Fused protein capable of inducing protective immunity against group b streptococcus and vaccine containing said protein
SUBSTANCE: described fused protein contains at least two amino acid sequences. The first amino acid sequence, having 90% sequence identity with an amino acid sequence represented in SEQ ID NO:2, is fused with a second amino acid sequence, having at least 90% sequence identity with an amino acid sequence represented in SEQ ID NO:4.
EFFECT: invention provides immunity against various clinically vital strains of group B streptococci.
9 cl, 5 dwg, 8 ex
The SCOPE of the INVENTION
The present invention relates to the field of Microbiology and technology of vaccines and refers to the development of a vaccine capable of generating immunity to streptococcal infections group C. More specifically, the present invention relates to a new fused protein, which produces immunity to invasive strains of Streptococcus group C. the Invention also relates to a selected nucleotide sequence that encodes a specified protein, the vector to the cell master to a vaccine and to a method for prevention or treatment of streptococcal infection group C.
Group b Streptococcus (Streptococcus agalactiae) (GBS) is the leading cause of invasive bacterial infections, including meningitis in the neonatal period. Only in the United States at the present time there are approximately 5,000 cases per year of invasive disease caused by this bacterium. These infections have an overall mortality rate of approximately 10%, and many babies who survive have permanent neurological consequences. In this regard, efforts are made to find ways of prevention and treatment and to determine the mechanisms by which GBS causes infection.
GBS also causes mastitis in cows, mad cow disease, which is of great economic importance. So develo the TKA vaccine against GBS infections is of interest also in veterinary medicine.
About 20% of all women are vaginal carriers of GBS, and the vertical transport from the genital tract of the mother in all probability is the most common source of infection in the neonatal disease caused by this bacterium. However, only approximately 1% of infants are colonised by GBS at birth, one is struck by a serious infection. Therefore, factors other than exposure to bacteria during birth, should contribute to the development of neonatal disease.
Streptococcal strains of group b are divided into nine serotypes (Ia, Ib and II-VIII) on the structure of the polysaccharide capsule (Baker, J.Inf. Dis. 1990, 161: 917). Four "classic" serotype Ia, Ib, II and III are found in about equal proportions among strains in the normal flora, but type III is clinically the most important serotype, in particular due to the fact that he is the cause of most cases of meningitis.
Because the capsule is a known virulence factor, it has been studied in great detail, in particular strains of type III. Attempts were made to develop a vaccine, in which the main component would be a polysaccharide capsule type III.
In EP 0866133 disclosed is a vaccine that can protect the recipient from infection caused by Streptococcus group C. This invention relates to the use of the combination policy is arid and fragment protein Epsilon. This document also revealed that epidemiological evidence suggests that typespecific capsule plays an important role in immunity to streptococcal infections group (see p.7, lines 2-3). In addition, this document provides a number of different combinations of different proteins, polysaccharide referred to in this application, but all the claims include polysaccharide, indicating the importance of this particular component. However, the application of polysaccharide capsules as vaccines can cause problems due to cross-reactions with human tissues (Pritchard et al., Infect. Immun. 1992, 60: 1598). Therefore, it would be very valuable to develop a vaccine protein-based, and not based on polysaccharides.
In the document Gravekamp et al., Infection and Immunity, Dec 1997, p 5216-5221, disclosed evaluation of immunogenicity and protective effect of number of repetitions alpha (α) With protein, and only the N-terminal part. It was found that the immunogenicity decreases with increasing number of repetitions (see Figv). However, in the analysis of protection was also found that, compared with antibodies against the N-terminal region for protection mainly responsible antibodies against the field repetitions (see C, left column, line 6 from below, and C, right column, lines 26-29).
In WO 9410317 described the use of the protein alpha, which is the surface of the s protein GBS, in the development of a conjugate vaccine. The disadvantage of this protein is that it is usually not expressed by strains of type III, which are the cause of many serious GBS infections. Therefore, a vaccine based on protein alpha will not induce protective immunity against these strains.
In WO 9421685 describes the use of protein Rib, which is the surface protein GBS, in the development of vaccines. This protein induces immunity in the introduction with alum. However, protein Rib has a drawback, which is that it does not induce protective immunity against all strains of GBS.
As stated above, currently, the vaccine is suitable for preventing the disease, GBS, is not yet available, although many works have been devoted to this problem. It is clear that there are currently overdue, but not satisfied the need to develop methods of prevention and treatment of GBS infections. Therefore, there remains a need for strategies for the use of vaccines, capable of generating protective immunity against a wide variety of strains of GBS.
Accordingly, the main objective of the present invention is to create a vaccine capable of inducing protective immunity against GBS infections.
The present invention is also to create a vaccine that induces protective is th immunity against many clinically important strains of GBS.
The present invention is also to create a vaccine consisting of a single fused protein, which induces protective immunity against GBS infections. One protein has several advantages over the vaccine, consisting of many proteins, such as the cost of production and safety.
Means for solving each of the above objectives and other objectives will become apparent from the following further description of the invention.
SUMMARY of the INVENTION
Unexpectedly it was found that the protein contains two different nimmanahaeminda area, such as a fragment of the N-terminal region of the protein Rib GBS, fused with a fragment of the N-terminal region of the protein alpha GBS, i.e. in the merger nimmanahaeminda plots of two different proteins expressed in two different GBS strains, produces a protein that provides very effective protection against infection by two different bacterial strains with the introduction of the fused protein to the mammal in the form of a vaccine. This protection shall antibodies.
In the first aspect of this invention relates to a fused protein containing at least one first fragment of the N-terminal region of the surface protein of group b Streptococcus or its analogue, homologue, derivative or immunologically related amino acid after outermost or fragments, which merged with at least one second fragment of the N-terminal region of the surface protein of group b Streptococcus or its analogue, homologue, derivative or immunologically related amino acid sequence, or fragments, where the specified at least one of the first and second fragments of the N-terminal region of surface proteins of group b Streptococcus obtained from different strains of group b Streptococcus, and where specified protein is able to induce protective immunity against Streptococcus group C.
The main advantage of the fused protein according to the invention is that it includes the area of the related surface proteins Rib and alpha, each of which is expressed by many clinically important strains of group b Streptococcus, and, most importantly, it was shown that it induces protective immunity against these clinically important strains.
This protein has the advantage that it is immunogenic even without adjuvant inducyruya protective immunity against Rib and alpha-expressing strains. Moreover, the vaccine based on the fused protein according to the invention can be entered with alum as adjuvant approved for use in humans. On the contrary, the previously described "universal vaccine" we only know that it works together with the adjuvant Blockers is that strongly irritating component that cannot be used in human medicine (Maione, D. et al, Science 2005, 309: 148-150).
Another advantage of the present invention is that the vaccine composition according to the invention may consist of a single fused protein and to induce protective immunity against different GBS infections. This gives several advantages compared with a vaccine consisting of a set of proteins. For example, one protein easier, safer and cheaper to manufacture than the mixture containing many proteins.
More specifically, the present invention relates to the specified fused protein, the selected nucleotide sequence, the vector to the cell master to a vaccine and to a method for prevention or treatment of streptococcal infection group C.
Below the present invention is described in more detail, inter alia, with reference to graphic materials.
A BRIEF DESCRIPTION of GRAPHIC MATERIALS
Figure 1 shows the proteins used in the examples. (A) shows proteins Rib and alpha, including their unique N-terminal region (N-region) and their areas of long repeats (R-region). The number of amino acid residues in different areas and identical residues are indicated. (C) Recombinant proteins of the Rib and alpha. (C) analysis of the purified proteins by the method of SDS-PAGE (polyacrylamide gel electrophoresis with dodecenal the volume of sodium). (D) Test for inhibition using mouse antibodies against the Rib. (E) Dot-blot analysis.
Figure 2 presents data from studies with passive immunization. (A) Reactivity of rabbit antisera against RibN or Rib2R with bacteria from the Rib-expressing strain VM (empty symbols) or Rib-negative mutant (filled symbols). (B) Passive immunization of mice rabbit antibody against RibN or against Rib2R.
3 shows: (A) the Analysis of cross-reactivity between the N-terminal regions of the Rib and alpha; (C) Characterization of rabbit antibodies against RibN-N and Rib2R-2R; (C) Passive immunization of mice with antibodies to the two fused proteins with subsequent stimulation of the Rib-expressing strain of type III BM110 or alpha-expressing strain of type Ia A909; (D) Passive vaccination fused protein anti-(RibN-N) followed by stimulation Rib-expressing strain of type II or alpha-expressing strain of type Ib; (E) Passive vaccination fused protein anti-(RibN-N) followed by stimulation Rib-negative mutant BM110.
Figure 4 presents the results of an active immunization fused protein RibN-N. (A) Immunogenicity RibN-N with the introduction of adjuvant or without adjuvant. (In) Active vaccination fused protein RibN-N.
