RU2232191C2 - Protein corresponding to neisseria meningitidis antigen protein and eliciting immunogenic properties (variants), antibody binding with it, nucleotide sequence (variants) and pharmaceutical composition comprising thereof - Google Patents

Abstract

FIELD: molecular biology, immunology, microbiology, pharmacy.

SUBSTANCE: invention represents proteins of meningococcus bacterium Neisseria meningitides (strains A and B) and Neisseria gonorrhoeae. Amino acid sequences of claimed proteins are represented in the description. Corresponding nucleotide sequences encoding the claimed proteins are represented also. Represented proteins are components of vaccines, immunogenic compositions as antigens and (or) can be used for diagnostic aims. Invention claims an antibody that binds with indicated protein. Invention provides enhancing the effectiveness of preparations used for diagnosis, treatment and prophylaxis of infections caused by Neisseria meningitides and Neisseria gonorrhoeae.

EFFECT: valuable medicinal properties of protein and composition.

12 cl, 2 tbl, 104 ex

Description

The present invention relates to antigens of bacteria of the genus Neisseria.

BACKGROUND OF THE INVENTION

The bacteria Neisseria meningitidis and Neisseria gonorrhoeae are immobile gram-negative diplococcal bacteria that are pathogenic to humans. N.meningitidis form colonies in the pharyngeal region and cause meningitis (and, in some cases, septicemia without meningitis); N.gonorrhoeae form colonies in the genital tract causing gonorrhea. Despite the fact that they form colonies in different parts of the body and cause completely different diseases, these two pathogens are very close to each other, although there is a clear difference between meningococcus and gonococcus associated with the presence of a polysaccharide capsule, which is found in all pathogenic meningococci.

Gonococcus N.gonorrhoeae causes approximately 800 thousand diseases per year for the period 1983-90. US only (chapter written by Meitzner & Cohen, 1997, "Vaccines Against Gonococcal Infection", In "New Generation Vaccines", 2d ed., ed. Levine, Woodrow, Kaper & Gobon, Marcel Dekker, NY, pp.817- 842). This disease is widespread, although mortality from it is low. Vaccination against the causative agent of gonorrhea is highly desirable, but numerous such attempts have been unsuccessful. The main “candidate antigens” for creating such vaccines are surface-located proteins, such as drank, porins, turbidity-associated proteins (Opas), and other surface proteins, such as Lip, Laz, IgAl protease, and transferrin-binding proteins. It was also proposed to use lipopolysaccharide (LOS) as a vaccine (Meitzner & Cohen, cited above).

Meningococcus N.meningitidis causes both endemic and epidemic forms of the disease. In the United States, the incidence rate is 0.6-1 per 100 thousand people per year, and this rate can increase in conditions of an outbreak of the disease (see Lieberman et al., 1996, "Safety and Immunogenicity of a Serogroups A / C Neisseria meningitidis Oligosaccharide- Protein Conjugate Vaccine in Young Children ", JAMA, 275 [19], 1499-1503; Schuchat et al., 1997," Bacterial Meningitis in the United States in 1995 ", New England J. Med., 337 [14], 970 -976). In developing countries, the frequency of endemic cases of the disease is significantly higher, and when epidemics occur, this indicator can reach 500 cases per 100 thousand people per year. The mortality rate is very high - approximately 10-20% in the United States and even higher in developing countries. Following the introduction of the Haemophilus influenzae combination vaccine, N. meningitidis meningococcus becomes the main causative agent of bacterial meningitis in all age groups in the United States (Schuchat et al., 1997, cited above).

Based on the parameters of the polysaccharide meningococcus capsule constituting the capsule, 12 N.meningitidis serogroups were identified. Group A includes a pathogen, which is mainly associated with epidemiological forms of the disease in the sub-Saharan Africa. Serogroups B and C are associated with the vast majority of cases of meningitis in the United States and most developed countries. Serogroups W135 and Y are associated with other cases in the United States and developed countries. The currently used meningococcal vaccine is a tetravalent polysaccharide vaccine containing factors of serogroups A, C, Y and W135. Being effective in adolescents and adults, this vaccine provides a weak immune response and short-term protection, and also cannot be used for young children (see, for example, the weekly report "Morbidity and Mortality weekly report., Vol.46, N RR -5, 1997) .This is due to the fact that polysaccharides are T-cell independent antigens that produce a very weak immune response that cannot be boosted (boosted) by re-immunization. Having succeeded in vaccinating against H. influenzae were developed comb vaccines against serogroups A and C - their clinical trials are currently being completed (WDZollinger, "New and Improved Vaccines Against Meningococcal Disease", In "New Generation Vaccines", supra, pp. 469-488; Lieberman et al., 1996 cited above; Constantino et al., 1992, "Development and phase I clinical testing of a conjugate vaccine against meningococcus A and C", Vaccine, 10, 691-698).

However, the serotype B of meningococcus remains problematic. Currently, this serotype accounts for approximately 50% of the total number of cases of meningitis in the USA, Europe and South America. The “polysaccharide approach” cannot be used because the menB capsular polysaccharide is a polymer of α (2-8) N-acetylneuraminic acids bound, which are also present in mammalian tissues. This determines the tolerance to this antigen: indeed, assuming the manifestation of an immune response, it will also be directed to its own organism - i.e. such an answer is undesirable. In order to exclude the induction of an autoimmune response and the induction of a protective immune response, the polysaccharide contained in the capsule was, for example, chemically modified by replacing N-acetyl groups with N-propionyl groups, whereby the specific antigenicity remains unchanged (Romero & Outschoorn, 1994, "Current Status of Meningococcal group In vaccine candidates: capsular or non-capsular? ", Clin. Microbiol. Rev., 7 [4], 559-575).

Alternative approaches to the development of vaccines against meningitis-B used complex mixtures of external membrane proteins (OMP), including the OMP or OMP proteins enriched in porins, or deleted class 4 OMPs, which are believed to induce antibody production, blocking bactericidal activity. In this approach, vaccines are obtained, the full characteristics of which have not yet been obtained. These vaccines are able to provide protection against a homologous strain, but at the same time they are essentially ineffective in cases where there are numerous antigenic variants of the outer membrane proteins. To overcome the antigenic variation factor, multivalent vaccines were prepared containing up to 9 different porins (see, for example, J.T. Poolman, 1992, "Development of a meningococcal vaccine", Infect. Agents Dis., 4, 13-28). Other proteins that are used to create “external membrane vaccines” are the ora and ors proteins — however, none of the approaches used overcome the antigenic variation factor (see, for example, Ala'Aldeen & Borriello, 1996, “The meningococcal transferrinbinding proteins 1 and 2 are both surface exposed and generate bactericidal antibodies capable of killing homologous and heterologous strains ", Vaccine, 14, 49-53).

Some data are available on the sequences of meningococcal and gonococcal genes and proteins (for example, according to patent applications EP A-0467714 and WO 96/29412), however, of course, they are incomplete. Obtaining additional data on the sequences will provide good prospects for the identification of secreted or located on the cell surface proteins that are promising targets for the immune system and which are not characterized by antigenic variation. For example, some of the identified proteins could be components of effective meningococcal B vaccines, some could be components of vaccines against all meningococcal serotypes, and others could be components of vaccines against all pathogenic forms of the genus Neisseria.

Invention

The present invention provides proteins comprising amino acid sequences belonging to the neisseries described in the following examples. These sequences relate to N.meningitidis or N..gonorrhoeae.

Also provided are proteins comprising sequences homologous (i.e., characterized by sequence identity) to the amino acid sequences of the neisseries shown in the examples. Depending on the particular sequence, the level of identity is preferably greater than 50% (for example, 65, 80, 90% or more). These homologous proteins include mutant and allelic variants of the sequences described in the examples. Typically, a 50% or higher identity of the two proteins is considered evidence of functional equivalence. The level of identity of the two proteins is preferably determined by the Smith-Waterman method, the algorithm of which is embedded in the computer program MPSRCH (Oxford Molecular): the search for “affine gaps” (that is, those that do not coincide in two sequences of sections) is used with the setting of “gap open penalty = 12 "and" gap extension penalty = 1 ".

The present invention further provides proteins comprising fragments of the amino acid sequences of the neisseries described in the following examples. These fragments must include at least n continuous amino acids from the base sequence, and, depending on the particular sequence, n is 7 or more (for example, 8, 10, 12, 14, 16, 18, 20 or more). Preferably, such fragments include an epitope from the sequence.

The proteins of the present invention can be obtained, of course, using various approaches (for example, by methods of recombinant expression, purification from cell cultures, chemical synthesis, etc.) and in various forms (for example, native, chimeric, etc.) . Preferably, they are prepared in substantially pure or isolated form (i.e., substantially free of other proteins of the neisseria or cellular proteins of the host organism).

In accordance with a further aspect of the present invention, antibodies are provided that bind to such proteins. These can be polyclonal or monoclonal antibodies, which can be obtained using suitable methods.

In accordance with a further aspect of the present invention, nucleic acids are provided comprising the nucleotide sequences of the neisseries described in the examples.

In addition, the present invention provides nucleic acids comprising homologous sequences (i.e., characterized by sequence identity) with respect to the nucleotide sequences of the neisseries described in the examples.

Further, the present invention provides nucleic acids that can hybridize with the neisseria nucleic acids described in the examples, more preferably under stringent hybridization conditions (for example, at 65 ° C in a solution of 0.1 × SSC, 0.5% SDS).

Nucleic acids including fragments of such sequences are also provided. They must include at least n consecutive nucleotides from the sequence of neisseries, and, depending on the particular sequence, n is 10 or more (for example, 12, 14, 15, 18, 20, 25, 30, 35, 40 or more )

In accordance with a further aspect, the present invention provides nucleic acids encoding the proteins and protein fragments of the present invention.

It should also be understood that the present invention provides nucleic acids comprising sequences complementary to those sequences described above (for example, for the purpose of generating antisense sequences or probes).

Nucleic acids in accordance with the present invention can, of course, be obtained in many ways (for example, using chemical synthesis, from genomic DNA or cDNA libraries, directly from the body, etc.) and can take various forms (for example, single-stranded, double-stranded , vector shape, probe shape, etc.).

Additionally, it should be said that the term "nucleic acid" includes DNA and RNA, as well as their analogues, such as those that include modified molecular skeletons, as well as nucleoproteins (PNA) and the like.

In accordance with another aspect of the present invention, vectors are provided comprising nucleotide sequences of the present invention (eg, expression vectors) and host cells transformed with such vectors.

In accordance with a further aspect of the present invention, compositions are provided comprising a protein, an antibody and / or nucleic acid in accordance with the present invention. These compositions can be used as, for example, vaccines or as diagnostic reagents, or as immunogenic compositions.

The present invention also provides a nucleic acid, protein, or antibody of the invention for use as medicaments (e.g., as vaccines) or as diagnostic reagents. The use of a nucleic acid, protein, or antibody of the invention in the manufacture of: (1) a medicament intended for the treatment or prevention of infection by bacteria of the genus Neisseria; (2) a diagnostic reagent designed to detect the presence of Neisseria bacteria or antibodies specific for them; and (or) (3) a reagent that can cause the production of antibodies against neisseria. Mentioned bacteria of the genus Neisseria can be represented by any species or strain (such as N..gonorrhoeae or any strain of N. meningitidis, such as strain A, strain B or strain C).

The present invention also provides a method of treating a patient, comprising administering to the patient a therapeutically effective amount of a nucleic acid, protein and / or antibody of the invention.

In accordance with other aspects of the present invention, various methods are provided.

A method of producing proteins of the present invention is provided, comprising the step of culturing a host cell in accordance with the present invention under conditions that stimulate protein expression.

A method for producing the protein or nucleic acid of the present invention is provided, although such a protein or such nucleic acid is synthesized in whole or in part using chemical techniques.

A method for detecting polynucleotides of the present invention is provided, comprising the following steps: (1) contacting a nucleotide probe of the present invention with a biological sample under conditions suitable for molecular hybridization to form duplexes; and (2) detection of said duplexes.

A method for detecting proteins of the present invention is provided, comprising the following steps: (1) contacting an antibody of the present invention with a biological sample under conditions suitable for forming an antigen-antibody complex; and (2) detection of said complexes.

The following is a review of standard methodologies and procedures that can be used to implement the present invention (for example, to use the claimed sequences for vaccination or for diagnostic purposes). This review is not a limitation of the present invention, but it is an example of such an implementation, which is not strictly required.

