Botulinum strain A661222.
FIG. 17 shows amino acid sequence of HA33 from Clostridium Botulinum strain A661222.
FIG. 18 shows nucleotide sequence encoding botR from Clostridium Botulinum strain A661222.
FIG. 19 shows amino acid sequence of botR from Clostridium Botulinum strain A661222.
FIG. 20 shows nucleotide sequence encoding NTNH from Clostridium Botulinum strain A661222.
FIG. 21 shows amino acid sequence of NTNH from Clostridium Botulinum strain A661222.



DESCRIPTION OF THE INVENTION
In General
The present invention relates to a novel subtype of type A botulinum neurotoxin (BoNT/A) and provides methods to purify the neurotoxin as well as uses thereof.
During the past few years, scientists have engaged in the identification and study of novel C. botulinum BoNT/A subtypes. A new subtype of BoNT/A was identified and named BoNT/A5 with five strains containing the neurotoxin (Carter, A. T., et al., 2009, BMC Genomics 10:115; Dover, N., et al., 2009, J. Clin. Microbiol. 47:2349-2350). Among these five strains, four have neurotoxin sequences that are identical and the fifth is 99.8% identical to the others on the amino acid level. The subtype features both a high degree of similarity to BoNT/A1 and a HA type gene cluster which is present in only BoNT/A1 clusters and none of the other BoNT/A subtypes. The Johnson laboratory identified one of the A5 strains, a strain of C. botulinum, A661222 (Jacobson, M. J. et al. 2008, Microbiology. 154(Pt 8):2408-2415, and U.S. provisional patent application 61/174,331, both of which are incorporated by reference herein).
The identification of A661222 is described in detail in Jacobson, M. J. et al. 2008. Briefly, a broad range of BoNT/A-producing bacteria were analyzed using Multi Locus Sequence Typing (MLST) followed by sequencing the bont/a and its associated neurotoxin cluster genes in strains demonstrating a unique MLST ST profile. The A661222 strain demonstrated a ST profile which placed it similar to the reference strain (ATCC 3502) but still retained significant divergence from the reference strain in a manner similar to strains known to possess a unique BoNT/A subtype. This led to the belief that the A661222 strain might possess a novel BoNT/A subtype.
Indeed, as described below in the Examples, we discovered a novel form of the C. botulinum encoded type A botulinum neurotoxin (BoNT/A) and identified it in the A661222 strain. Briefly, the neurotoxin gene and its associated genes were completely sequenced and analyzed on both the nucleotide and amino acid level. Thirty six amino acid differences were observed between BoNT/A1 and BoNT/A5, with most of the differences in the heavy chain. (Table 2, below, summarizes the changes.) 3-D molecular modeling was performed comparing this form of BoNT/A5 with the established BoNT/A1 subtype. These modeling studies on BoNT/A focused on determining if amino acid differences observed in BoNT/A5 would have an affect on known antibody epitope sites. The BoNT/A5 protein was then purified from culture and its toxicity was determined. The ability of BoNT/A1 specific antibodies to neutralize BoNT/A5 was also tested by mouse bioassay.
BoNT/A5 offers the advantages of possessing unique epitopes in the heavy chain, which are likely to affect the ability of antibodies capable of neutralizing BoNT/A1 to neutralize BoNT/A5. Thus, it may have significant advantages over existing commercial botulinum toxins used medicinally, particularly for patients that have immunity to BoNT/A1, which is a major limitation of current technology.
In one embodiment, the present invention is a substantially purified nucleotide sequence encoding BoNT/A5. Preferably, the sequence is SEQ ID NO:1 or substantially identical to SEQ ID NO:1, preferably 99% identical.
In another embodiment, the present invention is a substantially purified BoNT/A5 protein comprising SEQ ID NO:2, preferably at a purity rate of at least 95%, preferably encoded by the nucleotide sequence comprising SEQ ID NO: 1 or a nucleotide sequence substantially identical to SEQ ID NO:1.
In another embodiment, the present invention is a method of purifying BoNT/A5 complex and toxin. A preferable method comprises inoculating a starting culture with A661222 overnight and then using that culture to inoculate a carboy of media. Acid precipitation of a 96 hour culture would then be performed. Extraction of crude toxin complex from acid precipitate would then be performed. Toxin complex would be isolated from crude extract by chromatography at pH 5.5 using a DEAE-Sephadex A-50 column. 150 kDa toxin would typically be separated from complexing proteins by anion exchange chromatography on a DEAE-Sephadex A-50 column at pH 7.9. The final purification of the 150 kDa toxin would typically be via cation exchange chromatography on a SP sephadex column at pH 7.0.
In another embodiment, the present invention is a method of using the substantially purified BoNT/A5 complex. In one embodiment, a prophetic method comprises supplying BoNT/A5 to a patient in need of botulinum toxin therapy (refering to Table 1). Given that the complex is quite similar to the BoNT/A1 complex, BoNT/A5 is likely to be used as an alternative to BoNT/A1 for medicinal and cosmetic purposes but is likely to exert effects faster than BoNT/A1. Typically, one would use less than 100 LD50 of BoNT/A5 in one treatment.
In another embodiment, the present invention is a method of improving the medicinal use of BoNTs, comprising the steps of substituting BoNT/A5 for another BoNT/A subtype.