Figure 5 presents a comparison of bacteria (A) ability to envirovet cells che is bacescu cervical cell lines ME. (C) Inhibition of invasion of epithelial cells fused protein anti-(RibN-N).
DETAILED description of the INVENTION
The term "immunogenic" means "having the ability to induce an immune response. New protein according to the invention is immunogenic and is characterized by the fact that he has the ability to induce a protective immune response against at least GBS containing Rib and alpha protein.
The term "analog" means those proteins related Rib and alpha proteins in which one or more than one amino acid residue of the Rib - or alpha protein (SEQ ID NO: 2 and 4) is replaced by another amino acid residue, provided that the overall functionality and immunogenic properties of protein-analog or fused protein are preserved. Such analogs can be natural or can be produced synthetically or by recombinant DNA, for example by mutagenesis of one or both of the sequences SEQ ID NO: 1 and 3. Analogs fused protein have at least one epitope capable of inducing antibodies that interacts with Rib-protein, and at least one epitope that reacts with alpha protein. This analogue can have total homology or identity with fused protein represented in SEQ ID NO: 6, at least 80%, for example 80-99%homology or identity, or any range therein.
The percent homology can is to be determined, for example, by comparing sequence information using the computer program GAP, version 6.0, available from the University of Wisconsin Genetics Computer Group (UWGCG). The GAP program utilizes the alignment method of Needlman-Wunsch (J.Mol.Biol. 1970, 48: 443), adjusted by Smith and Waterman (Adv.Appl. Math. 1981, 2: 482). Briefly, the GAP defines similarity as the number of aligned symbols (i.e. nucleotides or amino acids), which are similar, divided by the total number of symbols in the shorter of the two sequences. The preferred default parameters for the GAP program include: (1) unitary matrix comparison (contains a numeric value of 1 for identities and 0 for neodenticula) and weighted matrix comparison Gribskov and Burgess (Nucl. Acids Res. 1986, 14: 6745), as described in Schwartz and Dayhoff, eds. Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, Washington, D.C. 1979, pp.353-358; (2) a penalty of 3.0 for each gap and an additional penalty of 0.10 for each symbol in each gap; and (3) no penalty for end gaps.
Used herein, the term "homologue" refers to the specified fused protein or Rib and alpha protein of the species Streptococcus agalactiae, where one or more than one amino acid residue in the amino acid sequence (SEQ ID NO: 2 or 4) is replaced by another amino acid residue, provided that the overall functionality and immunogenic properties of protein-homologue saved. Such th the Chelyabinsk can be natural or can be produced synthetically or by recombinant DNA. Homologs of SEQ ID NO: 2 or 4 will have at least one epitope capable of activating antibodies, which interact with the Rib or alpha protein. Such homologs can have total homology (i.e. similarity or identity with Rib - or alpha protein at least 80%, for example 80-99%homology (i.e. similarity or identity, or any range therein.
Used herein, "derivative" is a polypeptide that changes one or more physical, chemical or biological properties. Such changes include, but are not limited to: amino acid substitution, modification, addition or deletion; changes in the pattern of epidemiologia, glycosylation or phosphorylation; reaction of free amino, carboxyl or hydroxyl side groups of amino acid residues present in the polypeptide, with other organic and inorganic molecules; and other changes, each of which may lead to changes in primary, secondary, or tertiary structure.
"Fragments" in this invention have at least one immunogenic epitope. Preferred fragments in this invention will induce an immune response sufficient to prevent or diminish the severity of the infection.
The term "pharmaceutically acceptable carrier" refers to any suitable when memy excipient, adjuvant, carrier, diluent conventionally used in pharmaceutical preparations.
The invention relates to a vaccine that protects against infections caused by group b Streptococcus (GBS), which is the most important cause of life-threatening bacterial infections in newborns. The present invention is based on the knowledge of the inventor and the realization that protein from cobblestay in the two major surface proteins of group b Streptococcus, namely proteins Rib and alpha, induces protective immunity.
With a long-term goal to develop a single-component vaccine on the basis of group b Streptococcus (GBS) is the author of the invention have analyzed whether the protein of the Rib and alpha to induce protective immunity. The large size of the Rib and alpha and genetic instability regions repeats allowed to come to the conclusion that protein should be obtained from cobblestay. However, the choice of cobblestay was not obvious, since the protective epitopes are present in the field of repetitions of proteins the alpha and the Rib. It has been unexpectedly discovered that a protein of N-terminal regions has properties superior to the properties of the fused protein from other regions of these proteins, i.e. repetitions, and induces good protective immunity.
In this description, unless specifically stated otherwise, the use of the noun in the unity of nom the number means "one or more".
Throughout the description of the invention any and all of the specific sources of the information included in this description by reference.
In the first aspect of the present invention relates to a fused protein containing at least one first fragment of the N-terminal region of the surface protein of group b Streptococcus, which merged with at least one second fragment of the N-terminal region of the surface protein of group b Streptococcus, where these first and second fragments of the N-terminal region of the surface protein of group b Streptococcus obtained from different surface proteins of group b Streptococcus, and where specified protein is able to induce protective immunity against Streptococcus group C.
Different surface proteins of Streptococcus, which can be part of a fused protein of the present invention, include, but are not limited to, the Rib-protein of group b Streptococcus, alpha-protein of group b Streptococcus, beta-protein of group b Streptococcus, Epsilon-protein of group b Streptococcus and/or R28 protein of Streptococcus group C.
According to one embodiments of the present invention relates to a fused protein containing a fragment of the N-terminal region of the Rib-protein of group b Streptococcus, which is fused with a fragment of the N-terminal region of alpha-protein of group b Streptococcus, where the specified merged the th protein is able to induce protective immunity against Streptococcus group C.
According to another embodiment of the present invention relates to a merger where the specified protein contains at least one first amino acid sequence SEQ ID NO: 2 or its analogue, homologue, derivative or immunologically related amino acid sequence, or fragments, fused with at least one second amino acid sequence SEQ ID NO: 4 or its analogue, homologue, derivative or immunologically related amino acid sequence or fragments. The specified at least one first amino acid sequence contains the amino acid sequence having at least 80, 85, 90, 95, 96, 97, 98 or 99%sequence identity with the amino acid sequence presented in SEQ ID NO: 2. The specified at least one second amino acid sequence contains the amino acid sequence having at least 80, 85, 90, 95, 96, 97, 98 or 99%sequence identity with the amino acid sequence presented in SEQ ID NO: 4. One example of a fused protein represented in SEQ ID NO: 6, and another example is a protein that contains a combination of three or more amino acid sequences selected from the group consisting of SEQ ID NO: 2 and SEQ ID NO: 4 or its parts.
Protein Rib with which peptococci group, which is also referred to in this description as the Rib and Rib-protein is a surface protein known in this field and are described, for example, in WO 9421685. The designation "Rib" stands for: resistance to proteases (R), immunity (i) and group (b). Protein Rib was first isolated from streptococcal strain of group b serotype III as a separate protein with a molecular mass of 95 kDa. Protein Rib is expressed by almost all streptococcal strains groups In clinically important serotypes III, which cause most cases of meningitis, and some strains of other serotypes, such as serotype II. Moreover, the Rib is expressed by all strains hypervirulent clone type III. Was developed the method of purification protein Rib, and it was demonstrated that antibodies to this protein protects against lethal infection caused by strains expressing the protein Rib (additional details, such as DNA and protein sequence, see WO 9421685).
Protein alpha group b Streptococcus, also referred to herein as alpha, alpha-protein and alpha antigen is a surface protein of group b Streptococcus, known in this area. In WO 9410317 described conjugate vaccine composition containing protein alpha. Native the precursor protein alpha Streptococcus group b, as described in WO 9410317 has molekularnu the mass of 108 kDa. The proposed splitting the signal sequence of 41 amino acids gives a Mature protein with a molecular mass of 104 kDa. (It should be noted, however, that it was subsequently shown that this signal sequence has a length of 56 amino acid residues (Stalhammar-Carlemalm et al., J.Exp.Med. 177, 1593; 1993)). N-terminal region 20 kDa alpha-antigen does not show homology with the previously described protein sequences, and it is followed by a series of nine tandem repeating units that make up 74% of the Mature protein. Each repeating unit (here designated as "R") is identical and consists of 82 amino acids with a molecular mass of approximately 8500 daltons, and is encoded 246 nucleotides. C-terminal region of the alpha antigen contains a motif anchor domain of the cell wall present in a wide range of gram-positive surface proteins.
Each of the proteins Rib and alpha GBS includes a unique N-terminal region (N) and the area of long repeat (R). These proteins are expressed by strains of GBS BM110 and I are 12 and 9 repetitions, respectively, as indicated on Figa. The area of fixation in the cell wall localized to the C-terminal ends.
Tandem repeats in the Rib and alpha are identical within each protein, but not between proteins, and vary in number between isolates. In addition to this variation, the sequence of the Rib and alpha hundred is strong among strains. Despite the substantial identity of amino acid residues (Figa), these two proteins show a slight cross-reactivity or may not show cross-reactivity.