General Provisions

The practical implementation of the present invention is based, except as otherwise specified, on standard techniques of molecular biology, microbiology, recombinant DNA and immunology, which are known to specialists in this field of technology. Such techniques are described in detail in the scientific literature: for example, Sambrook, 1989, "Molecular Cloning: A Laboratory Manual", 2d Ed .; "DNA Cloning", Vol. I & II, ed. D.N. Glover, 1985; "Oligonucleotide Synthesis", ed. M.J. Gait, 1984; "Nucleic Acid Hybridization", eds. B. D. Hames & S. J. Higgins 1984; "Transcription and Translation", eds. B. D. Hames & S. J. Higgins 1984; "Animal Cell Culture", ed. R.I. Freshney, 1986; "Immobilized Cells and Enzymes", IRL Press, 1986; B. Perbal, 1984, "A Practical Guide to Molecular Cloning"; Methods in Enzymology series of tutorials (published by Academic Press Inc.), especially Volumes 154 and 155; "Gene Transfer Vectors for Mammalian Cells", eds. J. H. Miller & M. P. Calos, Cold Spring Harbor Lab., 1987; "Immunochemical Methods in Cell and Molecular Biology", eds. Mayer & Walker, Acad. Press, London, 1987; Scopes, 1987, "Protein Purification: Principles and Practice", 2d ed., Springer-Verlag, NY; and "Handbook of Experimantal Immunology", Vol. I-IV, eds. D.M. Weir & C. C. Blackwell, 1986).

In the present description, standard abbreviations are used to refer to amino acids and nucleotides.

All publications, patents, and patent applications cited in this text are included in their entirety in the form of bibliographic references. In particular, British Patent Applications No. 9723516.2, 9724190.5, 9724386.9, 9725158.1, 9726147.3, 9800759.4 and 9819016.8 are included in this text for information.

Definitions

A composition comprising X is “substantially free of Y” when at least 85% by weight of the sum of X + Y is in component X. Preferably X is at least about 90% by weight of the total of X + Y in this composition, more preferably at least about 95% or even 99% by weight.

The term “comprising” means “consisting”, as well as “comprising”: for example, a composition “comprising” X may consist solely of component X or may include something additional to X, for example, a combination of X + Y.

The term “heterologous” refers to two biological components that are not found together in nature. Such components may be host cells, genes, or regulatory segments, such as promoters. Although heterologous components are not found together in nature, they can have joint functionality, for example, when a promoter heterologous with respect to a gene is functionally linked to it. Another example is a situation in which the neisseria sequence is heterologous with respect to the mouse host cell. Additional examples can be two epitopes from the composition of the same or different proteins that are combined in a single protein in a combination that is never found in nature.

“Replication origin” means a polynucleotide sequence that initiates and regulates the replication of polynucleotides, such as an expression vector. The point (or site) of the beginning of replication behaves as an autonomous unit of polynucleotide replication in the cell, providing the ability to replicate under its control. The presence of an origin of replication may be necessary to allow replication of the vector in a particular host cell. If there are several points of origin of replication, the expression vector can be reproduced in a large number of copies in the presence of suitable proteins within the cell. Autonomously replicating sequences that are effective in yeast cells, as well as viral T-antigens effective in COS-7 cells are examples of replication origin points.

The term "mutant sequence" defines a DNA, RNA or amino acid sequence that differs from the native or declared sequence, but at the same time having similarities with it. Depending on the particular sequence, the level of sequence identity when comparing the native or claimed sequence and the mutant sequence preferably exceeds 50% (amounting, for example, 60, 70, 80, 90, 95, 99% and more: the calculation is carried out using the Smith-Waterman algorithm, described above). As used in this text, the term "allelic variant" of a nucleic acid molecule or region for which a nucleotide sequence is presented is a nucleic acid molecule or segment that is essentially located in the same locus of a particular genome of another or second isolate, despite the fact that, in view of natural variation, caused, for example, by mutational or recombination processes, is characterized by a similar but not identical nucleotide sequence. The coding segment of an allelic variant usually encodes a protein having a similar level of activity compared to that of a protein encoded by the gene with which this comparison is made. Allelic variant may also include the alternation of 5'- or 3'-untranslated regions of a particular gene, such as regulatory control segments (see, for example, US patent No. 5753235).

Expression Systems

The nucleotide sequences of the neisseries can be expressed using various expression systems: for example, mammalian cells, baculoviruses, plants, bacteria and yeast are used.

I. Mammalian systems

Mammalian expression systems are known in the art. The mammalian promoter can be any DNA sequence capable of binding mammalian RNA polymerase, thereby initiating transcription of the downstream (i.e., 3 ′) coding sequence (e.g., structural gene) to form mRNA. The promoter should include a transcription initiation site, which is usually proximal to the 5'-end of the coding sequence, as well as a TATA box, usually located 25-30 nucleotides above the transcription initiation site. It is believed that the TATA box provides RNA polymerase II-controlled start of RNA synthesis at the correct site. The mammalian promoter should also include an upstream promoter element, typically located 100-200 nucleotides above the TATA box. The upper promoter element determines the rate at which transcription is initiated and can be active in any orientation (Sambrook et al., 1989, "Expression of Cloned Genes in Mammalian Cells", In "Molecular Cloning: A Laboratory Manual", 2d ed.) .

The genes of mammalian viruses are usually characterized by intense expression and are characterized by a wide range of hosts: therefore, sequences encoding the genes of mammalian viruses are particularly promising for use as promoter sequences. Examples are the SV40 early gene promoter, the mouse mammary tumor virus LTR promoter, the main late adenovirus gene promoter (AdMLP), and the herpes simplex virus promoter. In addition, sequences derived from non-viral genes, such as the mouse metallothionein gene, also represent useful promoter sequences. Expression can be either constitutive or regulated (inducible), which depends on the possibility of inducing the promoter by glucocorticoids in cells controlled by such hormones.

The presence of an enhancer element (enhancer) together with the promoter elements described above usually leads to increased levels of expression. An enhancer is a regulatory DNA sequence that is capable of stimulating transcription up to a thousand-fold level if it is bound to homologous or heterologous promoters, despite the fact that the synthesis starts at the normal RNA initiation site. Enhancers are also active when they are placed either above or below the site of transcription initiation, both in normal and inverted by the phase switching mechanism, or at a distance of even more than 1000 nucleotides from the promoter (Maniatis et al., 1987, Science, 236, 1237; Alberts et al., 1989, "Molecular Biology of the Cell", 2d ed.). In particular, enhancer elements derived from viral genomes can be used, since they are usually characterized by a wide range of potential hosts. Examples are the enhancer of the early gene of the SV40 virus (Dijkema et al., 1985, EMBO J., 4, 761) and the enhancer + promoter derived from the Routh sarcoma virus long terminal repeats (LTR) (Gorman et al., 1982b, Proc. Natl. Acad. Sci. USA, 79, 6777) and human cytomegalovirus (Boshart et al., 1985, Cell, 41, 521). In addition, some enhancers are adjustable and only become active in the presence of an inducer such as a hormone or metal ion (Sassone-Corsi & Borelli, 1986, Trends Genet., 2, 215; Maniatis et al., 1987, Science, 236, 1237 )

A DNA molecule can be expressed within mammalian cells. The promoter sequence can be directly attached to the DNA molecule, taking into account the fact that the first amino acid from the N-terminus of the recombinant protein should always be methionine, which is encoded by the ATG start codon. If desired, the N-terminal portion can be cleaved from the protein by in vitro incubation with cyanogen bromide.

On the other hand, foreign proteins can also be secreted from the cell directly into the culture medium as a result of the creation of chimeric DNA molecules that would encode a chimeric protein that includes the leader sequence of a segment that secrets the foreign protein in mammalian cells. It is preferable to have processing sites encoded between the leader segment and the foreign gene, which could be cleaved either in vivo or in vitro. The leader segment usually encodes a signal peptide consisting of hydrophobic amino acids that secrete protein from the cell. The adenovirus tripartite leader segment is an example of a leader sequence that provides secretion of a foreign protein by mammalian cells.

Typically, transcription termination and polyadenyl sequences recognized by mammalian cells are regulatory segments located on the 3 ′ side of the stop codon: i.e., together with promoter elements, they are motifs flanking the coding sequence. The 3'-end of mature mRNA is formed by site-specific post-transcriptional cleavage and polyadenylation (Birnstiel et al., 1985, Cell, 41, 349; Proudfoot & Whitelaw, 1988, "Termination and 3 'end processing of eukaryotic RNA", In " Transcription and splicing ", ed; BDHames &DMGlover; Proudfoot, 1989, Trends Bio-chem. Sci., 14, 105). These sequences provide for transcription of mRNA, which can then be translated into a polypeptide encoded by the original DNA. Examples of transcription termination and polyadenylation termination signals are those motifs that originate from the SV40 virus genome (Sambrook et al., 1989, "Expression of cloned genes in cultured mammalian cells". In "Molecular Cloning: A Laboratory Manual").

Typically, the components described above, including the promoter, polyadenyl signal, and transcription termination site, are simultaneously included in expression constructs. Enhancers, introns, including functional donor and acceptor splicing sites, and leader sequences may also be included in the expression construct, if desired. Expression constructs are often maintained as a replicon, such as an extrachromosomal element (e.g., plasmid), which can stably remain in the host organism, such as a mammalian cell or bacterium. Mammalian replication systems include those derived from animal viruses that require the participation of trans-regulatory factors to replicate. For example, plasmids including the replication systems of papovaviruses, such as the SV40 monkey virus (Gluzman, 1981, Cell, 23, 175), or poliomaviruses, replicate in an extremely large number of copies in the presence of a suitable viral T-antigen. Additional examples of replicons for mammalian cells are those derived from bovine papillomavirus and Epstein-Barr virus. In addition, such a replicon can carry two replication systems, thereby providing the ability to maintain them, for example, in mammalian cells for expression and in prokaryotic cells for cloning and amplification. Examples of such bifunctional vectors for mammals / bacteria include pMT2 constructs (Kaufman et al., 1989, Mol. Cell. Biol., 9, 946) and pHEBO (Shimizu et al., 1986, Mol. Cell. Biol., 6, 1074 )

The choice of transformation procedures used depends on the type of host organism that will be transformed. Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art: they include dextran-mediated transfection, precipitation of calcium phosphate, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of polynucleotide (s) into liposomes and direct DNA microinjection.

Mammalian cell lines suitable as hosts for expression are well known and include a large number of immortalized cell lines available from the American Type Culture Collection (ATCC), including but not limited to Chinese hamster ovary (CHO) cells, HeLa cells human cells, neonatal hamster kidney cells (BHK), green monkey kidney (COS) cells, human hepatocytic carcinoma cells (e.g., HepG2), and many other cell lines.

2. Baculovirus systems

A polynucleotide encoding a protein can also be integrated into a suitable expression vector for insect cells: it is functionally combined with regulatory elements within such a vector. When constructing a vector, techniques are used that are well known in the art. In General, the components of the expression system include the actual vector for transfer, usually a bacterial plasmid, which includes a fragment of the baculovirus genome, and a standard restriction site for the insertion of a heterologous gene or genes that will be expressed; wild-type baculovirus, characterized by sequence similarity with a baculovirus-specific fragment of the transfer vector (this ensures homologous recombination of the heterologous gene in the baculovirus genome); and suitable insect host cells and culture medium.

After the DNA sequence encoding the specific protein is introduced into the transfer vector, this vector and the wild-type viral genome are used to transfect into an insect host cell in which the vector and the viral genome can recombine. The packaged recombinant virus is expressed, and recombinant plaques can be identified and purified. Materials and methods for the formation of expression systems "baculovirus / insect cells" are available in the form of special kits on a commercial basis, for example, inter alia, from the company Invitrogen (San Diego, USA): the set of reagents "MahVas". These techniques are generally known to those skilled in the art and are fully described in the Summers & Smith manual (Summers & Smith, 1987, Texas Agricult. Exper. Stat. Bull., N 1555) (hereinafter referred to as "Summers & Smith") .

Before embedding a DNA coding sequence for a protein in the baculovirus genome, the components described above, including the promoter, the leader segment (if desired), the coding sequence of interest, and the transcription termination site are usually arranged in an intermediate construct (transfer vector). Such a design may include a single gene and regulatory elements functionally attached to it; or multiple genes, each of which has its own set of functionally attached regulatory elements; or multiple genes under the control of the same regulatory elements. Intermediate interleaved constructs are often maintained in the form of a replicon, such as an extrachromosomal element (eg, a plasmid), which can stably remain in the host organism, such as a bacterium. Such a replicon should include a replication system, thereby ensuring maintenance in a suitable host for the purpose of cloning and amplification.