It is also envisioned that the present invention is a method of obtaining an antibody specific to BoNT/A5. Methods of producing antibodies is well known in the art. One could obtain either monoclonal or polyclonal antibodies specific to BoNT/A5. Typically, one would obtain purified BoNT/A5 either in its complete form or its separate domain parts, inactivate the purified BoNT/A5, administer the inactivated BoNT/A5 to a mammalian host in a standard process and obtain an antibody.
If monoclonal antibodies are needed, one would typically generate hybridomas by fusing spleen cells, which are from the mammalian host administered with the inactivated BoNT/A5, with myeloma cells from the same mammalian species and harvest monoclonal antibodies from the hybridomas. One would typically use mice as the mammalian host, although other mammals, such as rabbits, can also be used.
If polyclonal antibodies are needed, one would typically increase antibody yield against inherent epitopes by administering booster injections to the mammalian host receiving the inactivated BoNT/A5. One would then collect serum from the mammalian host and purify antibody specific to BoNT/A5 by affinity chromatography. Preferably, one would test the purified antibody for specificity to BoNT/A5. The knowledge to select a mammalian host to produce polyclonal antibodies is well known in the art. Mammals can be used as mammalian host include rabbits, mice, chickens, goats, guinea pigs, hamsters, horses rats, sheep and donkeys. Preferably, rabbits are used.
In one embodiment, the present invention is an antibody obtained by the method described above.
The present invention has been described above with respect to its preferred embodiments. Other forms of this concept are also intended to be within the scope of the claims.
EXAMPLES
Example 1
Results
Identification and Sequencing of the Neurotoxin Gene and its Associated Cluster Genes
PCR and sequencing reactions were performed on the neurotoxin and the associated neurotoxin cluster genes of C. botulinum A661222, and the results were compiled using the VectorNTI Suite Program. These studies involved a process of amplifying overlapping pieces of the neurotoxin and its associated cluster. Based on this work, it was determined that the A661222 strain contained only one neurotoxin gene cluster consisting of a complete HA cluster with ha70, ha17, ha33, botR, ntnh and bont/a. This arrangement is consistent with the cluster arrangement identified in other BoNT/A5 producing strains (Carter, A. T., et al., 2009, BMC Genomics 10:115; Dover, N., et al., 2009, J. Clin. Microbiol. 47:2349-2350).
Comparison of the Neurotoxin and Associated HA Cluster Proteins Between Strain A661222 and Two Other A5 Strains IBCA94-0216 and H04402 065
When the neurotoxin gene cluster of strain A661222 was compared to that of the A5 strains IBCA94-0216 and H04402 065, it was observed that the neurotoxin cluster genes and neurotoxin gene from A661222 strain were identical to those from the IBCA94-0216 strain (Dover, N., et al., 2009, J. Clin. Microbiol. 47:2349-2350). But there was 1% difference between A661222 strain and H04402 065 strain (Carter, A. T., et al., 2009, BMC Genomics 10:115) for all the genes except the ha17 and botR genes which were identical on the nucleotide level. On the amino acid level, the neurotoxin protein for the two strains was 99.8% similar and identical. HA70 was 99.2% similar and identical and HA33 was 99.7% similar and identical between the two strains. The NTNH proteins of the two strains were 99.9% similar and identical. Sequence comparisons of the BoNT/As demonstrated the high degree of homology among the BoNT/A5 strains as they grouped together and were clearly separated from the other Type A subtypes for both the entire length of the protein and the heavy chain portion (FIGS. 1A and 1B).
Comparison of the Neurotoxin and Associated HA Cluster Proteins Between Strain A661222 and the C. botulinum A1 Strain ATCC 3502
Analysis of the bont/a gene sequences of C. botulinum strains A661222 and the A1 strain ATCC 3502 demonstrated significant homology with the amino acid sequences having 97.1% and 97.9% identity and similarity respectively. There were only thirty-six amino acid differences between the two strains and the differences are mainly located in the heavy chain of the toxin, which were spread between the translocation domain and the binding domain (FIG. 2). Only four differences were located in the light chain (LC) (Table 2). This high degree of homology made it possible to generate a model for the BoNT/A5 subtype based on already known BoNT/A structures (Lacy, D. B., et al., 1998, Nat. Struct. Biol. 5:898-902; Stenmark, P., et al., 2008, PLoS Pathog. 2008, 4(8):e1000129).
The HA cluster genes demonstrated high homology between ATCC 3502 and A661222 (Table 3). The ha70 genes were 98% identical on the nucleotide level and 97.4% similar and 97.3% identical on the amino acid level. The ha17 gene was 97.7% identical on the nucleotide level and 97.3% similar and identical on the amino acid level. Also, the botR gene was 98.3% identical on the nucleotide level between the two strains and 97.2% similar and 96.6% identical on the amino acid level. The ntnh gene was 98.4% identical between the two strains and was 98.2% and 97.8% similar and identical on the amino acid level respectively. The ha33 gene demonstrated 95.0% nucleotide identity between the two strains, but only 91.5% similarity and 90.5% identity on the amino acid level.