Protein R28 represents a surface protein of group b Streptococcus, which provides protective immunity and stimulates binding to epithelial cells (Stalhammar-Carlemalm et al. Molecular Microbiology 1999, 33, 208-219).
Protein Epsilon is a similar alpha protein protein of group b Streptococcus (Creti et al. Clin. Environ. 2004, 42: 1326-9).
The term "N-terminal region" for the purposes of this invention relates to N-Terminus (N) protein. Examples of amino acid sequences of the Rib and alpha N-terminal regions such as shown in SEQ ID NO: 2 and SEQ ID NO: 4.
For the purposes of the present invention, the term "fused protein" refers to the associate of two or more regions of the protein or its fragments, containing, for example, a fragment of the N-terminal region of the protein Rib of group b Streptococcus and a fragment of the N-terminal region of the protein alpha Streptococcus group C. for Example, it may be a fragment of the N-terminal region of the Rib-protein and a fragment of the N-terminal region of alpha-protein, or 2, 3, 4 or 5 fragments of the N-terminal region of the Rib - and alpha-proteins, where the number of fragments of the two proteins are not equal.
Examples of fragments of the N-terminal region of the protein Rib Streptococcus group In fragmental N-terminal region of the protein alpha Streptococcus group include peptides, encoding the native amino acid sequence N-terminal regions of natural proteins alpha and Rib (for example SEQ ID NO: 2 and SEQ ID NO: 4), or it can be functional derivatives of the native sequences of these regions, and these functional derivatives retain the ability to induce protective immunity against Streptococcus group C. the Term "functional derivatives" covers a part of the sequences and fragments of the N-terminal regions. It also covers variants of natural proteins (such as proteins, which are changes in amino acid sequence but retain the ability to be immunogenic property, virulence or antigenic property, which shows the natural molecule), for example variants with altered flanking sequence.
According to the present invention can be used fragments of the N-terminal region from different strains of Streptococcus group C. It also involves mild variability in the sequence of fragments of the N-terminal region, but does not alter the biological properties and their functional ability to induce protective immunity. For example, mean that the antigens alpha and the Rib of group b Streptococcus isolated from different strains of group b Streptococcus, but not those which are disclosed in SEQ ID NO: 2 and SEQ ID NO: 4 that is fixed in the amount of us who Otsego of the invention.
The Association of polypeptides with obtaining the fused protein can be accomplished by several methods, for example: chemical combination, conjugation or cross-linking, either directly or through an intermediate structure; physically combination through capture or macromolecular structure; or molecular biological merge by combining molecules of recombinant nucleic acids, which contain fragments of nucleic acids that can encode each of the two, so in the end formed a single continuous expressional product.
For the purposes of the present invention, the term "protein" refers to a molecular chain of amino acids. Protein does not have a specific length and can, if required, be modified in vivo or in vitro, for example, by glycosylation, amidation, carboxylation or phosphorylation. Among other things, this definition encompasses peptides, oligopeptides and polypeptides. The protein or peptide can be of natural or synthetic origin. In this context, fused protein refers to two or more polypeptides covalently linked to each other either directly or indirectly in several ways, such as the methods mentioned above. The term "fused" refers to the creation of a fused protein, as mentioned above.
Strains with whom peptococci group, also referred to here as GBS, is well known and can be isolated from the blood of infected creatures. GBS is the most common cause of neonatal sepsis in the United States and is responsible for approximately 5,000 cases per year.
The designation "group b Streptococcus" stems from the fact that streptococci are divided into immunological groups based on the presence of specific carbohydrate antigens on their cell surfaces. Currently known group a-O (Davis, B.D. et al., In: Microbiology, 3rd. Edition, page 609, (Harper & Row, 1980)).
The term "protective immunity" with respect to this invention refers to the ability of serum antibodies and/or cytotoxic T-cell response that is called for immunization to protect (partially or totally) from diseases caused by an infectious agent such as Streptococcus group C. that is, vertebrate animal subjected to immunization vaccines according to the invention, growth and distribution of group b Streptococcus will be limited. To determine whether induced protective immunity fused protein or vaccine, you can use methods known to the person skilled in the art. For example, to determine induces whether immunization fused protein or vaccine according to the invention protective immunity against infections caused by group b Streptococcus,test immunized animals exposed to Streptococcus group b, and measure the growth and spread of Streptococcus group C. For example, to determine whether induced protective immunity, can be used the methods described in the examples below.
In one of the embodiments of the present invention protein further comprises a fragment of the N-terminal region R28 protein of group b Streptococcus (Gene bank acc no: AAD39085.1) and/or a fragment of the N-terminal region of the protein Epsilon Streptococcus group C.
In one of the embodiments of the present invention fused protein of the present invention contains a repeating peptide sequence fragments of N-terminal regions of proteins of group b Streptococcus (i.e. alpha and Rib).
According to one embodiments of the present invention protein contains an amino acid sequence having at least 80%, 85%, preferably 90%, more preferably 95%sequence identity with the amino acid sequence presented in SEQ ID NO: 6.
The term "sequence identity" indicates a quantitative measure of the degree of homology between two amino acid sequences of equal length, or between two nucleotide sequences of equal length. If two sequences that compare not equal length, they must be aligned to the best possible match. The sequence identity can be calculated, for example, using the program BLAST, for example, the programs BLASTP or BLASTN program (Pearson W.R and D.J. Lipman (1988) PNAS USA 85:2444-2448) (www.ncbl.nlm.nlh.gov/BLAST).
According to another embodiment of the present invention protein contains the amino acid sequence of which is presented in SEQ ID NO: 6.
The selected DNA and expression systems
In the second aspect of the present invention proposed a selected nucleotide sequence/DNA molecule containing the nucleotide sequence/sequence of DNA which encodes a protein according to the invention. One example is a nucleotide sequence containing at least one first nucleotide sequence represented in SEQ ID NO: 1, or its fragments, fused with at least one second nucleotide sequence represented in SEQ ID NO: 3, or its fragments.
Also proposed recombinant expression system comprising the vectors and cells of the host.
In ekspressirovannoj nucleotide sequences of this invention can be used with a wide variety of gene-expression combinations of host/vector. Useful expression vectors for eukaryotic hosts include, for example, vectors containing sequences controlling the expression of SV40 virus, bovine papillomavirus, adenovirus, adeno-associated virus, cytomegalovirus and retroviruses. Useful expression vectors for bacterial hosts include bacterial plasmids, such as plasmids from E. coli, including pBluescript, pGEX2T, pUC vectors, col El, pCRI, pBR322, pMB9 and their derivatives, plasmids for a wider range of hosts, such as RP4, phage DNA, such as the numerous derivatives of phage lambda, e.g. lambda GTIO and lambda GTI 1, NM989, and other DNA phages, such as M13 and filamentous single-stranded phage DNA. Useful expression vectors for yeast cells include plasmid 2.mu. and its derivatives. Useful vectors for insect cells include pVL 941.
In addition, any of a wide variety of sequences controlling the expression can be used in these vectors for the expression of nucleotide sequences/DNA sequences according to this invention. Useful sequences controlling the expression, include sequences controlling the expression associated with the structural genes of the above expression vectors. Examples of useful sequences controlling the expression include, for example, the early and late promoters of SV40 or adenovirus, the lac system, the trp system, the TAC or TRC system, the promoters, the T3 and T7, the major operator and promoter sites of phage lambda, the control plots fd top coat protein, the promoter for 3-phosphoglycerate or other glycol is critical enzymes, the promoters of acid phosphatase, e.g Pho5, the promoters of the yeast alpha-coupled systems and other constitutive and inducible promoter sequences known that they control the expression of genes of prokaryotic or eukaryotic cells or their viruses, and various combinations thereof.
Cell host transformed by the above vectors, constitute another aspect of the present invention. A variety of unicellular hosts useful in ekspressirovannoj nucleotide sequences/DNA sequences according to this invention. These hosts may include well known eukaryotic and prokaryotic hosts, such as gram-negative and gram-positive strains, such as strains of E.coli, Pseudomonas, Bacillus, Streptomyces, Streptococcus, Staphylococcus, Lactobacillus, Aspergillus, Shigella, Salmonella, Listeria, fungus, yeast, insect cells such as Spodoptera frugiperda (SF9), animal cells such as Cho (cells Chinese hamster ovary) cells and mouse cells, African green monkey, such as COS 1, COS 7, BSC 1, BSC 40, and BMT 10, and human cells and cell plants in tissue culture. Preferred organisms-hosts include bacteria such as E. coli and .subtilis, and mammalian cells in tissue culture.