Currently, the most common transfer vector for introducing foreign genes into AcNPV is pAC373. Many other vectors known to those skilled in the art can also be formed. These include, for example, pVL985 (which changes the start codon of the polyhedrin gene from ATG to ATT and which introduces a BamHI cloning site at 32 nucleotides below the ATT codon: see Luckow & Summers, 1989, Virology, 17, 31.

A commonly used plasmid also includes the polyadenylation signal of the polyhedrin gene (Miller et al., 1988, Ann. Rev. Microbiol., 42, 177) and the prokaryotic ampicillin resistance gene (amp) and the origin of replication necessary for selection and reproduction in E cells .coli.

Baculovirus transfection vectors typically include a baculovirus promoter. A baculovirus promoter is any DNA sequence capable of binding to baculovirus RNA polymerase and initiating transcription of a coding sequence (e.g., a structural gene) in the 5'-3 'direction to form mRNA. The promoter should include a transcription initiation site, which is usually placed proximal to the 5'-end of the coding sequence.

This transcription initiation site typically includes an RNA polymerase binding site and a transcription initiation site itself. The baculovirus transfection vector may also include a second domain, defined as an “enhancer," which, if present, is usually located distal to the structural gene. Expression can be either inducible or constitutive.

Structural genes intensively transcribed in the late stages of the viral infectious cycle allow us to isolate specifically applicable promoter sequences. Examples are sequences derived from a gene encoding a viral protein polyhedrine (Friesen et al., 1986, "The Regulation of Baculovirus Gene Expression", In "The Molecular Biology of Baculoviruses", ed. W.Doerfler; European Patent Publications No. 127839 and 155476), and a gene encoding the p10 protein (Vlak et al., 1988, J. Gen. Virol., 69, 765).

DNA encoding suitable signal sequences can be derived from genes encoding secreted insect or baculovirus proteins, such as the baculovirus polyhedrin gene (Carbonell et al., 1988, Gene, 73, 409). On the other hand, while the signals of post-translational modifications in mammalian cells (such as cleavage of the signal segment, proteolytic cleavage and phosphorylation) are apparently recognized by insect cells, the signals necessary for secretion and nuclear accumulation also Apparently, they are conservative in the cells of vertebrate and invertebrate animals; leader segments that are not related to the origin of insects, such as those that are derived from genes encoding human α-interferon (Maeda et al., 1985, Nature, 315, 592), human gastrin releasing factor (Lebacq-Verheyden et al., 1988, Mol. Cell. Biol., 8, 3129) human interleukin-2 (Smith et al., 1985, Proc. Natl. Acad. Sci. USA, 82, 8404), mouse interleukin-3 (Miyajima et al., 1987, Gene, 58, 273).

A recombinant polypeptide or polyprotein can be expressed intracellularly or, if expressed with the participation of special regulatory sequences, it can be secreted. Effective intracellular expression of non-chimeric carbon proteins usually requires heterologous genes, which ideally include a short leader sequence including suitable translation initiation signals prior to the ATG start codon. If desired, the N-terminal methionine residue can be cleaved from the mature protein by in vitro incubation with cyanogen bromide.

On the other hand, recombinant polyproteins or proteins that are not natively secreted can be secreted from insect cells by creating chimeric DNA molecules that encode a chimeric protein comprising a leader segment that secures the secretion of a protein foreign to insects. The leader sequence segment usually encodes a signal peptide consisting of hydrophobic amino acids that control the movement of the protein into the endoplasmic reticulum.

After embedding the DNA sequence and / or the gene encoding the expression product, which is the precursor of a specific protein, the insect host cell is simultaneously transformed with the heterologous DNA vector of the transfer and wild-type baculovirus genomic DNA - cotransfection methods are usually used. The promoter and transcription termination sequence of this construct usually should include a segment of the baculovirus genome with a length of 2-5 thousand pairs of nucleotides. Methods for introducing heterologous DNA at a desired site into a baculovirus are known in the art (see Summers & Smith, cit. Above; Ju et al., 1987; Smith et al., 1983, Mol. Cell. Biol., 3, 2156 ; Luckow & Summers, 1989). For example, embedding can be done inside a gene, such as the polyhedrin gene, for which homologous double recombination is used; embedding can also be carried out at a restriction enzyme site artificially created within the desired baculovirus gene (Miller et al., 1989, BioEssays, 4, 91). The DNA sequence, in the case when it is cloned into a region of the polyhedrin gene of the expression vector, is flanked on both sides (5 'and 3') by the sequences characteristic of the polyhedrin gene and placed below the polyhedrin promoter.

The newly formed baculovirus expression vector is sequentially packaged in an infectious recombinant baculovirus. Homologous recombination occurs at a low frequency (from about 1% to about 5%): therefore, the vast majority of viruses produced by co-transgranulation is wild-type. This means that a special approach is needed to identify recombinant viruses. An advantage of this expression system is the possibility of visual screening to detect recombinant viruses. The polyhedrin protein, characteristic of natural viruses, is produced in very large quantities in the nuclei of infected cells in the late stages of the viral infection cycle. The accumulated polyhedrine protein forms incorporated bodies, which also contain particles embedded in them. These included bodies up to 15 microns in size cause a significant refraction of light: this gives them a bright glow that can be easily visualized in a conventional light microscope. In cells infected with a recombinant virus, the included bodies are absent. To detect recombinant viruses and wild-type viruses, a transfection supernatant is applied to a monolayer culture of insect cells using techniques well known to those skilled in the art. Namely, plaques are analyzed under a light microscope to detect the presence (as a sign of a wild-type virus) or absence (as a sign of a recombinant virus) of the included bodies ("Current Protocols in Microbiology", Vol.2, eds. Ausubel et al., Section 16.8 (Supp.10, 1990); Summers & Smith, cited above; Miller et al., 1989).

Recombinant baculovirus expression vectors were designed to infect various types of insect cells. For example, among other things, recombinant baculoviruses were formed for: Aedes aegypti mosquito, Autographa californica scoops, Bombyx mori silkworm, Drosophila melanogaster, Dodophtera frugiperda and Trichoplusia ni scoops (patent application WO 89/04 al6699; Virol., 56, 153; Wright, 1986, Nature, 321, 718; Smith et al., 1983, Mol. Cell. Biol., 3, 2156; for a review, see Fraser et al., 1989, In Vitro Cell . Develop. Biol., 25, 225).

Cells and culture media are available commercially for both direct and chimeric expression of heterologous polypeptides in a baculovirus expression system; cell culture technology as a whole is well known to those skilled in the art (see, for example, Summers & Smith, cit. above).

The modified insect cells can then be grown in a suitable nutrient medium that will provide stable maintenance of the plasmid present in the modified insect host. In the case where the expression product is under the control of inducible regulators, the host organism can be grown at very high densities with expression induction. On the other hand, when expression is constitutive, the desired product will be constantly expressed in the culture medium, and the nutrient medium can be continuously circulated - this allows you to highlight the desired product of interest and replenish the depleted nutrient medium. This product can be purified using techniques such as chromatography, for example, HPLC, affinity chromatography, ion exchange chromatography and the like; electrophoresis; density gradient centrifugation; extraction from solution; or the like. If necessary, this product can be further purified to ensure substantial removal of any insect proteins that are also secreted into the culture medium or are formed as a result of lysis of insect cells, resulting in a product that is essentially free of debris from the hosts, for example, proteins, lipids and polysaccharides.

In order to ensure protein expression, recombinant host cells derived from the obtained transformants are incubated under conditions that allow expression of the sequence encoding the recombinant protein. These conditions may vary depending on the selected host cell. However, such conditions can easily be selected by a person skilled in the art based on known scientific data.

3. Plant systems

Numerous genetic expression systems based on plant cells and whole plants are known in the art. Examples of such genetic expression plant systems can be found in US patents Nos. 5693506, 5659122 and 5608143. Additional examples of genetic expression in cultured plant cells are described by Zenk (Zenk, 1991, Phytochemistry, 30, 3861-3863). Descriptions of the signal peptides of plant proteins can be found, in addition to the above sources, in Vaulcombe et al., 1987, Mol. Gen. Genet., 209, 33-40; Chandler et al., 1984, Plant Mol. Biol., 3, 407-418; Rogers et al., 1985, J. Biol. Chem., 260, 3731-3738; Rothstein et al., 1987, Gene, 55, 353-356; Whittier et al., 1987, Nucl. Acids Res., 15, 2515-2535; Wirsel et al., 1989, Mol. Microbiol., 3, 3-14; Yu et al., 1992, Gene, 112, 247-253. A description of the parameters for regulating the expression of plant genes involving phytohormones, gibberellic acid, and secreted enzymes induced by gibberellic acid can be found in the monograph by R. L. Jones & J. MacMillin, 1984, "Gibberellins", In "Advanced Plant Physiology", ed. M.B. Wilkins, Pitman Publ. Ltd, London, pp. 21-52. Materials describing other genes regulated by metabolic mechanisms: Sheen, 1990, Plant Cell, 2, 1027-1038; Maas et al., 1990, EMBO J., 9, 3447-3452; Benkel & Hickey, 1987, Proc. Natl. Acad. Sci. USA 84, 1337-1339.

Typically, using methodologies known in the art, the desired polynucleotide sequence is integrated into an expression cassette comprising genetic regulatory elements designed to function in plants. The expression cassette was inserted into the desired expression vector along with sequences located above and below the expression cassette suitable for expression in the host plant. The concomitant sequences may be of plasmid or viral origin and provide the necessary characteristics for the vector, which will provide the vectors with the ability to transfer DNA from the original host organism used for cloning, such as a bacterium, to the desired host plant. The basic bifunctional ("bacterial-plant") vector construct should preferably provide a wide range of hosts, include a prokaryotic origin of replication, a prokaryotic selective marker, and, in the variant with the transformation of Agrobacterium bacteria, a T-DNA sequence that allows gene transfer based on transformation with using Agrobacterium. When identification of a heterologous gene is difficult, this construct should preferably also include a selectable marker suitable for detecting whether a plant cell has been transformed. A review of selective markers, for example, members of the grass family, can be found in Wilmink & Dons, 1993, Plant Mol. Biol. Reptr., 11 (2): 165-185.

Sequences suitable for ensuring the integration of a heterologous sequence into the plant genome are also recommended. These may include transposon sequences and the like, which are associated with homologous recombination processes, as well as Ti sequences, which cause the heterologous expression cassettes to be accidentally inserted into the plant genome. Suitable prokaryotic selective markers include signs of resistance to antibiotics such as ampicillin or tetracycline. Other DNA sequences encoding additional functions may also be part of the vector, being known in the art.

The nucleic acid molecules of the present invention can be incorporated into an expression cassette designed to allow expression of the protein (s) of interest. Usually a single expression cassette is used, although two or more such cassettes can be used. The recombinant expression cassette must include, in addition to the sequence encoding the heterologous protein, the following elements: the promoter segment, 5'-untranslated sequences of the plant genome, the initiation codon, the presence or absence of which depends on whether it is present in the analyzed structural gene, and sites termination of transcription and translation. Unique restriction enzyme recognition sites at the 5'- and 3'-ends of this cassette provide convenient embedding into a pre-acting vector.

The heterologous coding sequence may be associated with any protein of the present invention. A sequence encoding a protein of interest will encode a signal segment that will process and move the protein, if necessary, and typically should be devoid of any sequence that could cause binding of the desired protein of the present invention to the membrane. While in most cases the transcription initiation site will be for a gene that is expressed and translocated during germination, by using a signal segment that allows for movement, the desired protein can also be moved. In this case, the protein (s) of interest will be transferred from the cells in which it has been expressed: therefore, it can be efficiently assembled. Usually, secretion in the seeds is provided from the aleuron region or corymbal epithelium into the endosperm of the seed. Although there is no specific need for the secretion of protein from the cells in which it was produced, this approach facilitates the isolation and purification of the recombinant protein.

Since the final expression of the desired gene product must occur in a eukaryotic cell, it is desirable to determine if any of the sections of the cloned gene contains sequences that could be lost as introns by the splicing mechanism of the host organism. If they are, then using the method of directed (site-specific) mutagenesis, such an “intron” region can be changed in such a way as to exclude the loss of part of the genetic material due to the presence of false-intron signals (Reed & Maniatis, 1985, Cell, 41 95-105).