TABLE 2







Designation of the thirty-six amino acid


differences found between BoNT/A1 and BoNT/A5.










LC
HCN
HCC






D102E
A567V
K897E
N1006A


 


E171D
R581S
V926I
V1017I


 


G268E
K592R
R948K
T1063P


 


K381E
D707G
N954S
H1064Q


 



D767G
S955K
D1103N


 



E775D
S957N
V1143I


 




M968I
M1144V


 




T990N
R1156M


 




Q991K
A1208V


 




E992Q
T1232R


 




I993N
A1259D


 




K994I
L1278F


 




I1005V
R1294S





The comparison was performed using the sequence of the BoNT/A1 and the first amino


acid written is from the sequence of that subtype. Light Chain (LC) comprises the amino


acid from #1 to #437, Heavy Chain N-terminal (HCN) comprises the amino acid from


#438 to #872 and Heavy Chain C-terminal (HCC) comprises the amino acid from #873 to #1296.



















TABLE 3





Genes
ha70
ha33
ha17
botR
ntnh
bont/a







Nucleotide
98.0
95.0
97.7
98.3
98.4
98.6


Amino
97.4/
91.5/
97.3/97.3
97.2/96.6
98.2/97.8
97.9/97.1


acid
97.3
90.5











Comparison of the bont/a and its associated cluster genes between C. botulinum strains A661222 and ATCC 3502 on both the nucleotide and amino acid levels. Nucleotide values are the % of identity, amino acid values are the % of similarity/identity respectively.

The sequence alignment between all the A subtypes has shown that the new A5 subtype is diverse compared to A3 and A4, somewhat close to A2, but is most similar to A1. The different domains of the toxin were compared and all the results show that the C-terminal part of the heavy chain is the most conserved domain through all subtypes (Table 4). This makes sense given the need to conserve areas of interaction between the protein receptor and ganglioside.














TABLE 4







Holo-
LC
HC
HCN
HCC-N
HCC-C



toxin
1-437
438-1296
438-872
873-1093
1094-1296






















BoNT/A1
97
99
96
98
92
96


BoNT/A2
90
95
88
87
87
92


BoNT/A3
85
81
87
85
87
91


BoNT/A4
87
89
87
86
80
94









Comparison between BoNT/A5 and the other BoNT/A subtypes. The toxin was split in several domains and subdomains to determine the % of identity between the subtypes. LC: Light Chain, HC: Heavy Chain, HCN: Heavy Chain N-terminal, HCC-N: Heavy Chain C-terminal N-portion, HCC-C: Heavy Chain C-terminal C-portion.

Antibody Recognition
An epitope comparison was done utilizing certain specific peptides of the A2 HC domain based on previous work. Although the identity between the A5 and A2 sequences for the overall HC domain were low, the specific identity for those peptide regions is around 98% between the A5 and A2 subtypes compared to only 83% between the A5 and A1 subtypes. Four different regions in the HC of the protein were selected (Table 5). These peptides are known to be important for antibody recognition since they were characterized as highly sensitive epitopes (Atassi, M. Z., et al., 2004, Protein J. 23:39-52, Dolimbek, B. Z., et al., 2007, Mol. Immunol. 44:1029-1041, Garcia-Rodriguez, C., et al., 2007, Nat. Biotechnol. 25:107-116, Lacy, D. B., et al., 1999, J. Mol. Biol. 291:1091-1104, Smith, T. J., et al., 2005, Infect. Immun. 73:5450-5457, Zarebski, L. M., et al., 2008, Expert Rev. Vaccines 7:55-74). The antibodies generated against A1 could be significantly affected by the differences in these regions even though the identity of the entire amino acid chains of A1 and A5 is close to 97%. Previous works have shown that the differences observed in A2 in this region are sufficient to disturb the binding of the antibodies (Garcia-Rodriguez, C., et al., 2007, Nat. Biotechnol. 25:107-116, Smith, T. J., et al., 2005, Infect. Immun. 73:5450-5457). 40% of the differences observed in BoNT/A5 compared to BoNT/A1 were found in these important areas (FIG. 3).







