Probiotic composition based on the enterococcus strain and used as a treatment means and method for the production thereof



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Accordingly, there is provided a polypeptide selected from the group consisting of SEQ ID NO : 2, SEQ ID NO : 4, SEQ ID NO : 6, and SEQ ID NO : 8, including fragments, variants and functional equivalents thereof as described below in more detail.
Functional equivalents and variants are used interchangeably herein. When being polypeptides, variants are determined on the basis of their degree of identity or their degree of homology with any predetermined sequence of consecutive amino acid

sequences of a fragment of SEQ ID NO : 2, SEQID NO : 4, SEQ ID NO : 6, AND SEQ ID NO : 8.


One therefore initially define a sequence of consecutive SEQ ID NO : 2, SEQ ID NO : 4, SEQID NO : 6, AND SEQ ID NO : 8 amino acid residues and then define variants and functional equivalents in relation thereto.
Accordingly, variants preferably have at least 75% sequence identity, for example at least 80% sequence identity, such as at least 85 % sequence identity, for example at least 90 % sequence identity, such as at least 91 % sequence identity, for example at least 91% sequence identity, such as at least 92 % sequence identity, for example at least 93 % sequence identity, such as at least 94 % sequence identity, for example at least 95 % sequence identity, such as at least 96 % sequence identity, for example at least 97% sequence identity, such as at least 98 % sequence identity, for example 99% sequence identity with the predetermined SEQ ID NO : 2, SEQ ID NO : 4, SEQ ID NO : 6, AND SEQ ID NO : 8 sequence of consecutive amino acid residues.
Sequence identity is determined in one embodiment by using the algorithm GAP, BESTFIT, or FASTA in the Wisconsin Genetics Software Package Release 7.0, using default gap weights.
The following terms are used to describe the sequence relationships between two or more polynucleotides : "predetermined sequence", "comparison window", "sequence identity","percentage of sequence identity", and"substantial identity". A"predetermined sequence"is a defined sequence used as a basis for a sequence comparison; a predetermined sequence may be a subset of a larger sequence.
Optimal alignment of sequences for aligning a comparison window may be conducted by the local homology algorithm of Smith and Waterman (1981) Adv. Appl.
Math. 2: 482, by the homology alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48: 443, by the search for similarity method of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. (U. S. A. ) 85: 2444, by computerised implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575

Science Dr. , Madison, Wis. ), or by inspection, and the best alignment (i. e. , resulting in the highest percentage of homology over the comparison window) generated by the various methods is selected.


The term"sequence identity"means that two amino acid sequences are identical over the window of comparison.
The term"percentage of sequence identity"is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which identical amino acid residues occur in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i. e., the window size), and multi- plying the result by 100 to yield the percentage of sequence identity.
As applied to polypeptides, a degree of identity of amino acid sequences is a func- tion of the number of identical amino acids at positions shared by the amino acid sequences. A degree of homology or similarity of amino acid sequences is a func- tion of the number of amino acids, i. e. structurally related, at positions shared by the amino acid sequences.
The term"substantial identity"means that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 75 percent sequence identity, such as at least 80 percent sequence identity, for example at least 85 percent sequence identity, such as e. g. at least 90 percent sequence identity, for example at least 95 percent sequence identity, such as at least 98 percent sequence identity, or even at least 99 percent sequence identity, compared to a predetermined sequence over a comparison window of at least 9 amino acid residues, such as 10 amino acid residues, for example 11 amino acid residues, such as 12 amino acid residues, for example 13 amino acid residues, such as 14 amino acid residues, for example 15 amino acid residues, such as 20 amino acid residues, for example 30 amino acid residues, such as 40 amino acid residues, for example 50 amino acid residues, such as 60 amino acid residues, for example 70 amino acid residues, such as 80 amino acid residues, for example 90 amino acid residues, such as 100 amino acid residues, for example 110 amino acid residues, such as 120 amino acid residues, for example 130 amino acid residues, such as

140 amino acid residues, for example 150 amino acid residues, such as 175 amino acid residues, for example 200 amino acid residues, such as 225 amino acid residues, for example 250 amino acid residues, such as 275 amino acid residues, for example 297 amino acid residues. Preferably, residue positions which are not identical differ by conservative amino acid substitutions.