The vector can be microinjected into plant cells using micropipettes, which will provide mechanical transfer of recombinant DNA (Crossway, 1985, Mol. Gen. Genet., 202, 179-185). Genetic material can also be transferred to plant cells using polyethylene glycol (Krens et al., 1982, Nature, 296, 72-74). Another method for introducing nucleic acid segments is through high-speed ballistic insertion of small particles (“shells”) inside or on the surface of which nucleic acid is located (Klein et al., 1987, Nature, 327, 70-73; Knudsen & Muller, 1991, Planta , 185, 330-336 - these works describe the application of the method of particle bombardment of barley endosperm with the aim of obtaining transgenic barley plants). Another method of incorporation may be fusion of protoplasts with other components, such as minicells, cells, lysosomes, or others capable of participating in fusion with protoplasts of the body having a lipid surface (Fraley et al., 1982, Proc. Natl. Acad. Sci. USA, 79, 1859-1863).

The vector can also be introduced into plant cells using electroporation (Fromm et al., 1985, Proc. Natl. Acad. Sci. USA, 82, 5824). In this technique, plant protoplasts are electroporated in the presence of plasmids including the gene construct. Electrical impulses, characterized by a high electric field strength, reversibly “perforate” biological membranes, thereby ensuring the penetration of plasmids inside. Electroporated plant protoplasts restore their cell wall, divide and form calluses.

All plants from which protoplasts can be isolated, followed by cultivation and production of whole regenerated plants, can be transformed in accordance with the present invention so that whole plants carrying the transferred gene are obtained. It is known that almost all plants can be regenerated from cultured cells or tissues, including, but not limited to, all major types of sugarcane, sugar beets, cotton, fruit and other trees, legumes and vegetables. Some suitable plants, for example, are the species of the genera Fragaria (strawberry), Lotus (lamb), Medicago (alfalfa), Onobrychis (sainfoin), Trifolium (clover), Trigonella (fenugreek), Vigna (vigna), Citrus (citrus), Linum (flax), Geranium (geranium), Manihot (cassava), Daucus (carrots), Arabidopsis (cucurbita), Brassica (cabbage, rutabaga, turnip), Raphanus (radish), Sinapis (mustard), Atropa (belladonna belladonna), Capsicum (vegetable pepper), Datura (datura), Hyoscyamus (belena), Lycopersion (tomato), Nicotiana (tobacco), Solanum (nightshade), Petunia (petunia), Digitalis (dewdrop), Majorana (marjoram), Cichorium (chicory) , Helianthus (sunflower), Lactuca (lettuce), Bromus (bonfire), Asparagus (asparagus), Antirrhinum (snapdragon), Hererocallis, Nemesia, Pelargonium (pelargonium), Panicum (millet), Pennisetum (American millet), Ranunculus (buttercup), Senecio (godson), Salpiglossis, Cucumis (cucumber, cantaloupe, melon) Growaine, Brow ), Lolium (chaff), Zea (corn), Triticum (wheat), Sorghum (sorghum) and Datura.

Regeneration methods vary depending on the type of plant, but in general, always first get a suspension of transformed protoplasts carrying copies of a heterologous gene. Then callus tissue is formed, in which shoots can be induced with the subsequent formation of roots. On the other hand, embryo formation can be induced in a protoplast suspension. These embryos germinate like ordinary embryos, forming plants. Culture media should usually contain various amino acids and hormones, such as auxin and cytokines. It is also preferred to add glutamic acid and proline to the culture medium, especially for species such as corn and alfalfa. Shoots and roots normally develop simultaneously. The efficiency of regeneration will depend on the culture medium, the genotype of the plant and on the parameters of a particular culture. If these three variables are under control, then regeneration is reproducible and repeatable.

In some plant cell culture systems, the desired protein of the present invention can be isolated on its own, or, on the other hand, such a protein can be extracted from a whole plant. In those cases where the desired protein of the present invention is secreted into the culture medium, it can be collected. Alternatively, embryos and half-seeds with the removed embryonic part or other plant tissues may be mechanically ground to isolate secreted protein from cells and tissues. The resulting mixture can be suspended in a buffer solution to re-isolate soluble proteins. Standard methods for the isolation and purification of proteins can be used to purify recombinant protein. The parameters of time, temperature, pH, oxygen content and volume can be adjusted using routine procedures to optimize expression and isolation of a heterologous protein.

4. Bacterial systems

Methods of expression in bacterial cells are well known. A bacterial promoter is any sequence. DNA capable of binding bacterial RNA polymerase and initiating transcription of the downstream (in the 3 'direction) coding sequence (e.g., structural gene) to form mRNA. The promoter should contain a transcription initiation site, which is usually proximal to the 5'-end of the coding sequence. This transcription initiation site typically includes an RNA polymerase binding site and a transcription initiation site itself. The bacterial promoter may also include a second domain called an “operator” that can overlap with an adjacent RNA polymerase binding site from which RNA synthesis begins. An operator provides negatively regulated (i.e., inducible) transcription: a repressor gene can bind to this operator, thereby inhibiting the transcription of a particular gene. Constitutive expression may occur in the absence of negative regulatory elements, such as an operator. In addition, positive regulation can be ensured by the sequence of binding of the gene-activating protein, which: if present, is located proximally (from the 5'-side) with respect to the binding site of the RNA polymerase. An example of a gene-activating protein is a catabolic activator (CAP), which is a helper to initiate transcription of the lac operon in the Escherichia coli Escherichia coli genome (E. coli) (Raibaud et al., 1984, Annu. Rev. Genet., 18, 173) . Thus, the regulation of expression can be negative or positive, thereby enhancing or weakening transcription.

Sequences encoding metabolic pathway enzymes represent specifically useful promoter sequences. Examples are promoter sequences derived from genes encoding enzymes involved in the metabolism of sugars such as galactose, lactose (lac) (Chang et al., 1977, Nature 198, 1056) and maltose. Further examples are promoter sequences derived from biosynthetic enzymes such as tryptophan biosynthesis enzymes (trp) (Goeddel et al., 1980, Nucl. Acids Res., 8, 4057; Yelverton et al., 1981, Nucl. Acids Res., 9, 731; U.S. Patent No. 4,738,921; European Patent Applications EP A-0036776 and EP A-0121775). The promoter system of g-laotamase (blа) (Weissmann, 1981, "The cloning of interferon and other errors". In "Interferon 3", ed. I. Gresser) and the promoter systems of PL λ-bacteriophage (Shimatake et al., 1981, Nature, 292.128) and T5 phage (US Pat. No. 4,689,406) can also be used as promoter sequences.

In addition, synthetic promoters that are not found in nature can also function as bacterial promoters. For example, transcription activation sequences of one of the bacterial or bacteriophage promoters can be linked to the operon sequences of another bacterial or bacteriophage promoter, resulting in the formation of a synthetic hybrid promoter (US patent No. 4551433). For example, the tac promoter is a trp-lac hybrid promoter consisting of the sequences of the trp promoter and the lac promoter, which are controlled by the lac repressor (Amann et al., 1983, Gene, 25, 167; de Boer et al., 1983, Proc. Natl. Acad. Sci. USA, 80, 21).

Further, the bacterial promoter may include naturally occurring promoters of non-bacterial origin that have the ability to bind bacterial RNA polymerase and initiate transcription. A naturally occurring promoter of non-bacterial origin can also be coupled with compatible RNA polymerase to provide a high level of expression of certain genes in prokaryotic cells. The RNA polymerase / promoter system of the T7 bacteriophage is an example of an integrated promoter system (Studier et al., 1986, J. Mol. Biol., 189, 113; Tabor et al., 1985, Proc. Natl. Acad. Sci. USA 82, 1074). In addition, the hybrid promoter may also consist of a bacteriophage promoter and an E. coli operator segment (EPO-A-0267851).

In addition to the functional promoter sequence, the efficient binding site on the ribosome is also useful for providing expression of foreign genes in prokaryotic cells. In E. coli, the ribosome binding site is called the Shine-Dalgarno (SD) sequence: it includes the ATG start codon and a sequence of 3-9 nucleotides located 3-11 nucleotides above the start codon (Shine et al., 1975, Nature, 254, 34). The SD sequence is thought to promote mRNA binding on the ribosome by base pairing as part of the SD sequence and the 3 ′ end of E. coli 163-rRNA (Steitz et al., 1979, “Genetic signals and nucleotide sequences in messenger RNA”, In "Biological Regulation and Development: Gene Expression", ed. RFGoldberger). A “weak” ribosome binding site is used to express eukaryotic genes and prokaryotic genes (Sambrook et al., 1989, “Expression of cloned genes in Escherichia coli.” In “Molecular Cloning: A Laboratory Manual”).

A DNA molecule can be expressed intracellularly. The promoter sequence can be directly attached to this DNA molecule: in this case, the first amino acid from the N-terminus must always be methionine, which is encoded by the ATG start codon. If desired, N-terminal methionine can be cleaved from the protein by in vitro incubation with cyanogen bromide or by in vivo or in vitro incubation with the bacterial enzyme N-terminal methionine peptidase (European patent application EPO-A-0219237).

Chimeric proteins are an alternative way to control expression. Typically, a DNA sequence encoding the N-terminal portion of an endogenous bacterial protein or other stable protein is fused to the 5'-end of the heterologous coding sequences. As a result of expression, this design leads to the formation of a chimera (hybrid) of two amino acid sequences. For example, the bacteriophage λ cell gene λ can be fused at its 5 ′ end to a foreign gene, followed by expression in bacteria. The resulting chimeric protein preferably retains an enzymatic processing site (factor XA), resulting in cleavage of the phage protein from the foreign gene product (Nagai et al., 1984, Nature, 309, 810). Chimeric proteins can also be formed using lacZ gene sequences (Jia et al., 1987, Gene, 60, 197), trpE (Allen et al., 1987, J. Biotechnol., 5, 93; Makoff et al., 1989 , J. Gen. Microbiol., 135, 11) and Chey (European Patent Application EP-A-0324647). The DNA sequence at the junction of two amino acid sequences may or may not encode a cleavage site. Another example is the chimeric protein ubiquitin. Such a chimeric protein is formed in such a way that within the actual ubiquitin segment the target site for the processing enzyme (for example, specific for the processing of ubiquitin protease), which is necessary for the cleavage of ubiquitin from a foreign protein, is preferably preserved. Using this approach, native foreign proteins can be isolated (Miller et al., 1989, Bio / Technol., 7, 698).

On the other hand, foreign proteins can also be secreted by cells due to the creation of chimeric DNA molecules that would encode a chimeric protein comprising a peptide fragment of a signal sequence that secrets the foreign protein by bacterial cells (US Patent No. 4,336,336). A signal sequence fragment typically encodes a signal peptide consisting of hydrophobic amino acids that secrete protein from the cell. This protein is secreted either into the culture medium (in variants with gram-positive bacteria) or into the periplasmic space located between the inner and outer membranes of the cell (gram-negative bacteria). Preferably, processing sites are present that can be cleaved either in vivo or in vitro in the region between the signal peptide fragment and the foreign gene product.

DNA encoding suitable signal sequences may be derived from genes encoding secreted bacterial proteins, such as the E. coli outer membrane protein gene (ompA) (Masui et al., 1983, In "Experimental Manipulation of Gene Expression"; Ghrayeb et al ., 1984, EMBO J., 3, 2437) and the signal sequence of the phoA gene encoding the alkaline phosphatase of E. coli (Oka et al., 1985, Proc. Natl. Acad. Sci. USA, 82, 7212). An additional example is the signal sequence of the α-amylase gene of various Bacillus rods, which can be used to secrete heterologous proteins from B. subtilis cells (Palva et al., 1982, Proc. Natl. Acad. Sci. USA, 79, 5582; European Patent Application EP-A-0244042).

Typically, transcription termination sequences recognized by bacteria are represented by regulatory segments located on the 3'-side of the stop codon: accordingly, along with the promoter, they flank the specific coding sequence. These sequences provide for mRNA transcription, which can then be translated into the polypeptide encoded by this DNA region. Transcription termination sequences often include DNA sequences of approximately 50 nucleotides capable of forming knitted structures that provide transcription termination. Examples are transcription termination sequences derived from genes having strong promoters, such as the E. coli trp gene, as well as other biosynthetic genes.