TABLE 5







Comparison of specific peptides of BoNT/A1, A2 and A5 that are 


known to be targets for antibody neutralization.











A1 vs
A5 vs
A5 vs



A2
A1
A2













925-957
88
85
97


 967-1013
85
83
98


1051-1069
79
90
85


1275-1296
86
91
96





All numbers are the percentage of identity between the specific subtypes analyzed.






Garcia-Rodriguez et al. have shown that several amino acids are important to optimize the interactions between certain antibodies and BoNT/A1 (Garcia-Rodriguez, C., et al., 2007, Nat. Biotechnol. 25:107-116). Some of these are different in BoNT/A5 making it similar to BoNT/A2, but several residues are also conserved (Table 6). The amino acids are listed according to their importance in the interactions between the BoNT/A1 and the two Fab species, AR2 and CR1. The different KD values were measured and proposed to affect the importance of these residues for antibody affinity. The most significant amino acid was H1064 which is deeply buried in the interface between the Fab and the toxin (FIG. 4A). The truncation of the side chain of H1064 to alanine reduces the affinity of AR2 and CR1 for BoNT/A1 by more than 200,000 fold (Garcia-Rodriguez, C., et al., 2007, Nat. Biotechnol. 25:107-116). The introduction of a mutated H1064R into the BoNT/A1 HC reduced the affinity for AR2 and CR1 by only 41- and 188-fold. This decrease is less profound probably due to the fact that the arginine can interact with the Fab amino acids as the modeling shows (FIG. 4B) (Garcia-Rodriguez, C., et al., 2007, Nat. Biotechnol. 25:107-116). In BoNT/A5, the difference is more pronounced because the H1064 is substituted for a glutamine, which is not able to pi-stack with F36. Furthermore, Q1064 has just one positive charge to interact with D102 and thus the stability of that loop decreases at the complex interface (FIG. 4C). The affinity would be lower compared to the mutated arginine form. That indicates that the antibodies developed against that BoNT/A1 area would be expected to have difficulty interacting with A5 and being able to neutralize it.







TABLE 6







Amino acids that are known to optimize


antibody binding with BoNT/A.











BoNT/A1
BoNT/A2
BoNT/A5







Ser 902
Asp
Ser



 



Phe 917
Ile
Phe



 



Asn 918
Asn
Asn



 



Leu 919
Leu
Leu



 



Glu 920
Glu
Glu



 



Phe 953
Phe
Phe



 



Asn 954
Ser
Ser



 



Ser 955
Lys
Lys



 



Ile 956
Ile
Ile



 



Lys 1056
Lys
Lys



 



Asp 1058
Asp
Asp



 



Arg 1061
Arg
Arg



 



Asp 1062
Asp
Asp



 



Thr 1063
Pro
Pro



 



His 1064
Arg
Gln



 



Arg 1065
Arg
Arg



 



Gly 1292
Gly
Gly



 



Arg 1294
Ser
Ser







The five BoNT/A1 amino acids that differ in BoNT/A5 and have direct contact with CR1



are in bold (Dineen, S. S., et al., 2003, Curr. Microbiol. 46: 345-352).