It is clear, of course, that not all vectors and sequences that control e is cpressey, function ekspressirovali nucleotide sequences/DNA sequences according to this invention will be equally good. Also not all owners will be equally good function with the same expression system. However, the person skilled in the art may make a selection among these vectors, sequences controlling the expression, and hosts without undue experimentation and without going beyond the scope of the invention. For example, the selection vector, it is necessary to take into account the host, because the vector to replicate in it. The number of copies of the vector, the ability to control this number of copies, and the expression of any other proteins encoded by this vector, such as antibiotic markers, should also be taken into account. When selecting a sequence controlling the expression, you should consider various factors. They include, for example, the relative strength of the sequence, its controllability, and its compatibility with nucleotide sequences/DNA sequences of this invention, especially in regard to potential secondary structures. Unicellular hosts should be selected by consideration of their compatibility with the chosen vector, the toxicity of the product encoded by the nucleotide sequences/DNA sequences by Dan the WMD to the invention, characteristics secretion, their ability to minimize protein, fermentation or cultivation and ease of purification of the products encoded by the nucleotide sequences/DNA sequences of this invention. Within these parameters, the person skilled in the art may choose different combinations of vector/sequence controlling the expression/owner that will Express the nucleotide sequences/DNA sequence according to this invention under cultivation or animal culture enlarged scale.
Polypeptides encoded by nucleotide sequences/DNA sequences according to this invention, can be isolated from microbiological cultures or cell culture and purified using various conventional methods, including liquid chromatography, such as normanniana or brasindiana HPLC (high performance liquid chromatography, FPLC (fast liquid chromatography of proteins) and the like, affinity chromatography (such as with inorganic ligands or monoclonal antibodies), ion-exchange chromatography, exclusion chromatography, metallokhelaty chromatography, gel electrophoresis, etc. the person skilled in the art can select the most suitable techniques for the isolation and purification, without going beyond the scope of the invention.
In addition, the polypeptides according to this invention can be obtained by any of several chemical methods. For example, they can be obtained using solid-phase synthetic method, originally described R..Merrifield (J.Am. Chem. Soc. 1963, 83: 2149-54), or they can be obtained by synthesis in solution. A description of the methods of peptide synthesis can be found in .Gross & .J.Meinhofer, 4 The Peptides: Analysis, Synthesis, Biology; Modern Techniques Of Peptide and Amino Acid Analysis, John Wiley & Sons, (1981); and .Bodanszky, Principles Of Peptide Synthesis, Springer-Verlag (1984).
Preferred compositions and methods of this invention include polypeptides having enhanced immunogenicity. Such polypeptides can be caused by modification of the native forms of the polypeptides or their fragments or as a result of processing, providing increased their immunogenic character of the intended recipient. Specialist in this field are available and known numerous techniques that can be used, without undue experimentation, to greatly increase the immunogenicity of the polypeptides described herein. For example, the polypeptides can be modified by binding dinitrophenolate groups or arsanilate acid, or by denaturation using heat and/or SDS (sodium dodecyl sulfate). In particular, if the floor is peptides are small polypeptides, chemically synthesized, it may be desirable to link them to immunogenic carrier. Of course, the linking should not affect the ability of either the polypeptide or the media to function properly. Review some General considerations concerning the strategies linking published in Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, ed. .Harlow and D.Lane (1988). Useful immunogenic carriers well-known in this field. Examples of such carriers are hemocyanin fissurella (KLH); albumins such as bovine serum albumin (BSA) and ovalbumin, PPD (purified protein derivative of tuberculin); red blood cells; tetanus toxoid; cholera toxoid; agarose beads; activated carbon; or bentonite.
The expression can be carried out in the so-called cell-free expression systems. Such systems contain all the necessary factors for the expression of the corresponding recombinant nucleic acid is functionally linked to the promoter, which will operate in this particular system.
Nucleotide sequence/DNA sequence N-terminal regions of the Rib and alpha such as represented in SEQ ID NO: 1 and SEQ ID NO: 3, and the nucleotide sequence/DNA sequence fused protein used in the examples below, such as the representation is prohibited in SEQ ID NO: 5.
In one of the embodiments of the invention relates to a method for the specified fused protein comprising the following stages: obtaining a host cell, as described above, containing the nucleotide sequence as described above; the reproduction of the specified host cell in a suitable medium a host, well-known specialist in this area; cleaning specified fused protein using one or more of the above methods; and obtaining the specified fused protein, which can then be used to prepare vaccines, as described below.
In the third aspect of the present invention proposed vaccine containing protein according to the invention and a pharmaceutically acceptable carrier.
Vaccine composition of the present invention may contain, in addition to the fused protein, other pharmaceutically acceptable ingredients, such as salts, buffers, immunoactive components, adjuvants, wetting agents, emulsifiers and suspendresume agents or sweeteners, corrigentov, flavorings, or other substances which are desirable for increasing the effectiveness of the composition. About the song saying that she "pharmacologically acceptable"if the individual is a recipient may transfer its introduction.
Polyvalent vaccine may also be prepared by volume is inane fused protein with other components, including, but not limited to, diphtheria toxoid or tetanus toxoid, or polysaccharides, using methods known in this field.
Other examples of preferred proteins multivalent vaccine of the present invention include additional surface proteins of group b Streptococcus or cash equivalents, such as the R28 protein and protein Epsilon.
In one embodiments, the vaccine composition of the present invention contains a fragment of the R28 protein of group b Streptococcus and/or protein fragment Epsilon Streptococcus group C.
The methods of preparation of vaccine compositions and their preparations well-known to specialists in this field. The choice of ingredients will vary, for example, depending on the route of administration of the composition. For example, compositions for parenteral administration include sterile or non-sterile solutions, suspensions and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters, such as etiloleat. Media or occlusive dressings can be used to increase permeability through the skin and enhance absorption of antigens. Liquid dosage forms for oral administration can contain mostly liposomal solution containing Jew the second dosage form. Suitable forms for the suspension of liposomes include emulsions, suspensions, solutions, syrups and elixirs containing inert diluents commonly used in this field, such as purified water.
Vaccine composition of the present invention may contain additional immunoactive component. Additional immunoactive component can be an antigen, enhances immunity substance and/or a vaccine, and they can all contain adjuvant.
Adjuvants are substances that can be used, in particular, for the increment of the specific immune response. Usually before the presentation in the immune system adjuvant and the composition is mixed, or represent them separately but in the same place of the immunized animal or human. Adjuvants can be divided on the basis of their composition approximately by several groups. These groups include oil adjuvants (for example, complete and incomplete beta-blockers), mineral salts (for example AIK(SO4)2, AINa(SO4)2, AINH4(SO4)), silicon dioxide, kaolin, and carbon, polynucleotide (for example acid poly IC (polyinosine: policitally acid) and poly AU (polyadenylate: poliuridilova acid), and certain natural substances (for example, wax D from Mycobacterium tuberculosis, as well as substances, obnaruzhivaemye Corynebacterium parvum, or Bordetella pertussis, and members of the genus Brucella). Substances, particularly useful as adjuvants include saponins, such as Quil A. Examples of substances suitable for use in vaccine compositions are provided in Remington''s Pharmaceutical Sciences (Osol, A, Ed, Mack Publishing Co, Easton, PA, pp.1324-1341 (1980). In yet another embodiment of the fused protein according to the invention can be used as a carrier for polysaccharide in a conjugate vaccine, In this embodiment, the vaccine contains a protein, i.e. a protein conjugated with a polysaccharide (such as capsular polysaccharide).
The use of the polypeptide, protein or fused protein as a carrier for polysaccharide in a conjugate vaccine known in the art (see, for example, US 6855321, WO 9410317 and US 4496538).
Under the polysaccharide refers to any linear or branched polymer consisting of monosaccharide residues, usually related glycosidic bonds, and, respectively, including oligosaccharides. Preferably, the polysaccharide contains from 2 to 50 monosaccharide units, more preferably from 6 to 30 monosaccharide units.
The polysaccharide component may be based on or derived from the polysaccharide components of the polysaccharide capsule of many gram-positive and gram-negative bacterial pathogens, such as .influenzae, N.meningitidis and S.pneumoniae. Other bacteria, polysaccharide components of which can be the ü conjugated to protein carriers of the present invention, include Staphylococcus aureus, Klebsiella, Pseudomonas, Salmonella typhi, Pseudomonas aeruginosa and Shigella dysenteriae. Polysaccharide components suitable for use according to this aspect of the present invention include Hib oligosaccharide, lipopolysaccharide from Pseudomonas aeruginosa (Seid and Sadoff, 1981), lipopolysaccharides from Salmonella (Konadu et al., 1996) and O-specific polysaccharide of Shigella dysenteriae (Chu et al, 1991). Other polysaccharide components suitable for use in accordance with the present invention, well-known experts in this field.
The fragments of bacterial capsular polysaccharide can be obtained by any suitable method, such as acid hydrolysis or ultrasonic irradiation (Szn et al, 1986). Other methods of obtaining the polysaccharide components of well-known experts in this field.