Typically, the components described above, including the promoter, the signal sequence (if desired), the coding sequence in question, and the transcription termination sequence are integrated into expression constructs. Expression constructs are often maintained as part of a replicon, such as an extrachromosomal element (e.g., a plasmid) capable of stably remaining in a host organism, such as a bacterium. Such a replicon should include a replication system that would ensure its ability to remain in a prokaryotic organism, designed either for expression or for cloning and amplification. In addition, the replicon can be either a high copy or low copy plasmid. The high copy plasmid should in principle have a copy number of from about 5 to about 200, and usually from about 10 to about 150. The host organism carrying the high copy plasmid should preferably carry at least about 10 plasmids, and more preferably at least about 20 plasmids. Both high copy and low copy plasmids can be selected based on the influence that the vector and the foreign protein have on the host organism.

Alternatively, expression constructs can be integrated into the bacterial genome using an integration vector. Integration vectors usually include at least one sequence that is homologous to the site of the bacterial chromosome: this provides the vector with the ability to integrate. It is believed that integration is due to recombination between homologous DNA in the vector and the bacterial chromosome. For example, integration vectors constructed from the DNA of various Bacillus strains are integrated into. Bacillus chromosome (European patent application EP-A-0127328). Integration vectors may also include phage or transposon sequences.

Typically, extrachromosomal and integration expression constructs may include selective markers allowing selection of transformed bacterial strains. Selective markers can be expressed in a bacterial host and can include genes that make bacteria resistant to drugs, such as ampicillin, chloramphenicol, erythromycin, kanamycin (neomycin) and tetracycline (Davies et al., 1978, Annu. Rev. Microbiol ., 32, 469). Selective markers may also include biosynthetic genes, such as genes for enzymes involved in the biosynthesis of histidine, tryptophan and leucine.

On the other hand, some of the components described above can be included together with each other in transformation vectors. Transformation vectors typically include a selectable marker that is either stored in a replicon or converted to an integrating vector as described above.

Expression and transforming vectors (either extrachromosomal or integrating vectors) were designed to transform many types of bacteria. For example, expression vectors were created, inter alia, for the following bacterial species: Bacillus subtilis hay bacillus (Palva et al., 1982, Proc. Natl. Acad. Sci. USA, 79, 5582; European patent applications EP-A-0036259 and EP-A-0063953; international patent application WO 84/04541), Escherichia coli E. coli (Shimatake et al., 1981, Nature, 292, 128; Amann et al., 1985, Gene, 40, 183; Studier et al. , 1986, J. Mol. Biol., 189, 113; European patent applications EP-A-0036776, EP-A-0136829 and EP-A-0136907), Streptococcus cremoris (Powell et al., 1988, Appl. Environ . Microbiol., 54, 655), Streptococcus lividans (Powell et al., 1988, Appl. Environ. Microbiol., 54, 655), Streptomyces lividans streptomycetes (US patent No. 4745056).

Methods for introducing exogenous DNA into host bacterial organisms are well known in the art and usually involve either transforming bacteria treated with calcium chloride or other agents such as divalent cations and dimethyl sulfoxide. DNA can also be introduced into bacterial cells by electroporation. Transformation procedures usually vary depending on the type of bacterium to be transformed: see, for example, Masson et al., 1989, FEMS Microbiol. Lett., 60, 273; Palva et al., 1982, Proc. Natl. Acad. Sci. USA 79, 5582; European patent applications EP-A-0036259 and EP-A-0063953; international patent application WO 84/04541 (bacteria of the genus Bacillus), Miller et al., 1988, Proc. Natl. Acad. Sci. USA, 85, 856; Wang et al., 1990, J. Bacteriol., 172, 949 (bacteria of the genus Campylobacter), Cohen et al., 1973, Proc. Natl. Acad. Sci. USA 69, 2110; Dower et al., 1988, Nucl. Acids Res., 16, 6127; Kushner, 1978, "An improved method for transformation of Escherichia coli with ColEl-derived plasmids." In "Genetic Engineering: Proc. Intern. Symp. Genet. On Engineering", eds. H. W. Boyer & S. Nicosia; Mandel et al., 1970, J. Mol. Biol., 53, 159; Taketo, 1988, Biochim. Biophys. Acta, 949, 318 (Escherichia), Chassy et al., 1987, FEMS Microbiol. Lett., 44, 173 (milk bacteria of the genus Lactobacillus), Fiedler et al., 1988, Anal. Biochem., 170, 38 (pseudomonads of the genus Pseudomonas), Augustin et al., 1990, FEMS Microbiol. Lett., 66, 203 (bacteria of the genus Staphylococcus), Barany et al., 1980, J. Bacteriol., 144, 698; Harlander, 1987, "Transformation of Streptococcus lactis by electroporation", In "Streptococcal Genetics", eds. J. Ferretti & R. Curtiss III; Perry et al., 1981, Infect. Immunol., 32, 1295; Powell et al., 1988, Appl. Environ. Microbiol., 54, 655; Somkuti et al., 1987, Proc. 4th European Congr. Biotechnol., 1, 412 (bacteria of the genus Streptococcus).

5. Expression in yeast

Yeast expression systems are also well known to those skilled in the art. A yeast promoter is any DNA sequence that can bind yeast RNA polymerase and initiate transcription of the downstream (3'-side) coding sequence (for example, a structural gene) to form mRNA. The promoter should include a transcription initiation site, which is usually placed proximal to the 5'-end of the coding sequence. This transcription initiation site typically includes an RNA polymerase binding site (ie, a “TATA box”) and a transcription initiation site itself. The yeast promoter may also include a second domain called the “Upper Activator Sequence” (UAS), which, when present, is usually located distally to the structural gene. The UAS segment provides regulated (inducible) expression. Constitutive expression occurs in the absence of a UAS segment. Regulated expression can be either positive or negative, which thereby either enhances or weakens transcription.

Yeast is a fermenter organism characterized by an active metabolism: therefore, sequences encoding the enzymes of the metabolic pathways are particularly promising promoter sequences. Examples are alcohol dehydrogenase (ADH) (European patent application EP-A-0284044), enolase, glucokinase, glucose-6-phosphatisomerase, glyceraldehyde-3-phosphate dehydrogenase (GAP or GAPDH), hexokinase, phosphofructokinase and phosphofructokinase 3, (European Patent EPO-A-0329203). The yeast gene PHO5 encoding acid phosphatase also represents applicable promoter sequences (Myanohara et al., 1983, Proc. Natl. Acad. Sci. USA, 80, 1).

In addition, synthetic promoters that are not found in nature can also function as yeast promoters. For example, the UAS sequences of one yeast promoter can be linked to the transcriptional activation site of another yeast promoter, resulting in a synthetic hybrid promoter.

Examples of such hybrid promoters are the regulatory sequence of the ADH gene linked to the GAP gene transcription activation site (US Pat. Nos. 4,876,197 and 4,880,734). Other examples of hybrid promoters include promoters that include regulatory sequences from the ADH2, GAL4, GAL10 or ORPHO5 genes combined with transcription activation sites of genes encoding glycolytic enzymes, such as GAP or Ruk genes (European patent application EP-A-0164556). Further, the yeast promoter may include naturally occurring promoters of non-yeast origin, characterized by the ability to bind yeast RNA polymerase and initiate transcription. Examples of such promoters include, but are not limited to, the elements described by Cohen et al., 1980, Proc. Natl. Acad. Sci., USA, 77, 1078; Henikoff et al., 1981, Nature, 283, 835; Hollenberg et al., 1981, Curr. Topics Microbiol. Immunol., 96, 119; Hollenberg et al., 1979, "The Expression of Bacterial Antibiotic Resistance Genes in the Yeast Saccharomyces cerevisiae", In "Plasmids of Medical, Environmental and Commercial Importance", eds. K.N. Timmis & A. Punler; Mercerau-Puigalon et al., 1980, Gene, 11, 163; Panthier et al., 1980, Curr. Genet., 2, 109).

A DNA molecule can be expressed within yeast cells. The promoter sequence can be directly attached to the DNA molecule: in this case, the first amino acid from the N-terminus of the recombinant protein should always be methionine, which is encoded by the ATG start codon. If desired, methionine from the N-terminus of the protein can be cleaved in vitro by incubation with cyanogen bromide.

Chimeric proteins represent an alternative approach for yeast expression systems to the same extent as for expression systems of mammalian cells, baculoviruses and bacteria. Typically, a DNA sequence encoding the N-terminal portion of an endogenous yeast protein or other stable protein is fused to the 5 ′ end of a heterologous coding sequence. After expression, this design will cause the formation of a chimera (hybrid) of two amino acid sequences. For example, yeast or human superoxide dismutase (SOD) genes can be linked to the 5'-end of a foreign gene, followed by expression in yeast. The DNA sequence in the region where two amino acid sequences are joined may or may not encode a cleavable site: see, for example, European patent application EP A-0196056. Another example is the chimeric ubiquitin protein. Such a chimeric protein is designed in such a way that preferably the ubiquitin segment retains the target site for the processing enzyme (for example, specific for the processing of ubiquitin protease), causing the cleavage of ubiquitin from a foreign protein. Using this method, therefore, native foreign proteins can be isolated (see, for example, international patent application WO 88/024066).

On the other hand, foreign proteins can also be secreted from the cell into the nutrient medium by creating chimeric DNA molecules that encode a chimeric protein comprising a leader segment sequence that secrets the foreign protein from the yeast cell. Preferably, encoded processing sites located between the leader segment and the foreign gene should be present, which could be cleaved in vivo or in vitro sequence. The leader segment usually encodes a signal peptide consisting of hydrophobic amino acids, which secures the secretion of this protein from the cell.

DNAs encoding suitable signal sequences can be derived from genes encoding secreted yeast proteins, such as the yeast invertase gene (European patent application EP-A-0012873; JPO 62096086) and the A-factor gene (US patent No. 4588684). On the other hand, there are leader segments of non-yeast origin, such as the leader segment of the interferon gene, which also provide secretion in yeast (European patent application EP-A-0060057).

The preferred class of secreting leader segments are those leaders who use fragments of the α-factor gene, which contains both the “pre” signal sequence and the “pro” segment. Such variants of the α-factor fragments that can be used are a full-sized α-factor preprolider (about 83 amino acid residues), as well as shortened variants of the α-factor signal segments (usually from about 25 to about 50 amino acid residues) (US Pat. No. 4546083 and 4870008; European patent application EP-A-0324274). Additional leader segments based on the α-factor leader, which provide secretion, are hybrid α-factor leader segments, assembled in such a way that the subsequences come from the first yeast α-factor, and the subsequences from the second yeast α-factor (see, for example, international patent application WO 89/02463).

Typically, the transcription termination sequences recognized by the yeast are represented by regulatory segments located on the 3 ′ side with respect to the translational stop codon: i.e. they along with the promoter flank the coding sequence. These sequences provide for mRNA transcription, which can then be translated into the polypeptide encoded by the DNA. Examples of transcription termination sequences and other yeast-recognized termination sequences are those elements that are part of genes encoding glycolytic enzymes.

Typically, the components described above, including the promoter, the leader (if desired), the coding sequence of interest and the transcription termination sequence, are introduced into expression constructs with each other. Expression constructs are often maintained in the form of a replicon, such as an extrachromosomal element (e.g., a plasmid) that can stably exist in a host organism, such as a yeast or bacterium. Such a replicon can include two replication systems: this will ensure maintenance, for example, in yeast for expression and in a prokaryotic host for cloning and amplification. Examples of such bifunctional bacterial yeast vectors are YEp24 (Botstein et al., 1979, Gene 8, 17-24), pCl / 1 (Brake et al., 1984, Proc. Natl. Acad. Sci. USA, 81, 4642- 4646) and YRpl7 (Stinchcomb et al., 1982, J. Mol. Biol., 158, 157). In addition, the replicon can be either a high copy or low copy plasmid. A high-copy plasmid will in principle have a copy number of from about 5 to about 200, and usually from about 10 to about 150. A host cell carrying a high-copy plasmid should preferably carry at least about 10, and more preferably at least about 20 copies . The presence of a high copy or low copy vector can be selected based on the effect that such a vector and a foreign protein have on the host organism (see, for example, Brake et al., Cited above).

On the other hand, expression constructs can be integrated into the yeast genome using an integrating vector. Typically, integrating vectors include at least one sequence homologous to the region of the yeast chromosome, which ensures the integration of this vector, and preferably includes two homologous sequences flanking the expression construct. Integration is thought to result from recombination between the homologous DNA of the vector and the yeast chromosome (Orr-Weaver et al., 1983, Meth. Enzymol., 101, 228-245). The integrating vector can be directed to a specific yeast locus by selecting the appropriate homologous sequence to be included in this vector (see Orr-Weaver et al., Cited above). One or more expression constructs can be integrated so that they are likely to result in a decrease in the production of recombinant protein (Rine et al., 1983, Proc. Natl. Acad. Sci. USA, 80, 6750). The chromosomal sequences included in the composition of this vector can either be in the form of a single segment of the vector, which determines the integration of the entire vector, or in the form of two segments that are homologous to adjacent segments of the chromosome and flank the expression construct of the vector, leading to stable integration of only the expression construct .