SNAP-25 Interaction
The LC structure of the BoNT/A5 was superimposed over the BoNT/A1 LC structure in complex with the SNAP25 (pdb code 1×TG) (Breidenbach, M. A., et al., 2004, Nature 432:925-929). The amino acids involved in the recognition of the peptide are mostly the same except for three of them. In A5 D102 was mutated to glutamic acid, E171 was transformed to aspartic acid (FIG. 5A) and V27 to alanine (FIG. 5B). The side chains are the same in term of charge and hydrophobicity but not in term of length. The impact on the specificity and the selectivity of the target should be weak.
Active Site
All catalytic residues in the active site are conserved in the new A5 subtype. The amino acids described as to perform hydrogen bonds with the substrate are the same in A5 (Silvaggi, N. R., et al., 2008, Biochemistry 47:5736-5745). Likewise, the different pockets involved in the substrate binding are conserved (S1, S1′, S2′, S3′, S4′ and S5′). The closest mutation is distant from the active site (FIG. 6).
The Ganglioside Binding Site of BoNT/A5
The structure of botulinum neurotoxin A1 in complex with the ganglioside GT1b has recently been determined (Stenmark, P., et al., 2008, PLoS Pathog. 2008, 4(8):e1000129). Amino acid alterations specific for A5 were then applied to this known structure to determine how they would affect ganglioside binding (FIG. 7). All of the amino acids that directly interact with GT1b are conserved between subtypes A1 and A5; these are Tyr 1117, Glu1203, Phe 1252, His 1253, Ser 1264, Trp 1266, Tyr 1267, Ser 1275 and Arg 1276. There are two amino acids in the second coordination sphere that are different between the two serotypes; Leu 1278 and Ala 1259 in BoNT/A1 correspond to Phe 1278 and Asp 1259 in BoNT/A5 (FIG. 7). Asp 1259 in BoNT/A5 is going to be located approximately 6 A from the ganglioside and is likely to interact with Lys 1121. This interaction is absent in BoNT/A1 and the difference will influence the electrostatics of the binding site, however, it is unlikely that this mutation will have any major effects on the ganglioside binding. In serotype B, E and the tetanus toxin the position corresponding to 1259 in BoNT/A5 is conserved as an aspartate (Rummel, A., et al., 2004, Mol. Microbiol. 51:631-643). Leu 1278 in BoNT/A1 is not conserved and is a phenylalanine in BoNT/A5; subtypes A2 and A4 also have a phenylalanine in this position (Arndt, J. W., et al., 2006, J. Mol. Biol. 362:733-742). Leu 1278 is located between Tyr 1117 and Ser 1275 in the BoNT/A1 GT1b complex; both Tyr 1117 and Ser 1275 form hydrogen bonds to SiaS of GT1b (Stenmark, P., et al., 2008, PLoS Pathog. 2008, 4(8):e1000129). The substitution of Leu 1278 (BoNT/A1) for a phenylalanine (BoNT/A5) is likely to influence the ganglioside affinity and specificity. The position of the Tyr 1117 and Ser 1275 can be changed and the position of the somewhat flexible loop harboring Ser 1275 could be affected (Lacy, D. B., et al., 1998, Nat. Struct. Biol. 5:898-902). A variety of mutations of Tyr 1117 has recently been shown to increase the affinity between the heavy chain of the toxin and nerve cells (U.S. patent publication US2007/0299008 A1), indicating that changes in this area are important for the ganglioside binding properties of the toxins.
Structural Analysis of the HA33 Protein from the BoNT/A5 Gene Cluster
HA33 is one of the major components of the progenitor toxin complex and is important for its immunogenicity and carbohydrate binding properties (Arndt, J. W., et al., 2005, J. Mol. Biol. 346:1083-1093; Sharma, S. K., et al., 2000, J. Nat. Toxins 9:357-362). The sequence identity between HA33/A1 and HA33/A5 is 90.5%; this makes it the component of the progenitor complex proteins with the least degree of conservation between A1 and A5 (Table 3). The structure of HA33/A1 has been solved and it consists of two β-trefoil fold domains (Arndt, J. W., et al., 2005, J. Mol. Biol. 346:1083-1093), with most of the sequence differences in this section located on the surface of the protein. There is a significant difference in the degree of conservation of the two domains between HA33/A5 and HA33/A1; the N-terminal domain is 93.9% identical and the C-terminal domain is 87.1% identical. This pattern of conservation has also been observed in an analysis of the sequence conservation between other serotypes (Arndt, J. W., et al., 2005, J. Mol. Biol. 346:1083-1093). The higher degree of conservation of the N-terminal domain indicates that it is mediating contacts to the rest of the progenitor toxin complex. The less conserved C-terminal domain is likely to be solvent exposed and to mediate binding to carbohydrates. The residues that have been suggested to be involve in carbohydrate binding are conserved between HA33/A1 and HA33/A5 (Arndt, J. W., et al., 2005, J. Mol. Biol. 346:1083-1093). Most of the immunogenic response of the progenitor complex has been attributed to the C-terminal part of HA33 (Sharma, S. K., et al., 2000, J. Nat. Toxins 9:357-362); the relatively high sequence variability of HA33/A5 could lead to a different immunological response of the A5 progenitor toxin complex than the A1 progenitor complex that is used extensively for medical applications.