Conservative amino acid substitutions refer in one embodiment to the interchangeability of residues having similar side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine ; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine, a group of amino acids having amide-containing side chains is asparagine and gluta- mine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains is cysteine and methionine. Preferred conservative amino acids substitution groups are : va ! ine- ! eucine-iso) eucine, phenyiaianine-tyrosine, iysine-arginine, afanine- valine, and asparagine-glutamine.
Additionally, variants are also determined based on a predetermined number of conservative amino acid substitutions as defined herein below. Conservative amino acid substitution as used herein relates to the substitution of one amino acid (within a predetermined group of amino acids) for another amino acid (within the same group), wherein the amino acids exhibit similar or substantially similar characteris- tics.
Within the meaning of the term"conservative amino acid substitution"as applied herein, one amino acid may be substituted for another within the groups of amino acids indicated herein below : Amino acids having polar side chains (Asp, Glu, Lys, Arg, His, Asn, Gin, Ser, Thr, Tyr, and Cys,) Amino acids having non-polar side chains (Gly, Ala, Val, Leu, Ile, Phe, Trp, Pro, and Met)

Amino acids having aliphatic side chains (Gly, Ala Val, Leu, Ile) Amino acids having cyclic side chains (Phe, Tyr, Trp, His, Pro) Amino acids having aromatic side chains (Phe, Tyr, Trp) Amino acids having acidic side chains (Asp, Glu) Amino acids having basic side chains (Lys, Arg, His) Amino acids having amide side chains (Asn, Gin) Amino acids having hydroxy side chains (Ser, Thr) Amino acids having sulphor-containing side chains (Cys, Met), Neutral, weakly hydrophobic amino acids (Pro, Ala, Gly, Ser, Thr) Hydrophilic, acidic amino acids (Gin, Asn, Glu, Asp), and Hydrophobic amino acids (Leu, Ile, Val) Accordingly, a variant or a fragment thereof according to the invention may comprise at least one substitution, such as a plurality of substitutions introduced independ- ently of one another. It is clear from the above outline that the same variant or fragment thereof may comprise more than one conservative amino acid substitution from more than one group of conservative amino acids as defined herein above.


The addition or deletion of at least one amino acid may be an addition or deletion of from preferably 2 to 250 amino acids, such as from 10 to 20 amino acids, for example from 20 to 30 amino acids, such as from 40 to 50 amino acids. However, additions or deletions of more than 50 amino acids, such as additions from 50 to 100 amino acids, addition of 100 to 150 amino acids, addition of 150-250 amino acids, are also comprised within the present invention. The deletion and/or the addition

may-independently of one another-be a deletion and/or an addition within a se- quence and/or at the end of a sequence.


The polypeptide fragments according to the present invention, including any func- tional equivalents thereof, may in one embodiment comprise a sequence of con- secutive SEQ ID NO : 2, SEQ ID NO : 4, SEQ ID NO : 6, AND SEQ ID NO : 8 amino acid residues of less than 250 amino acid residues, such as less than 240 amino acid residues, for example less than 225 amino acid residues, such as less than 200 amino acid residues, for example less than 180 amino acid residues, such as less than 160 amino acid residues, for example less than 150 amino acid residues, such as less than 140 amino acid residues, for example less than 130 amino acid resi- dues, such as less than 120 amino acid residues, for example less than 110 amino acid residues, such as less than 100 amino acid residues, for example less than 90 amino acid residues, such as less than 85 amino acid residues, for example less than 80 amino acid residues, such as less than 75 amino acid residues, for example less than 70 amino acid residues, such as less than 65 amino acid residues, for example less than 60 amino acid residues, such as less than 55 amino acid resi- dues, for example less than 50 amino acid residues, such as less than 45 amino acid residues, for example less than 30 amino acid residues, such as less than 25 amino acid residues, for example less than 20 amino acid residues, for example 10 consecutive amino acid residues of any of SEQ ID NO : 2, SEQ ID NO : 4, SEQ ID NO : 6, AND SEQ ID NO : 8.
"Functional equivalency"as used in the present invention is according to one pre- ferred embodiment established by means of reference to the corresponding func- tonality of a predetermined fragment of the sequence.
Functional equivalents or variants or fragments of SEQ ID NO : 2, SEQ ID N0 : 4, SEQ ID N0 : 6, AND SEQ ID NO : 8 as described herein will be understood to exhibit amino acid sequences gradually differing from preferred, predetermined sequences, as the number and scope of insertions, deletions and substitutions including conser- vative substitutions, increases. This difference is measured as a reduction in homol- ogy between a preferred, predetermined sequence and the SEQ ID NO : 2, SEQ ID NO : 4, SEQID NO : 6, AND SEQ ID N0 : 8 fragment or SEQID NO : 2, SEQ ID N0 : 4, SEQ ID N0 : 6, AND SEQ ID NO : 8 variant or functional equivalent.