In one of the embodiments of the present invention, the polysaccharide is a capsular polysaccharide derived from group b Streptococcus, or its equivalent.
Polysaccharide component of the conjugate vaccine is preferably linked to a protein carrier covalent bond. Especially preferred method of linking a polysaccharide and protein is reductive amination. Other methods include activation of the polysaccharide labronica with subsequent interaction with dihydrazide adipic acid (spacer) and conjugation with carbon the ilen groups of carrier protein with soluble carbodiimide (Shneerson et al, 1986); funktsionalizirovannyi of carrier protein by dihydrazide adipic acid with subsequent binding to activated labronica polysaccharides (Dick et al, 1989); chemical modification as of carrier protein and polysaccharide their subsequent binding (Kim et at, 1986; Kim et al, 1987 and 1989).
Polysaccharide molecule may be linked to a protein carrier through the molecule is a spacer, such as adipic acid. This molecule is a spacer may be used to facilitate binding protein with a polysaccharide. After the reaction of binding of the conjugate can be purified by diafiltration or other known methods to remove unreacted unreacted protein or polysaccharide components.
If the polysaccharide obtained from the bacterial pathogen other than GBS, the conjugate can induce immunity against two or more pathogens, such as many types of bacteria. This is a potentially important application of the fused protein. For the preparation of conjugate vaccines considerable advantage may be that the protein consists of a single fused protein.
For the person skilled in the art it is obvious that the vaccine composition of the present invention may contain other substances or compounds that are not mentioned above, for example, other diluents, emulsifiers or stabilizer is, or other proteins or polysaccharides. Such substances or compounds should give the composition the desired properties.
Methods of prevention and treatment of infections caused by group b Streptococcus
In another aspect of the present invention proposed ways to prevent or treat infections caused by Streptococcus group C. These methods include the introduction of individual pharmaceutically effective amount of the vaccine according to the invention. According to the invention also proposed the use of immunogenic compositions according to the invention for the manufacture of a vaccine for the prevention or treatment of infections caused by Streptococcus group C.
Immunoprophylaxis mother vaccine for protection against infection by group b Streptococcus in both mother and offspring has long been proposed as a possible path.
The term "preventing or treating" in its various grammatical forms in the context of the present invention relates to the prevention, cure, reverse, reduce, relief, improvement, inhibiting, minimizing, suppressing or stopping (1) the harmful effects of disorders associated with infection caused by group b Streptococcus, (2) the progression of the disorder or (3) of the pathogen disorders (group b Streptococcus). The term "prevention or treatment" also encompasses what formirovanie the individual's total or partial immunity to infection, caused by Streptococcus group C.
According to one embodiments of this method of prevention or treatment includes the introduction of females effective amount of the vaccine according to the invention is able to develop immunity to infections caused by group b Streptococcus, is not begotten offspring specified females. According to this embodiment the vaccine is not injected pregnant females or pregnant females in temporary and quantitative conditions sufficient to induce the production of antibodies, which serve to protect both females and fetus or newborn (through passive transfer of antibodies through the placenta).
In yet another embodiment of the method of prevention or treatment of infections caused by group b Streptococcus, includes an introduction to the individual an effective amount of antisera induced by the impact of the vaccine according to the invention for the second individual. According to this embodiment, the resistance to group b Streptococcus is formed from the individual passive immunization, i.e. the vaccine is administered to the host-volunteer (i.e. human or mammal), and induced anticigarette isolated and injected directly to the recipient, who suspect infection caused by Streptococcus group C. it is Assumed that such as is taylored you can enter the pregnant females (during birth or before birth) in temporary and quantitative terms, sufficient for anticavity could serve to protect and fetus, and the newborn (through passive incorporation of antibodies through the placenta).
Thus, the vaccine or anticigarette can be entered either before infection (to protect or reduce the anticipated infection)or after the initiation of a real infection.
Vaccine composition or anticigarette according to the invention it is possible to introduce people or animals, including mammals and birds, such as rodents (mouse, rat, Guinea pig or rabbit), poultry (Turkey, duck or chicken), other farm animals (cow, horse, pig, or pig), animals (dog, cat and other Pets and people. Although the preparation according to the invention can be treated many animals, preferred to treat the individual is a man or having commercial value of the animal and cattle.
Vaccine composition or anticigarette according to the invention it is possible to enter individual methods known in this field. Such methods include the introduction of, for example parenterally, such as the introduction of any by injection into or through the skin, such as intramuscular, intravenous, intraperitoneal, intradermal, mucosal, submucosal or subcutaneous. They can also be used by the local application in the de drops, spray, gel or ointment on the epithelium of the mucous membranes of the eyes, nose, mouth, anus or vagina, or on the epidermis of the outer integument in any part of the body. Other possible applications are the use of a spray, aerosol or powder inhalation through the respiratory tract. In this latter case, the used, the particle size will determine how deep the particles will penetrate into the respiratory tract. Alternatively, you can enter through the digestive tract, by combining with pizza, food or drinking water, for example in the form of a powder, liquid or tablets, or by injection directly into the oral cavity in the form of liquid, gel, tablets or capsules, or in the anus in the form of a suppository. The vaccine can also be entered in the form of DNA vaccines.
For the distribution of immunization in time there are many different techniques. The composition of the invention can be used more than once to increase the level and diversification of the expression of the immunoglobulin repertoire expressed subjected to immunize animals. Typically, if multiple immunization, it is carried out with an interval of one to two months.
The term "effective amount" in the context of the present invention refers to that amount which provides a therapeutic effect for a given status is I and mode of administration. This amount is a predetermined amount of the active substance, calculated to produce the desired therapeutic effect together with the required additives and diluents, i.e. a carrier or solvent for injection. This term also means a quantity sufficient to reduce and, most preferably, to prevent clinically significant deficit in the activity and response of the host. Alternatively, therapeutically effective amount is sufficient to cause an improvement in a clinically significant condition in the host. As is well known to specialists in this field, the number of connections may vary depending on its specific activity. Appropriate dose quantity can contain a predetermined amount of the active composition calculated to produce the desired therapeutic effect together with the required diluent, i.e. a carrier or additive. In addition, the insertion dosage will vary depending on the active principle or principles, according to the age, weight and other characteristics of the individual, which are treated.
Generally, the dosage will consist of the initial injection, most likely with adjuvant, from about 0.01 to 10 mg, and preferably from 0.1 to 1.0 mg fused protein-antigenre of individuals with the most likely one, or perhaps more booster injections of doses. Preferably, booster doses administered approximately 1 and 6 months after the initial injection.
In order that this invention could be better understood, the following examples. It should be borne in mind, however, that the following examples are given to illustrate the present invention, and the invention is not limited to the specific conditions and details described in these examples.
The following examples used the following strains of group b Streptococcus (GBS): A (type Ia), SB35sed1 (type Ib), 1954/92 (type II) and VM (type III) (Larsson et al. Infect. Immun. 1996, 64:3518-3523; Stalhammar-Carlemalm et al. J.Exp.Med. 1993, 177: 1593-1603). Strain VM is a member hypervirulent clone ST-17. All GBS strains were grown in broth Todd-Hewitt in 3°C without shaking.
All the above mentioned strains were obtained by the inventors of the Lund University and Lund University hospital (Dr. Gunnar Lindahl, Department of medical Microbiology [University of Lund and the Lund University Hospital (Doctor Gunnar Lindahl, Department of Medical Microbiology, Solvegatan 23, SE-22362 Lund, Sweden)].
Example 1: Design of the Rib - and alpha-negative bacterial mutants
Rib-negative mutant was obtained from WM. A fragment of ~7 TPN (thousand base pairs)that carries the gene rib and flanking sequences, was subcloned into the pJRS233 (Perez-Casal et al, Mol. Environ. 193, 8: 809-819).
Gene rib was deleterule inverse PCR (polymerase chain reaction) was replaced with the kanamycin-resistant cassette. After transformation in VM Rib-negative mutant was isolated homologous recombination (Perez-Casal, J. et al, 1993). A gene rib is absent in this mutant, in contrast to previously described (Waldemarsson et al. J.Bacteriol. 2006, 188: 378-388). The structure of the mutant was confirmed by PCR. This mutant lacked reactivity with anti-Rib serum, but this did not affect the expression of the capsule. Alpha-negative mutant A designed in a similar way. This mutant lacked reactivity with anti-alpha serum, but this did not affect the expression of capsules or beta-protein.
Example 2: construction of the fused proteins and other derivatives Rib and alpha
In the examples described here used the intact proteins and a series of recombinant proteins (see Figv). Fragments of the gene Rib (SEQ ID NO: 1) in VM and bca gene encoding alpha-protein (SEQ ID NO: 3) in A cloned in pGEX-6P-2 (Amersham) and used to obtain GST-fusions (GST-glutathione-S-transferase). After removal of the GST purified derivative had the N-terminal sequence GPLGS. RibN and Rib2R correspond to amino acid residues respectively 1-174 and 175-332 Rib protein, and aaN corresponds to residues 1-170 alpha protein (numbering from Wastfelt et al. J.Biol. Chem. 1996, 271: 18892-18897). RibN-aaN contains amino acids 1-174 Rib-protein and AMI is ocelote 1-170 alpha protein and Rib2R-2R 12 contains amino acids 175-332 Rib-protein and amino acids 171-334 alpha protein. As a result of procedures each protein includes a sequence of EF between the two areas. Rib and alpha was purified from VM and A respectively.