Typically, extrachromosomal and integrating expression constructs can carry selective markers that allow selection of yeast strains that have been transformed. Selective markers may be biosynthetic genes that will be expressed in yeast host cells, such as ADE2, HIS4, LEU2, TRP1, as well as ALG7 and G418 resistance genes that provide yeast resistance to tunicamycin and G418, respectively. In addition, a suitable selective marker can also give the yeast the ability to grow in the presence of toxic substances, such as metal ions. For example, the presence of the CUP1 gene provides yeast growth in the presence of copper ions (Butt et al., 1987, Microbiol. Rev., 51, 351).

On the other hand, some of the components described above can be included together with each other in the composition of transforming vectors. Transforming vectors typically include a selectable marker that is either maintained as a replicon or converted to an integrating vector as described above.

Expression and transforming vectors (in the form of either extrachromosomal replicons or integrating vectors) were designed to transform many types of yeast. For example, expression vectors were designed, inter alia, for the following yeast species: Candida albicans (Kurtz et al., 1986, Mol. Cell. Biol., 6, 142), Candida maltosa (Kunze et al., 1985, J. Basic Microbiol., 25, 141), Hansenula polymorpha (Gleeson et al., 1986, J. Gen. Microbiol., 132, 3459; Roggenkamp et al., 1986, Mol. Gen. Genet., 202, 302), Kluyveromyces fragilis (Das et al., 1984, J. Bacteriol., 158, 1165), Kluyveromyces lactis (De Louvencourt et al., 1983, J. Bacteriol., 154, 737; Van den Berg et al., 1990, Bio / Technol ., 8, 135), Pichia guillerimondii (Kunze et al., 1985, J. Basic Microbiol., 25, 141), Pichia pastoris (Cregg et al., 1985, Mol. Cell. Biol., 5, 3376; patents USA No. 4837148 and 4929555), Saccharomyces cerevisiae (Hinnen et al., 1978, Proc. Natl. Acad. Sci. USA, 75, 1929; Ito et al., 1983, J. Bacteriol., 153, 163), Schizosaccharomyces pombe (Beach & Nurse , 1981, Nature, 300, 706) and Yarrowia lipolytica (Davidow et al., 1985, Curr. Genet., 10, 39; Gaillardin et al., 1985, Curr. Genet., 10, 49).

Methods for introducing exogenous DNA into yeast host cells are well known in the art and typically involve the transformation of either spheroplasts or intact yeast cells treated with alkaline cations. Transformation procedures usually vary depending on the type of yeast whose transformation is carried out: see, for example, Kurtz et al., 1986, Mol. Cell. Biol., 6, 142; Kunze et al., 1985, J. Basic Microbiol., 25, 141 (Candida); Gleeson et al., 1986, J. Gen. Microbiol., 132, 3459; Roggenkamp et al., 1986, Mol. Gen. Genet., 202, 302 (Hansenula); Das et al., 1984, J. Bacteriol., 158, 1165; De Louvencourt et al., 1983, J. Bacteriol., 154, 1165; Van den Berg et al., 1990, Bio / Technol., 8, 135 (Kluyveromyces); Cregg et al., 1985, Mol. Cell. Biol., 5, 3376; Kunze et al., 1985, J. Basic Microbiol., 25, 141; U.S. Patent Nos. 4,837,148 and 4,929,555 (Pichia); Hinnen et al., 1978, Proc. Natl. Acad. Sci. USA 75, 1929; Ito et al., 1983, J. Bacteriol., 153, 163 (Saccharomyces); Beach & Nurse, 1981, Nature, 300, 706 (Schizosaccharomyces); Davidow et al., 1985, Curr. Genet., 10, 39; Gaillardin et al., 1985, Curr. Genet., 10, 49 (Yarrowia).

Antibodies

As used in this text, the term "antibody" refers to a polypeptide or group of polypeptides comprising at least one antigen-binding site. The term "antigen-binding site" refers to the spatial structure, surface parameters and charge distribution in which are complementary to the parameters of the antigen epitope: this provides the process of binding of the antibody to the corresponding antigen. By the term “antibody” is meant, for example, vertebrate antibodies, chimeric antibodies, hybrid antibodies, humanized antibodies, altered antibodies, monovalent antibodies, Fab proteins and monodomain antibodies.

Antibodies specific for the proteins of the present invention are used for affinity chromatography, immunological tests and the identification and identification of proteins of the neisseries.

Antibodies specific for the proteins of the present invention, both polyclonal and monoclonal, can be obtained using standard methods. In general, a protein is firstly used to immunize a suitable animal, preferably a mouse, rat, rabbit or goat. Rabbits and goats are the preferred objects for obtaining polyclonal sera due to the significant volume of blood serum obtained, as well as the availability of anti-rabbit and anti-goat antibodies. Immunization is usually carried out by mixing or emulsifying a particular protein in saline (physiological) solution, preferably with an adjuvant, such as Freund's complete adjuvant, followed by injection of the resulting mixture or emulsion parenterally (usually by subcutaneous or intramuscular injection). Usually, 50-200 micrograms per injection are sufficient doses. Immunization is usually boosted (i.e., repeated) after 2-6 weeks by one or more additional injections of the protein in saline, preferably with the inclusion of an incomplete Freund adjuvant. Antibodies can also be obtained in an alternative way using in vitro immunization using methods known in the art which, from the point of view of the objectives of the present invention, may be equivalent to in vivo immunization methods. Polyclonal antisera are obtained by taking blood from immunized animals in a glass or plastic container, followed by incubation of blood at 25 ° C for 1 hour and then incubation at 4 ° C for 2-18 hours. The serum is then isolated by centrifugation (for example, at 1000 g for 10 minutes). In rabbits, 20-50 blood cubes can be taken in one sampling.

Monoclonal antibodies are obtained using the standard Kohler-Milstein method (Kohler & Milstein, 1975, Nature, 256, 495-496) or its modifications. Typically, mice or rats are immunized as described above.

However, unlike taking blood from animals to obtain serum, the spleen (and, optionally, several large lymph nodes) is removed and dissolved into a single cell. If desired, spleen cells can be screened (after removal of non-specific adhesive cells) by applying the cell suspension to a plate or to a separate plate well coated with an antigen protein. B lymphocytes expressing a membrane-bound immunoglobulin specific for the test antigen binds on the plate in such a way that it does not wash off the remainder of the suspension from the plate. Then, the resulting B-lymphocytes or all dissolved splenocytes are fused with myeloma cells, resulting in the formation of hybridomas, which are then cultured in selective medium (for example, in HAT medium containing hypoxanthine, aminopterin and thymidine). The resulting hybridomas are seeded in limited dilution and tested for the production of antibodies that specifically bind to the antigen used for immunization (and which do not bind to extraneous antigens). Selected monoclonal antibody-secreting hybridomas (mAb) are then cultured either in vitro (for example, in hollow fiber fermenters or glass tissue culture containers) or in vivo (in ascites fluid in mice).

If desired, antibodies (both polyclonal and monoclonal) can be labeled using standard methods. Suitable labels include fluorophores, chromophores, radioactive atoms (in particular ³² P and ¹²⁵ I), electron-dense reagents, enzymes and ligands for which binding partners are known. Enzymes are usually detected by their catalytic activity. For example, horseradish peroxidase is usually detected by its ability to convert 3,3 ', 5,5'-tetramethylbenzidine into a blue pigment, quantified on a spectrophotometer. The term "specific binding partners" refers to a protein capable of binding a ligand molecule, while exhibiting a high level of specificity, as, for example, in the case of an antigen and a monoclonal antibody specific for it. Other examples of specific binding partners are biotin and avidin (or streptavidin), immunoglobulin-G and A-protein, as well as numerous pairs of receptors and their ligands known in the art. It should be clear that the above description does not pretend to classify different markers with dividing them into any classes, since the same label can be used in different modes. For example, the ¹²⁵ I isotope can serve as a radioactive label or as an electron-dense agent. Horseradish peroxidase can be either an enzyme or an antigen for a monoclonal antibody. In addition, various labels can be combined to achieve the desired effect. For example, mAb and avidin equally need labeling in connection with the present invention: therefore, it is possible to label the mAb with biotin and detect its presence using avidin labeled in turn with the ¹²⁵ I isotope or with the anti-avidin monoclonal antibody labeled with horseradish peroxidase . Other options and possibilities will be apparent to those skilled in the art and are considered equivalent in the scope of the present invention.

Pharmaceutical Compositions

The pharmaceutical compositions may contain the polypeptides, antibodies or nucleic acids of the present invention. These pharmaceutical compositions should contain a therapeutically effective amount of the polypeptides, antibodies or polynucleotides of the invention.

The term “therapeutically effective amount” as used herein means an amount of a therapeutic agent for treating, alleviating symptoms or preventing a particular disease or condition, or for providing a detectable therapeutic or prophylactic effect. Such an effect can be detected, for example, by the levels of chemical markers or antigens. Therapeutic effects also include a decrease in physical symptoms, such as a decrease in body temperature. The exact effective amount for a particular individual will depend on body weight and state of health, on the nature and severity of the disease state, as well as on the types of drugs or their combinations selected for treatment. Therefore, there is no need for an initial accurate determination of the effective amount. However, the effective amount for a particular situation can be determined using routine experiments: this approach is within the competence of the attending physician.

For the purposes of the present invention, an effective dose should be in the range of from about 0.01 mg / kg to 50 mg / kg, or from 0.05 mg / kg to about 10 mg / kg of the DNA construct in the application to the particular individual to whom it will be administered.

The pharmaceutical composition may also contain a pharmaceutically acceptable carrier. The term “pharmaceutically acceptable carrier” means a carrier used to administer a drug, such as an antibody or polypeptide, genes, or other drugs. This term refers to any pharmaceutical carrier that does not, by itself, induce the production of antibodies that are harmful to the individual receiving the pharmaceutical composition, i.e. which can be administered without causing toxicity. Suitable carriers can be large, slowly metabolized macromolecules, such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers and inactivated viral particles. Such carriers are well known to those skilled in the art.

Pharmaceutically acceptable salts may be used: for example, salts of inorganic acids such as hydrochlorides, hydrobromides, phosphates, sulfates and the like, or salts of organic acids such as acetates, propionates, malonates, benzoates and the like. A detailed discussion of pharmaceutically acceptable excipients is available in Remington's Pharmaceutical Sciences (Mack Publ. Co., NJ, 1991).

Pharmaceutically acceptable carriers in the therapeutic compositions may contain liquids such as water, saline, glycerol and ethyl alcohol. In addition, excipients such as wetting or emulsifying agents, buffering agents used to adjust pH, and the like may be present in such formulations. Typically, therapeutic compositions are formulated for injection in the form of either solutions or suspensions; solid forms suitable for dissolution or suspension to form liquid excipients prior to injection may also be prepared. A liposome is also included in the definition of a pharmaceutically acceptable carrier.

Delivery methods

After preparing the pharmaceutical compositions of the present invention, they can be administered directly to the patient. The patients being treated may be animals; in particular, treatment may be carried out in relation to a person.

Direct delivery of compositions should, in principle, be achieved by injection — subcutaneous, intraperitoneal, intravenous or intramuscular — or by delivery to the interstitial region. Also, compositions may be administered through damage (wound). Other routes of administration include oral and intrapulmonary, in the form of suppositories, and transdermal or transdermal administration (see, for example, international patent application WO 98/20734), using needles, "gene guns" or hypospray. Dosages may include single or multiple doses.

Vaccines

Vaccines in accordance with the present invention can be either prophylactic (i.e., to prevent infection) or therapeutic (i.e., treat an existing disease).

Such vaccines include an immunizing antigen (s), an immunogen (s), a polypeptide (s), a protein (s) or a nucleic acid, usually in combination with “pharmaceutically acceptable carriers”, which are any carriers that do not themselves induce antibody production dangerous to the individual receiving this composition. Suitable carriers are typically large, slowly metabolized macromolecules, such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, lipid aggregations (such as oil drops or liposomes) and inactivated viral particles. Such carriers are well known to those skilled in the art. In addition, these carriers can function as immunostimulants (“adjuvants”). Moreover, the antigen or immunogen can be combined with a bacterial toxoid, such as diphtheria toxoid, tetanus toxoid, cholera toxoid, toxoid H. pylori and other pathogens.