All SEQ ID NO : 2, SEQ ID NO : 4, SEQ ID NO : 6, AND SEQ ID NO : 8 fragments comprising or consisting of consecutive SEQ ID NO : 2, SEQ ID NO : 4, SEQ ID NO : 6, AND SEQ ID NO : 8 amino acid residues as well as variants and functional equivalents thereof are included within the scope of this invention, regardless of the degree of homology they show to a predetermined sequence. The reason for this is that some regions of the SEQ ID NO : 2, SEQ ID NO : 4, SEQ ID NO : 6, AND SEQ ID NO : 8 fragments are most likely readily mutatable, or capable of being completely deleted, without any significant effect on e. g. the binding activity of the resulting fragment.


A functional variant obtained by substitution may well exhibit some form or degree of native binding activity, and yet be less homologous, if residues containing functionally similar amino acid side chains are substituted. Functionally similar in this respect refers to dominant characteristics of the side chains such as hydrophobic, basic, neutral or acidic, or the presence or absence of steric bulk. Accordingly, in one embodiment of the invention, the degree of identity is not a principal measure of a fragment being a variant or functional equivalent of a preferred predetermined fragment according to the present invention.
The homology between amino acid sequences may be calculated using well known algorithms such as any one of BLOSUM 30, BLOSUM 40, BLOSUM 45, BLOSUM 50, BLOSUM 55, BLOSUM 60, BLOSUM 62, BLOSUM 65, BLOSUM 70, BLOSUM 75, BLOSUM 80, BLOSUM 85, and BLOSUM 90.
Fragments sharing homology with fragments comprising or consisting of consecu- tive SEQ ID NO : 2, SEQ ID NO : 4, SEQ ID NO : 6, AND SEQ ID NO : 8 amino acid residues are to be considered as failing within the scope of the present invention when they are preferably at least about 90 percent homologous, for example at least 92 percent homologous, such as at least 94 percent homologous, for example at least 95 percent homologous, such as at least 96 percent homologous, for example at least 97 percent homologous, such as at least 98 percent homologous, for example at least 99 percent homologous with a predetermined SEQ ID NO : 2, SEQ ID NO : 4, SEQ ID NO : 6, AND SEQ ID NO : 8 fragment. According to one embodiment of the invention the homology percentages indicated above are identity percentages.

Additional factors that may be taken into consideration when determining functional equivalence according to the meaning used herein are i) the ability of antisera to detect a SEQID NO : 2, SEQID NO : 4, SEQ ID NO : 6, AND SEQ ID NO : 8 fragment according to the present invention, or ii) the ability of a functionally equivalent SEQ ID NO : 2, SEQ ID NO : 4, SEQ ID NO : 6, AND SEQID NO : 8 fragment to compete with a predetermined SEQ ID NO : 2, SEQID NO : 4, SEQID NO : 6, AND SEQ ID NO : 8 fragment in an assay. One method for determining a sequence of immunogenically active amino acids within a known amino acid sequence has been described by Geysen in US 5,595, 915 and is incorporated herein by reference.


A further suitably adaptable method for determining structure and function relationships of peptide fragments is described by US 6,013, 478, which is herein incorporated by reference.
In addition to conservative substitutions introduced into any position of a preferred SEQ ID NO : 2, SEQ ID N0 : 4, SEQ iD N0 : 6, AND SEQ ID NO : 8 fragments, it may also be desirable to introduce non-conservative substitutions in any one or more positions of such a fragment. A non-conservative substitution leading to the formation of a functionally equivalent fragment would for example i) differ substantially in polarity, for example a residue with a non-polar side chain (Ala, Leu, Pro, Trp, Val, Ile, Leu, Phe or Met) substituted for a residue with a polar side chain such as Gly, Ser, Thr, Cys, Tyr, Asn, or Gin or a charged amino acid such as Asp, Glu, Arg, or Lys, or substituting a charged or a polar residue for a non-polar one; and/or ii) differ substantially in its effect on polypeptide backbone orientation such as substitution of or for Pro or Gly by another residue; and/or iii) differ substantially in electric charge, for example substitution of a negatively charged residue such as Glu or Asp for a positively charged residue such as Lys, His or Arg (and vice versa); and/or iv) differ substantially in steric bulk, for example substitution of a bulky residue such as His, Trp, Phe or Tyr for one having a minor side chain, e. g. Ala, Gly or Ser (and vice versa).
Variants obtained by substitution of amino acids may in one preferred embodiment be made based upon the hydrophobicity and hydrophilicity values and the relative similarity of the amino acid side-chain substituents, including charge, size, and the like. Exemplary amino acid substitutions which take various of the foregoing char-

acteristics into consideration are well known to those of skill in the art and include: arginine and lysine ; glutamate and aspartate; serine and threonine; glutamin and asparagine; and valine, leucine and isoleucine.