Example 3: Analysis of purified proteins
On Figs presents the analysis of the purified proteins by the method of SDS-PAGE (polyacrylamide gel electrophoresis with sodium dodecyl sulfate). The image combined two of the gel. Numbers on the left indicate molecular mass in kDa. Because of the Rib and alpha migrate in gels aberrante the apparent sizes of the proteins do not correspond exactly to the dimensions derived from amino acid sequences.
Example 4: Testing immunodominant areas of repetitions Rib-protein and alpha-protein
Rabbit anticigarette received subcutaneous immunization approximately 35 µg protein in CFA (complete beta-blockers), and then two booster doses of about 18 µg protein in IFA (incomplete beta-blockers). Mice were immunized subcutaneously with 25 μg of protein with adjuvant or without adjuvant, as indicated, were immunized again after 4 weeks 12 µg protein and two weeks later, the blood was collected. For CFA-mice booster dose was injected with IFA.
Tests on the binding and inhibition of antibody (Fig.1D) was performed essentially as described in Stalhammar-Carlemalm et al., J.Exp.Med. 1993, 177: 1593-1603; Wastfelt et al. J.Bio. Chem. 1996, 271: 18892-18897 to Analyze whether murine anti-Rib antibodies induced using alum as adjuvant, directed against the N-terminal region and/or area of repetitions. Antibodies induced using alum as adjuvant, was used to detect pure Rib immobilized in microtiter wells, and the binding is inhibited by adding the specified pure protein (2 μg). Related rabbit antibodies were detected using protein G with a radioactive label, and the bound mouse antibodies were detected by incubation with rabbit antibody against mouse Ig, then protein G with a radioactive label. Binding was calculated in % protein G bound peroxidase at the lowest dilution of antisera. Sensitivity tests on inhibition (Fig.1D) optimized using the coating solution 0.05 mg/ml and mouse serum diluted 1000-fold. All tests were conducted at least three times, and the values of SD (standard deviation) are shown. For dot-blot analysis, membranes were incubated with the indicated mouse serum and bound peroxidase antibodies were detected by incubation with rabbit antibody against mouse Ig, then protein G with a radioactive label and autoradiography.
As expected, the Rib completely inhibited binding of the Rib, and nearly complete Engibarov the tion was also observed with Rib2R, while RibN had very little effect. Thus, almost all antibodies were directed against replays. Inhibition of protein Rib2R was nonspecific, since it does not inhibit the binding of the antibody to an unrelated GBS antigen (data not shown).
In the alpha system, the dot-blot analysis showed that anti-alpha reacted with intact alpha, not alfam (File, left). The lack of reactivity of N is not a property inherent to this construction, since the anti-N reacted with the alpha, and the N (File, right).
The reason immunodominant areas of repetitions in the Rib and alpha is not known. Multivalent interactions between repeats and Ig receptors on b cells can contribute, but Rib and alpha are not T-cell-independent antigens, because they induce IgG responses. Noteworthy is the fact that a weak immune response to the NH2-terminal region was not the result of masking, since these areas are accessible to antibodies (see below).
Example 5: Passive vaccination
It would seem that as antibodies to the Rib and alpha is directed almost exclusively against replays, and provide protection, a vaccine based on the fused protein should be obtained from these repetitions. However, available data did not rule out the fact that the selected N-terminal region could provide the best the th protection, than repetitions, and be suitable for the construction of the fused protein. To analyze this hypothesis, the authors present invention used Rib system for direct comparison of the protective capacity of antibodies directed against the N-terminal region or repetitions. In the analysis used rabbit antibodies induced RibN or Rib2R, and a murine model of passive vaccination.
Passive immunization was performed as described in Stalhammar-Carlemalm et al., J.Exp.Med. 1993, 177: 1593-1603, using mice SN/HeN, rabbit antisera and lethal dose LD90bacteria in log phase (105-106CFU (colony forming unit), depending on the strain). Survival was recorded over a time period of 96 hours For active immunization mice were immunized subcutaneously with 10 μg of protein, mixed with alum. 5 µg realnaya dose was administered after 4 weeks, with alum. Control mice received PBS (phosphate buffered saline) and alum. Two weeks after the second immunization, the mice were subjected to lethal dose LD90bacteria and recorded survival. All experiments were approved by the local oversight Committee for research on animals.
Antibodies reacted with Rib-expressing bacteria, but not with Rib-negative mutant, demonstrating that they recognize epitopes, exponere is installed on the native form of the Rib (Figa). Because anti-RibN serum was approximately 7-fold higher titer than anti-Rib2R serum, diluted, respectively, to be able to directly compare in murine models. In this model, each anticavity protected from lethal infection (Pigv), and diluted anti-RibN protected at least as well as undiluted anti-Rib2R. By p-values refer to comparisons with non-immune control at 96 hours. The test results in Rib was allowed to assume that a protein of N-terminal regions of the Rib and alpha should be compared with the fused protein of repetitions. However, it is not clear that a protein of N-terminal regions are required, since these regions are 61%identical residues (Figa), which indicates that they may respond to a crosshair. The cross-reactivity could pass unnoticed in previous studies, which used antibodies against the intact protein, i.e. antibodies directed primarily against replays.
This hypothesis was analyzed using anti-RibN and anti-N (Figa). Each anticavity reacted with whole bacteria from the Rib-expressing strain WM (left unfilled symbols), but not with Rib-negative mutant (left, shaded symbols). Similarly, each anticavity reacted with bacterial alpha-Express the dominant strain A (right, unfilled symbols), but not with alpha-negative mutant (right, shaded symbols). Similar data were obtained using two rabbit sera of each type. This indicates that the N-terminal region does not have cross-reactivity. So was designed protein RibN-N, and it was compared with the fused protein of similar size from the repeats, Rib2R-2R. Judging by reactivity with Rib - or alpha-expressing bacteria (Pigv), rabbit protein RibN-N evoked the best response of antibodies than Rib2R-2R. For comparison of passive protection in the mouse model of anti-(RibN-N) serum diluted to the same titer, and anti-(Rib2R-2R) serum. Each anticavity protected from Rib-expressing strain of type III and alpha-expressing strain of type Ia (Figs). Thus, each of the two fused proteins induced protective antibodies directed against the Rib and alpha.
Example 6: Passive vaccination for multiple serotypes of GBS
To analyze whether the protection provided by anti-(RibN-N) serum, independent of capsular serotype, was used to model passive immunization. Good protection was observed in experiments with Rib-expressing strain of type II and alpha-expressing strain of type Ib (Fig.3D). So, anti-(RibN-N) protected from Rib and al is a-expressing strains of the four classic serotypes, Ia, Ib, II and III. This protection was not nonspecific, because anti-(RibN-N) not protected from Rib-negative mutant (Fige). It is noteworthy that this analysis can be used Rib-negative mutant, because he did not show reduced virulence in the mouse model. Antibodies to RibN-N also recognized strains expressing two proteins related proteins Rib and α proteins R28 and Epsilon, which is expressed by many strains of serotypes V and Ia, respectively (Lindahl et al., Clin. Environ. Rev. 2005, 18: 102-127; Brimil et al. Int. J.Med. Environ. 2006, 296: 39-44). However, for protection against strains expressing R28 or Epsilon, you may need to design fused protein comprising N-terminal region of these proteins.
Example 7: Active vaccination
Figure 4 presents the results of an active immunization fused protein RibN-altham. (A) Immunogenicity RibN-N with the introduction of adjuvant or without adjuvant. Groups of four mice were subjected to immunization RibN-N mixed with CFA, alum or PBS, were subjected to repeated immunization 4 weeks and the blood was collected 2 weeks later. Mouse serum was analyzed for reactivity with purified antigen, immobilized in microtiter wells. Bound peroxidase mouse antibodies were detected by incubation with rabbit antibody against lg mouse, then protein G with radioactive match the th. (In) Active vaccination fused protein RibN-N. Mice (in the amount indicated on the y-axis) were subjected to immunization pure RibN-N, mixed with alum, was subjected to repeated immunization 4 weeks and 2 weeks later was introduced to them Rib-expressing strain VM type III (left), or alpha-expressing strain A type Ia (right). Control mice received PBS and alum. Data for the alpha strain were collected from two experiments. By p-values refer to comparisons of 96 hours.