Purification of BoNT/A5 and Determination of Toxicity
The BoNT/A5 was able to be purified by using the purification method previously used to isolate BoNT/A1. This was expected because BoNT/A5 is most closely related to BoNT/A1 among the type A subtypes and they are the only subtypes to have a HA cluster arrangement associated with the bont/a. Purified BoNT/A5 was confirmed by SDS-PAGE under reducing condition (FIG. 8) and mouse bioassay. SDS-PAGE data showed that 95% pure BoNT/A5 was obtained after the final chromatography step. Specific toxicity of 150 kDa protein was determined to be  ̃1.25×108 LD50/mg.
Neutralization of BoNT/A1 and BoNT/A5 Using an anti-BoNT/A1 Antibody
To assess the effect of the variations at specific epitopes of BoNT/A5 from BoNT/A1 on antibody neutralization, it was necessary to use a mouse model with a high titer of both toxin and antibody raised against a highly immunogenic BoNT/A1 toxin. This approach was used as it would more effectively identify how epitope differences between the two toxins affect neutralization. As disclosed below, our results indicate that the differences at key epitopes between the two toxins may affect the ability of antibodies to bind to and neutralize the individual toxins.
More specifically, the neutralization results showed that 2 μl anti-BoNT/A1 antibody was able to fully neutralize 10,000 LD50 of either BoNT/A1 or BoNT/A5, but was not able to completely neutralize 12,000 LD50 of either toxin subtype. The data indicate that both BoNT/A1 and BoNT/A5 have very similar binding reactions with anti-BoNT/A1 antibody. However, there were some differences in the time to death of mice between the ones injected with the BoNT/A1+antibody mixture and the ones injected with the BoNT/A5+antibody mixture. The mice injected with a BoNT/A5+antibody mixture died one day faster than those injected with a BoNT/A1+antibody mixture at a 12,000 LD50 dose. At the higher dose of 16,000 LD50, it took mice injected with BoNT/A1+antibody mixture 2 days to die while mice injected with BoNT/A5+antibody mixture died within 4 hours, suggesting the antibodies were less effective at neutralizing BoNT/A5 compared to BoNT/A1 at dosages of 12,000 LD50 and 16,000 LD50. Additionally, mice injected with 10,000 LD50 BoNT/A5+antibody mixture exhibited more severe symptoms than those injected with 10,000 LD50 BoNT/A1+antibody even though all of the mice survived for 4 days.
Taken as a whole, the above data indicate that, although the ability of anti-BoNT/A1 antibody to bind to lower levels of BoNT/A1 and BoNT/A5 are similar, the structural differences between BoNT/A1 and BoNT/A5 are more visible when higher levels of toxins are used, which is consistent with our prediction (see paragraph [0047]).
Discussion
Prior publications have identified a novel BoNT/A subtype termed BoNT/A5. This designation was based on the level of divergence compared to known BoNT/A subtypes but did not identify the importance of these differences on a biochemical level. Our laboratory identified a strain producing this neurotoxin and analyzed it using protein modeling to assess the effect of its amino acid differences compared to BoNT/A1. There are thirty six amino acids differences between the neurotoxins of strains A661222 and ATCC 3502 (BoNT/A1) with thirty-two of them present in the heavy chain which is responsible for binding to neural cells and a target for antibody neutralization. BoNT/A5 was able to be purified by using the method previously to purify BoNT/A1. The neutralization experiment also performed to compare the binding reaction to a BoNT/A1 specific antibody between the BoNT/A1 and the BoNT/A5.
BoNT/A5 is unique compared to the other BoNT/A subtypes as it is highly similar to BoNT/A1 but also exhibits similarities to BoNT/A2 in areas that have been previously shown to affect antibody binding. The most important of these changes was at residue 1064. Previous studies have shown that mutating this residue from its original histidine to an alanine has the effect of decreasing binding by 200,000 fold as the pi-stacking between the histidine and F36 from the antibodies. In BoNT/A5, this residue is substituted for a glutamine and the pi stacking is also not present based on protein modeling experiments. This led to the hypothesis that the BoNT/A5 might have a different antibody neutralization profile than BoNT/A1.