In a further embodiment the present invention relates to functional variants comprising substituted amino acids having hydrophilic values or hydropathic indices that are within +/-4.9, for example within +/-4.7, such as within +/-4.5, for example within +/-4. 3, such as within +/-4.1, for example within +/-3.9, such as within +/-3.7, for example within +/-3.5, such as within +/-3.3, for example within +/-3.1, such as within +/-2.9, for example within +/-2.7, such as within +/-2.5, for example within +/- 2.3, such as within +/-2.1, for example within +/-2.0, such as within +/-1.8, for example within +/-1.6, such as within +/-1.5, for example within +/-1.4, such as within +/-1.3 for example within +/-1.2, such as within +/-1.1, for example within +/- 1.0, such as within +/-0.9, for example within +/-0. 8, such as within +/-0. 7, for example within +/-0.6, such as within +/-0.5, for example within +/-0. 4, such as within +/-0.3, for example within +/-0. 25, such as within +/-0.2 of the value of the amino acid it has substituted.
The importance of the hydrophilic and hydropathic amino acid indices in conferring interactive biologic function on a protein is well understood in the art (Kyte & Doolittle, 1982 and Hopp, U. S. Pat. No. 4, 554, 101, each incorporated herein by reference).
The amino acid hydropathic index values as used herein are: isoleucine (+4.5) ; valine (+4.2) ; leucine (+3.8) ; phenylalanine (+2. 8) ; cysteine/cystine (+2.5) ; methionine (+1.9) ; alanine (+1. 8) ; glycine (-0.4) ; threonine (-0.7) ; serine (-0. 8) ; tryptophan (-0.9) ; tyrosine (-1.3) ; proline (-1.6) ; histidine (-3.2) ; glutamate (-3.5) ; glutamin (- 3.5) ; aspartate (-3.5) ; asparagine (-3.5) ; lysine (-3.9) ; and arginine (-4.5) (Kyte & Doolittle, 1982).
The amino acid hydrophilicity values are: arginine (+3.0) ; lysine (+3.0) ; aspartate (+3.0. +-. 1); glutamate (+3.0. +-. 1); serine (+0.3) ; asparagine (+0.2) ; glutamin (+0.2) ; glycine (0); threonine (-0.4) ; proline (-0.5. +-. 1); alanine (-0.5) ; histidine (-0.5) ; cysteine (-1.0) ; methionine (-1.3) ; valine (-1.5) ; leucine (-1.8) ; isoleucine (-1.8) ; tyrosine (-2.3) ; phenylalanine (-2.5) ; tryptophan (-3.4) (U. S. 4,554, 101).

In addition to the peptidyl compounds described herein, sterically similar compounds may be formulated to mimic the key portions of the peptide structure and that such compounds may also be used in the same manner as the peptides of the invention.


This may be achieved by techniques of modelling and chemical designing known to those of skill in the art. For example, esterification and other alkylations may be employed to modify the amino terminus of, e. g. , a di-arginine peptide backbone, to mimic a tetra peptide structure. It will be understood that all such sterically similar constructs fall within the scope of the present invention.
Variants and functional equivalents of SEQ ID NO : 2, SEQ ID NO : 4, SEQ ID NO : 6, AND SEQ ID NO : 8 also includes derivatives of SEQ ID NO : 2, SEQ ID NO : 4, SEQ ID NO : 6, AND SEQ ID NO : 8 or fragments thereof, for example fragments substituted with one or more chemical moieties.
Peptides with N-terminal alkylations and C-terminal esterifications are also encompassed within the present invention. Functional equivalents also comprise glycosyl- ated and covalent or aggregative conjugates formed with the same or other polypeptide selected from SEQ) D N0 : 2, SEQID NO : 4, SEQ) D N0 : 6, AND SEQ ID NO : 8, or a fragment thereof, including dimers and unrelated chemical moieties.

*Such functional equivalents are prepared by linkage of functionaiities to groups which are found in fragment including at any one or both of the N-and C-termini, by means known in the art.