Preferred adjuvants intended to enhance the effectiveness of a particular composition are, but are not limited to: (1) aluminum salts such as aluminum hydroxide, aluminum phosphate, aluminum sulfate, and the like; (2) emulsion water-oil preparations (together with other specific immunostimulants, such as muramyl peptides [see below], or components of bacterial cell walls, with or without them), such as, for example, (a) MF59 ™ preparation (application WO 90 / 14837; chapter 10 in Vaccine design: the subunit and adjuvant approach, eds. Powell & Newman, Plenum Press, 1995) containing 5% squalene, 0.5% Tween-80, and 0.5% Span-85 ( optionally including various amounts of MTP-PE [see below], although this is not necessary) prepared in the form of submicron particles using a microdisperser, such ak mikrodispergator Model 110Y (Microfluidics, Newton, MA); (b) an SAF preparation containing 10% squalane, 0.4% Tween-80, 5% blocked polymer L121 and threonyl-MDP (see below) either microdispersed in a submicron emulsion or mixed to form an emulsion with larger particles, and (c) an adjuvant Ribi ™ system (Ribi Immunochem, Hamilton, MT) containing 2% squalene, 0.2% Tween-80 and one or more components of the bacterial cell walls represented by a group comprising monophosphoryl lipid-A (MPL), dimicolattrehalose ( TDM) and cell wall skeleton preparation (CWS), preferably MPL + CWS (Detox ™); (3) saponin adjuvants such as Stimulon ™ (Cambridge Bioscience, Worcester, MA) can be used, as well as particles formed on their basis, such as ISCOM (immunostimulatory complexes); (4) Freund's complete adjuvant (CFA) and Freund's incomplete adjuvant (IFA); (5) cytokines, such as interleukins (e.g., IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, etc.), interferons (e.g., γ-interferon ), colony-stimulating macrophage factor (M-CSF), tumor necrosis factor TNF), etc .; and (6) other substances and compounds that act as immunostimulants that enhance the effectiveness of this composition. Preferred are aluminum salts and the preparation MF59 ™.

As indicated above, muramyl peptides include, but are not limited to, N-acetylmuramyl-L-threonyl-D-isoglutamine (threonyl-MDP), N-acetylnormuramyl-L-alanyl-D-isoglutamine (nor-MDP), N-acetylmuramyl -L-alanyl-D-isoglutaminyl-L-alanine-2- (1'-2'-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy) ethylamine (MTP-PE) and the like.

Immunogenic compositions (e.g., an immunizing antigen, or an immunogen, or a polypeptide, or a protein, or a nucleic acid together with a pharmaceutically acceptable carrier and adjuvant) should usually contain solvents such as water, saline, glycerol, ethanol and the like. In addition, auxiliary components such as wetting or emulsifying components, buffer components used to control pH, and the like may be included in such excipients.

Typically, immunogenic compositions are prepared as injectables or as liquid solutions or suspensions; solid forms, or suspensions in liquid excipients, immediately prior to injection can also be prepared. Also, the drug can be emulsified or encapsulated in liposomes in order to enhance the adjuvant effect, which was discussed above when considering pharmaceutically acceptable carriers.

The immunogenic compositions used as a vaccine contain an immunologically effective amount of antigenic or immunogenic polypeptides, as well as any other of the above components, if necessary. By "immunologically effective amount" is meant the administration of such an amount to a subject, either as a single dose or as a series of doses, thereby achieving effective treatment or prevention. This amount varies depending on the health and physical condition of the particular subject being treated, the taxonomic group to which the subject belongs (for example, inhuman monkeys, primates in general, etc.), the ability of an individual immune system to synthesize antibodies, the desired level of protection , the composition of the vaccine, the assessment of a specific situation by the attending physician and other related factors. It is assumed that the analyzed amount should fall in a fairly wide range, which can be quantified using routine methods.

Immunogenic compositions are routinely administered via the parenteral route, for example, by injection - subcutaneous, intramuscular or transdermal (“through the skin”) (for example, international patent application WO 98/20734). Additional formulations suitable for other routes of administration are oral and intrapulmonary formulations, suppositories, and transdermal formulations. Dosages may be in single doses or in multiple doses. The vaccine can be administered along with other immunoregulatory agents.

As an alternative to a protein-based vaccine, DNA vaccination can be used (see, for example, Robinson & Torres, 1997, Seminars in Immunol., 9, 271-283; Donnelly et al., 1997, Annu. Rev. Immunol., 15, 617-648; see below in this text).

Gene Delivery Devices

Gene therapy devices designed to deliver constructs bearing the coding sequence of a therapeutic value of the present invention, which is intended to be delivered to a mammal in which it is to be expressed, can be administered either locally or systemically. These constructs may be based on viral or non-viral vector approaches implemented in in vivo or ex vivo models. Expression of such a coding sequence can be induced using mammalian endogenous promoters or heterologous promoters. In vivo expression of the coding sequence can be either constitutive or inducible (regulated) in nature.

The present invention includes gene delivery systems capable of expressing representative nucleotide sequences. Such a delivery gene system is preferably based on a viral vector and, more preferably, a retroviral, adenovirus, adenovirus (AAV) viral, herpes virus and alphavirus vector. The viral vector may also be astrovirus, corona virus, orthomyxovirus, papovavirus, paramyxovirus, parvovirus, picornavirus, poxvirus or togavirus: for reviews, see Jolly, 1994, Cancer Gene Therapy, 1, 51-64; Kimura, 1994, Hum. Gene Therapy, 5, 845-852; Connelly, 1995, Hum. Gene Therapy, 6, 185-193; Kaplitt, 1994, Nature Genet., 6, 148-153.

Retroviral vectors are well known in the art, and applicants believe that any retroviral gene therapy vector is applicable in connection with the present invention, including type B, C and D retroviruses, xenotropic retroviruses (e.g., NZB-X1, NZB-X2 and NZB9- strains 1 [see (O'Neill, 1985, J. Virol., 53, 160]), polytropic retroviruses, for example, MCF and MCF-MLV (see Kelly, 1983, J. Virol., 45, 291), spumaviruses and lentiviruses; see "RNA Tumor Viruses", 2d ed., Cold Spring Harbor Lab., 1985.

Fragments of a retroviral gene therapy vector may be derived from various retroviruses. For example, the long terminal repeats (LTRs) included in the retroviral vector can come from the mouse sarcoma virus, the tRNA binding site from the Routh sarcoma virus, the “packaging signal” from the mouse leukemia virus, and the second-chain replication start site from the bird leukemia virus .

These recombinant retroviral vectors can be used to create transduction competent retroviral vector particles, which is achieved by introducing them into a suitable "packaging cell line" (see US patent No. 5591624). Retroviral vectors can be designed to provide site-specific integration within the host cell DNA, which is achieved by the inclusion of a chimeric integrase enzyme in the viral particle (see international patent application WO 96/37626). Preferably, such a recombinant viral vector is a replication defective recombinant virus.

"Packaging cell lines" suitable for use with the retroviral vectors described above are well known in the art and can be easily created (see international patent applications WO 95/30763 and WO 92/05266) and can be used to create production cell lines (also called "vector cell lines", or "VCL") necessary for the production of recombinant vector particles. Preferably, packaging cell lines are formed from parental human cells (e.g., HT1080 cell line) or parental mink cell lines in which inactivation of human serum is excluded.

Preferred retroviruses suitable for constructing retroviral gene therapy vectors are avian leukemia virus, cattle leukemia virus, mouse leukemia virus, mink cell induction virus, mouse sarcoma virus, reticuloendotheliosis virus and Routh sarcoma virus. Specifically preferred mouse leukemia viruses are strains 4070A and 1504A (Hartley & Rowe, 1976, J. Virol., 19, 19-25), Abelson sarcoma virus (ATCC No. VR-999), Freund sarcoma virus (ATCC No. VR-245) , Graffi and Gross sarcoma viruses (ATCC No. VR-590), Kirsten, Harvey and Rauscher sarcoma viruses (ATCC No. VR-998), and Moloni mouse leukemia virus (ATCC No. VR-190). These retroviruses can be obtained from depository collections such as the American Type Culture Collection (“ATCC”) located in Rockville, Maryland, or isolated from known sources using standard techniques.

Examples of known retroviral gene therapy vectors applicable within the scope of the present invention are those vectors described in patent applications - British GB 2200651, European EP 0415731, EP 0345242, EP 0334301, international WO 89/02468, WO 89/05349, WO 89 / 09271, WO 90/02806, WO 90/07936, WO 94/03622, WO 93/25698, WO 93/25234, WO 93/11230, WO 93/10218, WO 91/02805, WO 91/02825, WO 95 / 07994, U.S. Patent Nos. 5219740, 4405712, 4861719, 4980289, 4777127, 5591624; see also Vile, 1993, Cancer Res., 53, 3860-3864; Vile, 1993, Cancer Res., 53, 962-967; Ram, 1993, Cancer Res., 53, 83-88; Takamiya, 1992, J. Neurosci. Res., 33, 493-503; Baba, 1993, J. Neurosurgery, 79, 729-735; Mann, 1983, Cell, 33, 153; Cane, 1984, Proc. Natl. Acad. Sci. USA 81,6349; Milter, 1990, Hum. Gene Therapy, 1.

Viruses for gene therapy based on human adenoviruses are also well known in science and are applicable in connection with the present invention: see, for example, Berkner, 1988, Biotechniques, 6, 616 and Rosenfeld, 1991, Science, 252, 431, as well as international patent applications WO 93/07283, WO 93/06223 and WO 93/07282. Examples of known adenoviral gene therapy vectors applicable for the purposes of implementing the present invention are those vectors described in the sources cited above and in international patent applications WO 94/12649, WO 93/03769, WO 93/19191, WO 94/28938, WO 95 / 11984, WO 95/00655, WO 95/27071, WO 95/29993, WO 95/34671, WO 96/05320, WO 94/08026, WO 94/11506, WO 93/06223, WO 94/24299, WO 95 / 14102, WO 95/24297, WO 95/02697, WO 94/28152, WO 94/24299, WO 95/09241, WO 95/25807, WO 95/05835, WO 94/18922 and WO 95/09654. Alternatively, the introduction of DNA coupled to the killed adenovirus described in Curiel, 1992, Hum. Gene Therapy, 3, 147-154. Gene delivery systems of the present invention can also be based on adeno-associated viruses (AAV). Preferred examples of such vectors in connection with their use of the present invention are vectors based on the AAV-2 strain claimed by Srivastava in the international patent application WO 93/09239. Most preferred AAV vectors include two inverted terminal repeats from the AAV genome in which the native D sequences are modified by nucleotide substitutions so that at least five native nucleotides and up to 18 native nucleotides, and preferably at least 10 native nucleotides and up to 18 native nucleotides, and most preferably 10 native nucleotides are retained, and the remaining nucleotides from the D-sequence are delegated or replaced with non-native nucleotides.

Native D sequences from the composition of the inverted terminal AAV repeats are 20 nucleotide motifs present in each terminal AAV repeat repeat (i.e., there is one such motif in each terminal region) that are not involved in the formation of HP. A non-native replacement nucleotide can be any nucleotide other than the nucleotide present in the native D sequence at the same position. Other applicable examples of AAV virus-based vectors are pWP-19 and pWN-1 strains: both are described by Nahreini, 1993, Gene, 124, 257-262. Another example of such an AAV vector is the psub201 vector (see Samulski, 1987, J. Virol., 61, 3096). Another example of an AAV vector is the Double-D-ITR vector. The construction of the Double-D-ITR vector is described in US patent No. 5478745. Other vectors are those claimed by Carter in US Pat. No. 4,797,368 and Muzyczka in US Pat. No. 5,139,941, as well as Chartejee in US Pat. No. 5,474,935 and Kotin in International Application WO 94/288157. Another example of an AAV vector useful in connection with the present invention is the SSV9AFABTKneo construct: it includes an AFP enhancer and an albumin gene promoter and provides expression primarily in the liver. Its structure and construction are described in Su, 1996, Hum. Gene Therapy, 7, 463-470. A number of AAV vectors for gene therapy are described in US patent No. 5354678, 5173414, 5139941 and 5252479.