The ability of the BoNT/A5 to form a complex with the ganglioside GT1b was also analyzed. All of the amino acids that directly interact with GT1b are conserved between subtypes A1 and A5; these are Tyr 1117, Glu1203, Phe 1252, His 1253, Ser 1264, Trp 1266, Tyr 1267, Ser 1275 and Arg 1276. There are two amino acids in the second coordination sphere that are different between the two serotypes; Leu 1278 and Ala 1259 in BoNT/A1 correspond to Phe 1278 and Asp 1259 in BoNT/A5 (FIG. 7). Asp 1259 in BoNT/A5 is going to be located approximately 6 A from the ganglioside and is likely to interact with Lys 1121. This interaction is absent in BoNT/A1 and the difference will influence the electrostatics of the binding site, however, it is unlikely that this mutation will have any major effects on the ganglioside binding.
One of the toxin associated proteins was also analyzed via molecular modeling procedures. The amino acid sequence identity between HA33/A1 and HA33/A5 is 90.5% compared to the other genes which are highly homologous (97-98%). Some of these changes however appear to have little affect on the ability of HA33 to perform its role in the formation of a BoNT complex as the residues that have been suggested to be involve in carbohydrate binding are conserved between HA33/A1 and HA33. However, most of the immunogenic response of the progenitor complex has been attributed to the C-terminal part of HA33 which in HA33/A5 is only 87.1% identical compared to HA33/A1.
BoNT/A5 was able to be purified by using the method to purify the BoNT/A1, this was expected because of high degree of homologous between these two toxins. Both BoNT/A1 and BoNT/A5 were able to be neutralized by 2 μl anti-BoNT/A1 antibody at a dose of 1,000 LD50 but could not be completely neutralized at a dose of 12,000 LD50. The data indicated the binding between each toxin and the BoNT/A1 specific antibody was very similar but differences in the time to death of mice were observed between the two toxins. The mice injected with a BoNT/A5+antibody mixture died one day faster than those injected with a BoNT/A1+antibody mixture at a 12,000 LD50 dose. At the higher dose of 16,000 LD50, it took mice injected with the BoNT/A1+antibody mixture 2 days to die while only took mice injected with the BoNT/A5+antiboy mixture to die within 4 hours. Additionally, mice injected with 10,000 LD50 BoNT/A5+antibody mixture exhibited more severe symptoms than those injected with 10,000 LD50 BoNT/A1+antibody mixture even though all of the mice survived for 4 days. This indicates that the binding between BoNT/A1 and BoNT/A5 to anti-BoNT/A1 antibody might have slight differences which were consistent with comparative structure predictions.
An intriguing question is how this particular BoNT/A subtype arose. The strain itself is significantly different from the reference strain ATCC 3502 as it demonstrated a unique ST profile from MLST analysis implying that the strains are not closely related. While the two strains were significantly different under this analysis, they did bear some degree of similarity. Specifically, they had high degrees of similarity in most of their BoNT associated genes (between 97.7 and 98.4% identity on the nucleotide level). Only one of the genes (ha33) demonstrated significant divergence, as it was only 95% identical. This is interesting as it is located in the middle of the cluster arrangement, flanked by ha70 and ha17 on one side and botR and ntnh on the other. What makes this really interesting though is that these two flanking regions are expressed in opposite directions. Based on this, there is the possibility that the HA33 gene was so divergent between the two strains compared as it is the most likely area for a recombination event to occur at some point in the origin of the cluster. This would be in keeping with the results seen elsewhere that the complex originated from a mammalian virus, perhaps of a neurotropic source (DasGupta, B. R., 2006, J. Gen. Appl. Microbiol. 52:1-8; Johnson, E. A., et al., 2001, Toxicon 39:1703-1722). Additionally, it would explain the HA33 gene demonstrating significant sequence divergence but why few of these alterations affect the ability of the protein to engage in binding with the BoNT to help form the complex.
Example 2
Materials and Methods
Bacterial strains and growth conditions. Clostridium botulinum strains A661222 and ATCC 3502 included in this study were from the Eric A. Johnson (EAJ) strain collection. The A661222 strain was grown from a lyophilized culture which was received from the Lanzhou Institute in February 1981. Cultures were grown in 10 mL of sterile TPGY media (per liter: 50 g trypticase peptone, 5 g Bacto peptone, 4 g Δ-glucose, 20 g yeast extract, 1 g cysteine-HC1, at pH 7.4) for 2 days at 37° C. under anaerobic conditions.
Total genomic DNA isolation. Total genomic DNA was isolated from C. botulinum by lysozyme and proteinase K treatment as described previously (Dineen, S. S., et al., 2003, Curr. Microbiol. 46:345-352). DNA was then diluted to a concentration of 50 ng/μL and used for PCR amplification.