Functional equivalents may thus comprise SEQ ID NO : 2, SEQID NO : 4, SEQ ID NO : 6, AND SEQ ID NO : 8, or fragments thereof conjugated to aliphatic or acyl esters or amides of the carboxyl terminus, alkylamines or residues containing carboxyl side chains, e. g. , conjugates to alkylamines at aspartic acid residues; O-acyl derivatives of hydroxyl group-containing residues and N-acyl derivatives of the amino terminal amino acid or amino-group containing residues, e. g. conjugates with fMet-Leu-Phe or immunogenic proteins. Derivatives of the acyl groups are selected from the group of alkyl-moieties (including C3 to C10 normal alkyl), thereby forming alkanol species, and carbocyclic or heterocyclic compounds, thereby forming aroyl species. The reactive groups preferably are difunctional compounds known per se for use in cross-linking proteins to insoluble matrices through reactive side groups.

Covalent or aggregative functional equivalents and derivatives thereof are useful as reagents in immunoassays or for affinity purification procedures. For example, a SEQ ID NO : 2, SEQ ID NO : 4, SEQ ID NO : 6, AND SEQ ID NO : 8 or fragments thereof according to the present invention may be insolubilized by covalent bonding to cyanogen bromide-activated Sepharose by methods known per se or adsorbed to polyolefin surfaces, either with or without glutaraldehyde cross-linking, for use in an assay or purification of anti-SEQ ID NO : 2, SEQ ID NO : 4, SEQ ID NO : 6, AND SEQ ID NO : 8 antibodies or cell surface receptors. Fragments may also be labelled with a detectable group, e. g. , radioiodinated by the chloramine T procedure, covalently bound to rare earth chelate or conjugated to another fluorescent moiety for use in e. g. diagnostic assays.


SEQ ID NO : 2, SEQ ID NO : 4, SEQ ID NO : 6, AND SEQ ID NO : 8 or fragments thereof according to the invention may be synthesised both in vitro and in vivo. In one em- bodiment the SEQ ID NO : 2, SEQ ID NO : 4, SEQ ID NO : 6, AND SEQ ID NO : 8 frag- ments of the invention are synthesised by automated synthesis. Any of the commercially available solid-phase techniques may be employed, such as the Merrifield solid phase synthesis method, in which amino acids are sequentially added to a growing amino acid chain. (See Merrifield, J. Am. Chem. Soc. 85: 2149-2146,1963).
Equipment for automated synthesis of polypeptides is commercially available from suppliers such as Applied Biosystems, Inc. of Foster City, Calif., and may generally be operated according to the manufacturer's instructions. Solid phase synthesis will enable the incorporation of desirable amino acid substitutions into any of SEQ ID NO : 2, SEQ ID NO : 4, SEQ ID NO : 6, AND SEQ ID NO : 8, or a fragment thereof, according to the present invention. It will be understood that substitutions, deletions, insertions or any subcombination thereof may be combined to arrive at a final sequence of a functional equivalent. Insertions shall be understood to include aminoterminal and/or carboxyl-terminal fusions, e. g. with a hydrophobic or immunogenic protein or a carrier such as any polypeptide or scaffold structure capable as serving as a carrier.
Oligomers including dimers including homodimers and heterodimers of any of SEQ ID NO : 2, SEQ ID NO : 4, SEQ ID NO : 6, AND SEQ ID NO : 8 or fragments thereof

fragments according to the invention are also provided and fall under the scope of the invention. Functional equivalents and variants of SEQ ID NO : 2, SEQ ID NO : 4, SEQ ID NO : 6, AND SEQ ID NO : 8, or fragments thereof, can be produced as homodimers or heterodimers with other amino acid sequences or with native SEQ ID NO : 2, SEQ ID NO : 4, SEQID NO : 6, AND SEQ ID NO : 8 sequences. Heterodimers include dimers containing immunoreactive SEQ ID NO : 2, SEQID NO : 4, SEQ ID NO : 6, AND SEQ ID NO : 8 variants and fragments as well as SEQ ID NO : 2, SEQ ID NO : 4, SEQ ID NO : 6, AND SEQ ID NO : 8 variants and fragments that need not have or exert any biological activity.


Method for producing microbial cell surface polypeptide In another embodiment there is provided a method for producing a microbial cell surface polypeptide, or a fragment thereof capable of modulating an immune response in an individual or modulating the amount and/or composition of mucosal mucins, comprising the step of culturing a host cell as described herein under conditions suitable for the production of said polypeptide, or fragment thereof. The cell surface polypeptide is preferably selected from any of SEQ ID NO : 2, SEQ ID NO : 4, SEQ ID NO : 6, AND SEQ ID NO : 8, including functional equivalents and variants and fragments thereof.



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