Active immunization pure RibN-N this protein was equally immunogenic for mice with the introduction of CFA, alum or PBS (Figa). Moreover, active immunization RibN-N and alum protected mice from Rib and alpha-expressing strains (Pigv). Thus, RibN-N induced protective immunity with adjuvant suitable for use in humans.
Antibodies induced RibN-N, represented almost exclusively IgG antibodies (data not shown). Extrapolated to humans, these data suggest that the fetus may be protected by maternal anti-(RibN-N) antibodies. This conclusion is reinforced by the fact that it was found that antibodies to the intact Rib and alpha are transported across the human placenta.
In contrast to the results obtained with RibN-N, protein Rib2R-2R induced antibodies only one is the first of four CFA mice and not induced antibodies in mice who received antigen with alum or PBS (data not shown). So, Rib2R-2R was poorly immunogenic for mice, although the intact Rib and alpha caused a good immune responses to repetitions. These data confirm that RibN-N is of particular interest as a vaccine component.
Example 8: Antibodies to RibN-N prevent invasion of epithelial cells
Figure 5 shows that antibodies to RibN-N prevent invasion of epithelial cells. (A) the Role of the Rib and alpha in invasion of epithelial cells. Rib-negative mutant strain FM (left) and alpha-negative mutant strain A (right) compared with the corresponding bacterial wild-type (WT) according to their ability to infect cells of the human cervical cell line ME. (C) Inhibition of invasion of epithelial cells by protein anti-(RibN-N). Bacteria strain VM (left) or A (right) was preincubated with rabbit anti-(RibN-N) or with non-immune serum and then used in the analysis of invasion. All data in diagrams (a) and based on three different experiments. The values of SD and p are specified.
Overnight bacterial cultures were washed in PBS, resuspendable environment DME (supplemented with 10 mm Hepes and 4 mm L-glutamine) to 1×107CFU ml-1and the sample (500 µl) was added to a monolayer of human cervical cell line ME ATSS NTSS), wirawan the mu to 100%confluently in well 24-well plate. Added bacteria ranged from 6.7×106CFU to 2.7×107CFU. The tablet was centrifuged at 800×g for 10 minutes and incubated for 1 h at 37°C. After five washes with PBS to each well was added DME (1 ml)containing gentamicin (500 mcgm-1) and penicillin G (5 mcgm-1), and incubation was continued for 2 hours After 3 washes with PBS the cells were tsalala using trypsin-EDTA (ethylenediaminetetraacetic acid) and subjected to lysis of 0.025%Triton X-100, and intracellular bacteria was determined by cultivation in the cups. To analyze the inhibition of invasion by anticorodal, washed bacteria (500 μl) was mixed with anticorodal (50 ál) and incubated at room temperature for 30 minutes. The mixture was added to the monolayer ME. Determined the number of CFU before and after incubation with anticorodal. The analysis is then performed as described above. Non-immune rabbit serum was used as control. The fraction of bacteria that inhabit ME, in the absence of antisera was of 0.13-0.37 per cent of the inoculum.
Studies in Primate models showed that GBS invasive epithelial cells during infection. Because alpha stimulates invasion GBS in vitro, the authors of the present invention compared the role of the Rib and alpha in invasion, using GBS mutants (Figa). Invasion of human cells BE decreased 20 times for Rib-Mut the NTA and 4 times for the alpha mutant compared with the parent strains. Thus, the Rib and alpha have in common the ability to stimulate invasion. This is a potentially important function effectively blocked fused protein anti-(RibN-N) (Pigv). The decrease in invasion was not the result of antibody-mediated bacterial aggregation, which has not occurred in the conditions used (data not shown). This result suggests that the anti-(RibN-N) blocks of biologically important function.
Test data protection in mice were analyzed using two-sided Fisher's exact test. The analysis of data obtained in tests on the invasion of epithelial cells, was based on the standard normal approximation estimates of maximum probability for two independent, distributed according to a binomial law variables. Differences were considered statistically significant at p<0,05.
In conclusion, the authors of the present invention demonstrated that the N-terminal region of the Rib and alpha can be used to produce vaccines based on the fused protein, which exceeds the vaccine is derived from repetition. As for GBS vaccines for humans, the findings also indicate that protein RibN-N can induce protective immunity against many clinically important strains, including most strains causing meningitis.Although in the examples described in detail the preferred embodiment of the present invention, the experts in this field it is obvious that there may be modifications and adaptations of the present invention. However, it should be clearly understood that such modifications and adaptations are included in the scope of the present invention, which is defined by the claims.
1. Protein is able to induce protective immunity against group b Streptococcus, containing at least two amino acid sequences, where the two amino acid sequences consist of a first amino acid sequence having at least 90%sequence identity with the amino acid sequence presented in SEQ ID NO: 2, fused with a second amino acid sequence having at least 90%sequence identity with the amino acid sequence presented in SEQ ID NO: 4.
2. Fused protein according to claim 1, where the specified first amino acid sequence has at least 95, 96, 97, 98 or 99%sequence identity with the amino acid sequence presented in SEQ ID NO: 2, or where the specified second amino acid sequence has at least 95, 96, 97, 98 or 99%sequence identity with the amino acid is based sequence, presented in SEQ ID NO: 4.
3. Fused protein according to claim 1, which contains an amino acid sequence having at least 90%identity with the amino acid sequence presented in SEQ ID NO: 6.
4. Fused protein according to claim 3, which contains an amino acid sequence having at least 95%identity with the amino acid sequence presented in SEQ ID NO: 6.
5. Protein according to any one of claims 1, 2, which contains three or more amino acid sequences selected from the amino acid sequence as defined in claim 1 or 2.
6. Fused protein according to claim 1, modified by glycosylation, amidation, carboxylation or phosphorylation.
7. The vaccine containing a pharmaceutically effective amount of the fused protein according to any one of claims 1 to 6, is able to induce protective immunity against group b Streptococcus, containing a pharmaceutically acceptable carrier.
8. The vaccine according to claim 7, which further comprises adjuvant.
9. The vaccine according to any one of claims 7, 8, where the specified protein konjugierte with the polysaccharide with the formation of the conjugate vaccine.
SUBSTANCE: invention relates to biotechnology. Described is a plasmid which codes esterase Psychrobacter cryohalolentis K5T and contains Ndel/Xhol - a DNA fragment of plasmid pET32a with length of 5.366 thousand base pairs, which includes a promoter of bacteriophage T7, a translation enhancer of gene 10 of the bacteriophage T7, a transcription terminator of the bacteriophage T7, a bla β-lactamase gene which determines resistance of cells transformed by plasmid pPC023 to ampicillin, a replication initiation section ori; and Ndel/Sall - a DNA fragment with size of 0.869 thousand base pairs, which contains a gene which codes the mature form of esterase Psychrobacter cryohalolentis K5T with an amino acid sequence SEQ ID NO: 2, given in the description, having an C-end hexahestidine linker. Provided is an Escherichia coli bacteria strain which is modified by said plasmid, a producer of a polypeptide having esterase Psychrobacter cryohalolentis K5T activity.
EFFECT: invention increases the level of biosynthesis of esterase to 20% of total cell protein.
2 cl, 2 dwg, 5 ex
SUBSTANCE: method involves transformation of a Clostridium acetobutylicum cell by a vector containing: a replicon origin enabling its replication in C. acetobutylicum; a replacement cartridge containing a first marker gene surrounded by two sequences homologous to selected sites around the DNA target sequence, enabling recombination of said cartridge; a second marker gene representing upp counter-selection marker. The cells expressing the first marker gene are selected with the cartridge integrated in their genome. The cells not expressing the second marker gene with the eliminated said vector are selected.
EFFECT: invention enables producing the transformed Clostridium acetobutylicum cell which is genetically stable and marker-free.
31 cl, 6 dwg, 4 ex
SUBSTANCE: there are presented a polynucleotide coding an antibody, an expression vector, a host cell, compositions containing the antibody, and also a method for preparing the antibody and methods for using the antibody.
EFFECT: invention may be used for preparing therapeutic and diagnostic agents to be used for the purpose of detecting the pathological conditions associated with expression or activity of ephrine B2 ligand pathways.
27 cl, 10 dwg, 3 tbl, 6 ex
SUBSTANCE: polypeptide with phytase activity is proposed. The sequences are shown in the description. A polynucleotide coding the said polypeptide is described. The application of the said polypeptide for the use in a feed mix or a feed supplement is described.
EFFECT: allows to increase modified phytase resistance against temperature and humidity.
6 cl, 6 ex, 12 tbl, 10 dwg
SUBSTANCE: disclosed is procariotic recombinant host cell containing heterologous protein of replication activation, which activated condition-dependent replicon origin, and extrachromosomal DNA molecule, containing heterologous therapeutic gene and condition-dependent replicon origin. Also disclosed are method for producing of plasmid containing heterologous therapeutic gene and condition-dependent replicon origin, plasmid representing suicide vector for gene transferring, containing heterologous therapeutic gene and condition-dependent replicon origin.