PCR amplification and DNA sequencing. PCR amplifications were performed using the GeneAmp® High Fidelity PCR System (Applied BioSystems). PCR cycles were as follows: 95° C. for 2 minutes, followed by 25 cycles of 95° C. for 1 minute, an annealing step for 45 seconds at 48° C., 72° C. for extension, followed by 1 cycle of 72° C. extension for 10 minutes. Extension time depended on the length of the fragment being amplified. Following amplification, PCR products were isolated using the PureLinkTM PCR Purification Kit (Invitrogen). Sequencing was performed using conditions advised by the University of Wisconsin Biotechnology Center using the ABI PRISM® BigDyeTM Cycle Sequencing Kit (Applied BioSystems). Primers used for PCR and sequencing for the HA cluster, ntnh and the bont/a gene are the same as those used before (Jacobson, M. J., et al., 2008, Appl. Environ. Microbiol. 74:2778-2786). PCRs were performed in a staggered manner such that the amplicons produced overlapping products for each of the genes in the neurotoxin cluster. Appropriate primers were then used for sequencing each PCR product. Correct assembly of the contigs was verified using overlapping sequence data with each region of the sequence being analyzed at least four times. Sequencing analysis was performed at the University of Wisconsin Biotechnology Center and final sequencing results were analyzed using the Vector NTI Suite Program (Invitrogen). Sequences for the neurotoxin cluster genes and bont/a from A661222 were determined and disclosed in FIGS. 9-21. Specifically, FIGS. 9 and 10 disclose nucleotide and amino acid sequences of BoNT/A5, respectively. FIG. 11 discloses genomic DNA sequence of BoNT/A5 neurotoxin cluster with HA genes including HA70 (reverse), HA17 (reverse), HA33 (reverse), botR (forward), NTNH (forward) and BoNT/A5 (forward). FIGS. 12 and 13 disclose nucleotide and amino acid sequences of HA70, respectively. FIGS. 14 and 15 disclose nucleotide and amino acid sequences of HA17, respectively. FIGS. 16 and 17 disclose nucleotide and amino acid sequences of HA33, respectively. FIGS. 18 and 19 disclose nucleotide and amino acid sequences of botR, respectively. FIGS. 20 and 21 disclose nucleotide and amino acid sequences of NTNH, respectively.
Sequence alignment. Amino acid sequences of BoNT/A subtypes A1-A5 were aligned using Clustal W and MEGA software to produce a UPGMA phylogeny tree of the subtypes as a whole and for their heavy chains.
Molecular modeling. The first model comparing the BoNT/A's was generated with the program Coot (Emsley, P., et al., 2004, Acta Crystallogr. D Biol. Crystallogr. 60:2126-2132) using the crystal structures of the BoNT/A1 (PDB code 1BTA) (Lacy, D. B., et al., 1998, Nat. Struct. Biol. 5:898-902). Pymol was used to generate illustrations (Delano, W. L., The PyMOL Molecular Graphics System. In Anonymous. DeLano Scientific LLC, San Carlos, Calif., USA).
Purification of BoNT/A5 and determination of toxicity. The BoNT/A5 toxin was purified using the previously described protocol used for the purification of BoNT/A1 (DasGupta, B. R., et al., 1984, Toxicon 22:415-424). The purified BoNT/A5 was visualized on a 4-12% NuPage SDS-PAGE (Invitrogen) under reducing and non-reducing condition to assess protein purity.
The specific toxicity of the purified BoNT/A5 toxin was determined by IP injection using four toxin concentrations of 15 pg, 10 pg, 6.67 pg and 4.45 pg per mouse. The toxin was diluted in 0.5 ml gel phosphate buffer and four mice were injected per each concentration and observed for 4 days for symptoms. The LD50/mg of toxin was calculated using the method described in Reed and Muench (Reed, L. J., et al., 1938, Am. J. Hyg. 27:493-497).
Neutralization of BoNT/A5 using anti-BoNT/A1 antibody. Polyclonal anti-BoNT/A1 antibody was raised in rabbit and then protein A purified. The antibody was tittered so 1 μl of this antibody can neutralize 5000 LD50 of BoNT/A1. In this study, 2 μl antibody was used to neutralize 16,000 LD50, 12,000 LD50, 10,000 LD50, 5,000 LD50, 2,500 LD50, 1,250 LD50 respectively either with BoNTA1 or BoNT/A5 to compare antibody neutralization between BoN/A1 and BoNT/A5. Toxin was diluted with gel phosphate to achieve the appropriate LD50 concentrations. The different mixtures of toxin and antibody were incubated at 37° C. for 90 minutes prior to injection. Two mice were injected with 0.5 ml of the toxin+antibody mixture respectively and were observed for 4 days for symptoms.

## Claims

1. A substantially purified BoNT/Az5 protein, wherein the protein is encoded by the nucleotide comprising SEQ ID NO:1 or by a nucleotide sequence at least 99% identical with SEQ ID NO:1.

2. The BoNT/A5 protein of claim 1, wherein the purity of the protein is at least 90%.

3. The BoNT/A5 protein of claim 1, wherein the purity of the protein is at least 95%.