EFFECT: method for avoiding of non-controlled overexpression of therapeutic gene and resistance genes.
44 cl, 41 dwg, 10 tbl, 14 ex
FIELD: biotechnology, genetic engineering, pharmaceutical industry.
SUBSTANCE: plasmid DNA pET23-a(+)/PrxVIhumΔ178 with molecular weight of 19691.61 Da is constructed. DNA contains RNA-polymerase T7 promoter; replication initiation site; genetic marker which determinates resistance of cells transformed by said plasmid to ampicillin; and nucleotide sequence encoding N-terminal fragment of human peroxiredoxine VI containing 177 of amino acid residues. E.coli strain BL21/DE3/pET23-a(+)/PrxVIhumΔ178 being producer of N-terminal fragment of human peroxiredoxine VI is obtained by transformation of E.coli cells with plasmid DNA pET23-a(+)/PrxVIhumΔ178. Method of present invention makes it possible to obtain human peroxiredoxine VI fragment having reduced molecular weight, improved tissue permeability, and antioxidant activity of full-scale peroxiredoxine.
EFFECT: human peroxiredoxine VI fragment with improved tissue permeability.
2 cl, 3 dwg, 4 ex
FIELD: medicine, pharmaceutics.
SUBSTANCE: inventions refers to biotechnology and concern hypoallergic fused proteins. A presented hypoallergic fused protein consists of one hypoallergic molecule originated from an allergen, fused with a second non-allergic protein originated from a pathogen, or a fragment thereof with the hypoallergic molecule originated from the allergen showing 50% decreased ability to bind IgE, 30% decreased T-cell responsiveness as compared with a wild-type allergen. A nucleic acid molecule coding the fused protein, an expression vector, a host cell expressing the same protein, and a vaccine composition containing the protein are also presented.
EFFECT: presented invention enables preparing therapeutic and prophylactic drugs for an allergy or diseases caused by pathogens with using no toxic adjuvants, showing no side effects.
14 cl, 23 dwg, 17 tbl, 27 ex
FIELD: medicine, pharmaceutics.
SUBSTANCE: invention refers to biotechnology, more specifically preparing a tumour necrosis factor receptor and may be used in medicine. Genetic engineering technique is used to produce mutant TNFRp75 bound to a tumour necrosis factor and lymphotoxins substantially consisting of N-terminal 257 amino acid residues TNFRp75 wherein the N-terminal residue Glu92 is substituted by Asn, His, Ser or Ala and wherein the N-terminal residue Trp89 is optionally substituted by Tyr or Phe. The produced mutant is used to construct a fused protein with an additional amino acid fragment specified in a constant area of human immunoglobulin and one of five functional areas of albumin found on C-terminal of the soluble mutant TNFRp75. The produced mutant TNFRp75 and the fused protein is used as a part of a pharmaceutical composition for treating the diseases associated with TNFα overexpression which involve rheumatoid arthritis, psoriasis, sclerodermatitis, Sjogren syndrome, Strumpell-Marie disease, lupus erythematosus, acute disseminated myositis and syndrome similar to systemic lupus erythematosus.
EFFECT: invention enables producing soluble TNFRp75 mutant able to be bound to tumour necrosis factor and lymphotoxins at a high affinity degree.
9 cl, 12 dwg, 10 ex
SUBSTANCE: invention relates to biotechnology, specifically obtaining immunogens from Neisseria meningitides and can be used in medicine. Disclosed is protein which is 80% or more identical to amino acid sequence SEQ ID NO:19, and an immunogenic composition with said protein.
EFFECT: invention enables to use said protein for effective prevention or treatment of bacterial meningitis.
6 cl, 5 dwg, 28 tbl
SUBSTANCE: what is presented is a polynucleotide coding a fused protein of telomerase reverse transcriptase (TERT) wherein an inactive version of TERT polypeptide is combined with a considerable portion of a subunit B of a heat-labile enterotoxin (LTB). It is shown that the fused protein under the invention induces an immune response in a mammal which in the preferential versions of the implementation is stronger than that induced by isolated wild-type TERT.
EFFECT: invention discloses preparing the fused protein with the version of hTERT and necessary genetic structures, particularly adenovirus vectors and plasmids, as well as host cells providing expression of a polynucleotide coding the fused protein under the invention.
7 cl, 19 dwg, 14 ex
SUBSTANCE: there are presented two recombinant plasmid DNA pFastBac 1 -G2R-dSECRET and pQE-60-TNFR-CrmB-Ind-67 coding TNF-binding CrmB protein domain. Said recombinant plasmid DNA pQE-60-TNFR-CrmB-Ind-67 is designed for transformation in cells of the strain E.coli SG13009[pRep4] - a producer of TNF-binding CrmB BHO protein domain.
EFFECT: presented group of inventions is applicable for preparing drugs used in therapy of severe human diseases caused by hyperproduction of tumour necrosis factor and may be used in medicine.
3 cl, 3 dwg, 12 ex
SUBSTANCE: invention relates to biotechnology and can be used to obtain recombinant human interferon-gamma gene. Plasmid DNA pGD-14 is constructed, which determines expression in Escherichia coli bacteria cells of a genetically engineered structure consisting of an N-terminal sequence of a dextran-binding domain of betaccoci and the human interferon-gamma gene, which are joined in a single reading frame through an aspartate-proline acid-labile spacer.
EFFECT: invention provides stable expression of the dextran-binding domain of betacocci and human interferon-gamma gene at 20% of the total protein of the transformed Escherichia coli cell.
3 dwg, 3 ex
SUBSTANCE: invention relates to biotechnology and specifically to obtaining factor VII (FVII) and factor Vila (FVIIa) albumin linked polypeptides, and can be used in medicine. A polypeptide, which is a FVII or FVIIa polypeptide is obtained in a recombinant manner, said peptide being linked with albumins through a glycerine-serine peptide linker of a special structure, which separates part associated with FVII or FVIIa from the albumin part, wherein the FVII or FVIIa polypeptide lies on the N-end of the fused protein. The linked polypeptide or vector structure, which contains its coding nucleic acid, is used as a medicinal agent for treating or preventing blood-clotting disorders.
EFFECT: invention enables to obtain a protein with FVII or FVIIa biological activity and longer functional half-time in plasma in vitro compared to non-linked FVII or FVIIa.
12 cl, 4 dwg, 6 tbl, 6 ex
SUBSTANCE: hybrid polypeptide is a polypeptide 1 to which a polypeptide 2 is covalently bonded, where polypeptide 1 is a human endostatin sequence with 135-184 amino acid residues, wherein at position 173, Cis is replaced with Ala with a relatively native endostatin sequence, and polypeptide 2 is a human plasminogen sequence with 82-341 or 463-511 amino acid residues. Also disclosed is a method of obtaining the hybrid polypeptide using an E.coli producer, which involves methods for extraction and purification thereof.
EFFECT: polypeptide is capable of inhibiting human endothelial cell proliferation in vitro and can be used when producing nontoxic preparations for inhibiting angiogenesis.
4 cl, 4 dwg, 6 ex
SUBSTANCE: method involves providing a cell culture containing mammal cells which contain a gene coding rTNF-lg expression of which occurs in the cell culture medium. The medium containing glutamine and possessing the essential properties is used. Said culture is maintained in an initial growth phase at a first set of cultivation conditions during a first period of time. At least one of the cultivation conditions are modified to produce a second set of cultivation conditions with said cultivation condition at the specified stage of modification of at least one of cultivation conditions specified from the group consisting of: (i) temperature; (ii) pH; (iii) osmolality; (iv) a level of the chemical activator and their combinations. Said culture is maintained at the specified second set of conditions during a second period of time so that rTNF-lg is accumulated in the specified culture.
EFFECT: invention enables the scale rTNF-lg production in the cell culture.
48 cl, 76 dwg, 27 tbl, 17 ex
SUBSTANCE: fused proteins contain an endoglucanase nucleus amino acid sequence having at least 95% identity to SEQ ID NO:2, fused with an amino acid sequence containing a linker and a cellulose-binding domain (CBD), having at least 95% identity to SEQ ID NO:15. Such fused proteins can be obtained via a recombinant technique using suitable polynucleotides, expression vectors and host cells.
EFFECT: invention provides cellulase, having low activity with respect to restaining, and can be used to treat cellulose material; disclosed fused proteins and enzyme preparations based thereon can be used to prepare detergent compositions or for improving quality of animal feed.
26 cl, 8 dwg, 10 tbl, 10 ex
SUBSTANCE: disclosed are anti-5T4 antibodies, nucleic acids which encode variable regions of such antibodies, antibody/drug conjugates, a method of delivering a drug using such a conjugate, as well as a method of treating a subject with cancer which is characterised by 5T4 antibody expression, by administering the disclosed conjugate.
EFFECT: present invention can further be used in therapy of 5T4-associated diseases.
42 cl, 8 ex, 15 tbl, 15 dwg