The vectors for gene therapy in accordance with the present invention also include herpesvirus-based vectors. The most acceptable and preferred examples are herpes simplex vectors, comprising a sequence that encodes a thymidine kinase polypeptide, such as those described in US Pat. No. 5,288,641 and European Application EP 0176170 (Roizman). Further examples of herpes simplex vectors are HFEM / ICP6-LacZ disclosed in WO 95/04139 (Wistar Institute), pHSVlac described in Geller, 1988, Science, 241, 1667-1669 and in international patent applications WO 90/09441 and WO 92/07945; HSV vector Us3 :: pgC-lacZ described by Fink, 1992, Hum. Gene Therapy, 3, 11-19, and the vector HSV 7134, 2-RH-105 and GAL4, described in European application EP 0453242 (Breakefield), as well as those that are included in the ATCC collection under depositary No.ATCC VR-977 and ATCC VR-260.

Alphavirus-based gene therapy vectors that can be used in connection with the present invention are also provided. Sindbis alphavirus vectors are preferred. Togaviruses, Semliki Forest virus (ATCC VR-67; ATCC VR-1247), Middleberg virus (ATCC VR-370), Ross River virus (ATCC VR-373; ATCC VR-1246), Venezuelan horse encephalitis virus (ATCC VR-923 ; ATCC VR-1250; ATCC VR-1249; ATCC VR-532) and those togaviruses as described in US patents Nos. 5091309 and 5217879 and in international patent application WO 92/10578. More specifically, alphavirus vectors are used, which are described in US patent application No. 08/405627 of March 15, 1995, in international patent applications WO 94/21792, WO 92/10578 and WO 95/07994 and in US patent No. 5217879 and 5091309. Such alphaviruses may be available from depositories and collections, such as the ATCC collection located in Rockville, Maryland, or isolated from known sources using standard methods. Alphavirus vectors characterized by reduced cytotoxicity should preferably be used (see USSN No. 08/679640).

DNA vector systems, such as eukaryotic expression systems, can also be used for expression of nucleic acids of the present invention: see international patent application WO 95/07994 for a detailed description of eukaryotic expression systems. Preferably, the eukaryotic expression systems of the present invention are based on alphavirus vectors, and most preferably on a vector based on the Sindbis virus genome.

Other viral vectors suitable for use in the practice of the present invention include those derived from polioviruses, for example ATCC VR-58 and those described by Evans, 1989, Nature, 385 and Sabin, 1973, J. Biol. Standart., 1, 115; from rhinoviruses, for example ATCC VR-1110 and those described by Arnold, 1990, J. Cell. Biochem., L401; from poxviruses such as canary poxivirus and vaccinia virus, for example ATCC VR-111 and ATCC VR-2010 and those described by Fisher-Hoch, 1989, Proc. Natl. Acad. Sci. USA, 86, 317; Flexner, 1989, Ann. N.Y. Acad. Sci., 569, 86; Flexner, 1990, Vaccine, 8, 17; U.S. Patent Nos. 4,602,112 and 4,769,330 and International Patent Application WO 89/01973; from the SV40 monkey virus, for example ATCC VR-305 and those described by Mulligan, 1979, Nature, 277, 108 and Madzak, 1992, J. Gen. Virol., 73, 1533; from influenza virus, for example, ATCC VR-797 and recombinant influenza viruses, which were used in reverse genetics methods in accordance with the description of US patent No. 5166057 and Enami, 1990, Proc. Natl. Acad. Sci. USA 87, 3802-3805; Enami & Palese, 1991, J. Virol., 65, 2711-2713 and Luytjes, 1989, Cell, 59, 110 (see also McMichael, 1983, New England J. Med., 309, 13 and Yap, 1978, Nature , 273, 238 and Nature, 1979, 277, 108); from human immunodeficiency virus as described in EP 0386882 and Buchschacher, 1992, J. Virol., 66, 2731; from measles virus, for example, ATCC VR-67 and ATCC VR-1247 and those described in EP 0440219; from auraviruses, for example, ATCC VR-368; from Bebaru virus, for example, ATCC VR-600 and ATCC VR-1240; Cabassou virus, for example ATCC VR-922; Chikungunya virus, for example ATCC VR-64 and ATCC VR-1241; from Fort Morgan virus, for example, ATCC VR-924; from Getah virus, for example ATCC VR-369 and ATCC VR-1243; from the Kyzylgach virus, for example, ATCC VR-927; Mauro virus, for example ATCC VR-66; from the Mucambo virus, for example, ATCC VR-580 and ATCC VR-1244; from Ndumu virus, for example ATCC VR-371; from Pixuna virus, for example ATCC VR-372 and ATCC VR-1245; from Tonate virus, for example, ATCC VR-925; from Triniti virus, for example, ATCC VR-469; from Una virus, for example, ATCC VR-374; from Whataroa virus, for example, ATCC VR-926; from the virus Y-62-33, for example, ATCC VR-375; from O'Nyong virus (eastern encephalitis virus), for example, ATCC VR-65 and ATCC VR-1242; from Western encephalitis virus, for example, ATCC VR-70, ATCC VR-1251, ATCC VR-622 and ATCC VR-1252; and from coronaviruses, for example ATCC VR-740 and those described by Hamre, 1966, Proc. Soc. Exp. Biol. Med., 121, 190.

The delivery of the compositions of the present invention to cells is not limited to the use of the viral vectors described above. Other delivery methods and media may also be used, such as, for example, nucleotide expression vectors with polycation-packed DNA, whether or not coupled to killed adenovirus: see, for example, U.S. Patent Application No. 08/366787, filed December 30 1994 and Curiel, 1992, Hum. Gene Therapy, 3, 147-154; DNA linked to a ligand: see, for example, Wu, 1989, J. Biol. Chem., 264, 16985-16987; delivery system-cells for eukaryotic cells: see, for example, US patent application No. 08/240030, filed May 9, 1994, and US patent application No. 08/404796; depositing light-curing hydrogel material; the use of a "hand-held gene gun" as described in US Pat. No. 5,149,655; the use of ionizing radiation in accordance with described in US patent No. 5206152 and international patent application WO 92/11033; the use of "nucleotide charge neutralization" or fusion with the cell membrane. In addition, similar approaches are described in Philip, 1994, Mol. Cell. Biol., 14, 2411-2418 and Woffendin, 1994, Proc. Natt. Acad. Sci. USA, 91, 1581-1585.

Particle gene delivery may be used: see, for example, US Patent Application No. 60/023867. Briefly, the sequence can be integrated into standard vectors, which contain the usual regulatory sequences that provide high-intensity expression; they are then incubated with a synthetic gene delivery molecule, such as polymeric DNA-binding cations, for example, polylysine, protamine and albumin, associated with ligands marking cells, such as asialorosomcoid, as described in Wu & Wu, 1987, J. Biol. Chem., 262, 4429-4432, or insulin as described by Hucked, 1990, Biochem. Pharmacol., 40, 253-263, or galactose as described by Plank, 1992, Bioconjugate Chem., 3, 533-539, or lactose, or transferrin.

Open (naked) DNA can also be used. Examples of methods for introducing open DNA are those techniques that are described in international patent application WO 90/11092 and in US patent No. 5580859. The absorption efficiency can be improved by using latex beads, which are destroyed in the biological environment. DNA loaded into latex beads is efficiently delivered to cells as a result of endocytosis, which is initiated by these beads. This method can be improved by treating the beads to increase hydrophobicity, thereby facilitating the destruction of the endosome and the release of this DNA into the cytoplasm.

Liposomes that can be used as gene delivery systems are described in US Pat. No. 5,421,220, in international patent applications WO 95/13796, WO 94/23697 and WO 91/14445 and EP 524968. As described in US Patent Application No. 60/023867 non-viral delivery method, the nucleotide sequences encoding the polypeptide can be inserted into standard vectors, which include the usual regulatory sequences that provide intense expression, followed by incubation with a synthetic gene-delivery molecule, such as polymeric DNA-binding cations, for example, polylysine, protamine and albumin, associated with ligands marking cells, such as asialorosomcoid, insulin, galactose, lactose or transferrin. In other delivery systems, liposomes are used to encapsulate DNA that carries a particular gene under the control of tissue-specific or ubiquitously active (i.e., tissue-independent) promoters. Other non-viral delivery systems are mechanical delivery systems, such as those used in the method described by Woffendin et al., 1994, Proc. Natl. Acad. Sci. USA 91, 11581-11585. Moreover, the coding sequence and its expression product can be delivered by deposition of light curing hydrogel materials. Other standard gene delivery methods may be used, including coding sequences, for example, based on the use of a “hand-held gene gun” as described in US Pat. No. 5,149,655, on the use of ionizing radiation to enable the transfer of genes as described in the patent. US No. 5206152 and international patent application WO 92/11033.

Examples of liposomal and polycationic gene delivery systems are those systems that are described in US Pat. , "Biochemistry", ed. W.H. Freeman, San Francisco, pp. 236-240; Szoka, 1980, Biochem. Biophys. Acta, 600, 1; Bayer, 1979, Biochem. Biophys. Acta, 550, 464; Rivnay, 1987, Meth. Enzymol., 149, 116; Wang, 1987, Proc. Natl. Acad. Sci. USA, 84, 7851; Plant, 1989, Anal. Biochem., 176, 420.

The polynucleotide composition may include a therapeutically effective amount of a gene therapy system in accordance with the term defined above. For the purposes of the present invention, an effective dose should be in the range of from about 0.01 mg / kg to 50 mg / kg, or from 0.05 mg / kg to about 10 mg / kg of DNA constructs as applied to the individual to whom they are being administered.

Delivery methods

Once included in a pharmaceutical composition, such polynucleotide compositions of the present invention can be (1) directly administered to a patient; (2) delivered ex vivo to cells derived from this patient; or (3) used for expression of recombinant proteins in vitro. The subjects to which such compositions will be administered may be mammals or birds. Also, treatment can be given for people.

Direct administration of the compositions should in principle be by injection - subcutaneous, intraperitoneal, intravenous or intramuscular - or they must be introduced into the interstitial spaces. Also, these compositions can be introduced into injuries (wounds). Other routes of administration are oral and intrapulmonary administration, the use of suppositories, as well as transdermal administration and administration “through the skin” (see, for example, international patent application WO 98/20734), or administration using needles, “gene guns” or hyposprays. Dosages may be in single or multiple doses.

Methods for administering ex vivo followed by reimplantation of transformed cells to a subject are well known in the art and are described, for example, in international patent application WO 93/14778. Examples of cells useful for ex vivo administration are, for example, stem cells, in particular hematopoietic cells, lymphatic cells, macrophages, dendritic cells or tumor cells.

In general, delivery of nucleic acids in ex vivo and in vitro applications can be carried out using, for example, the following procedures: transfection mediated by dextran, precipitation with calcium phosphate, transfection with polybrene, fusion of protoplasts, electroporation, encapsulation of polynucleotide (polynucleotides) into liposomes and direct microinjection of DNA into nuclei - all of these methods are well known in science.

Pharmaceutical compositions containing polynucleotides and polypeptides

In addition to the pharmaceutically acceptable carriers and salts that have been described above, the following auxiliary components can be used that are included in compositions containing a polynucleotide and / or polypeptide.

A. Polypeptides

One example is polypeptides, which, without limitation, include: asialorosomucoid (ASOR); transferrin; asialoglycoproteins; antibodies; antibody fragments; ferritin; interleukins; interferons; granulocyte and macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), macrophage colony stimulating factor (M-CSF), stem cell factor and erythropoietin. Viral antigens such as enveloped proteins may also be used. Can also be used proteins of other pathogenic organisms, such as the 17-amino acid peptide of the malarial plasmodium round sporozoite stage, known as RII.

B. Hormones, vitamins, etc.

Other groups of compounds that may be included include, for example, hormones, steroids, androgens, estrogens, thyroid hormone or vitamins, for example, folic acid.

C. Polyalkylene, polysaccharides, etc.

Also, polyalkylene glycol may be included along with the desired polynucleotides or polypeptides. In a preferred embodiment, the polyalkylene glycol is polyethylene glycol. In addition, mono-, di- or polysaccharides may be included. In a preferred embodiment of this aspect, the polysaccharide is dextran or DEAE-dextran. Chitosan and poly (lactide-co-glycolide) may also be used.

D. Lipids and liposomes

The desired polynucleotides or polypeptides can be encapsulated in lipids or packaged in liposomes before delivery to a subject or cells derived from it.

Lipid encapsulation is usually carried out using liposomes that are capable of stably binding or incorporating nucleic acid. The ratio of the packaged polynucleotide to the lipid material may vary, but in principle is close to 1: 1 (mg of DNA to μmol of lipid), or the amount of lipid is slightly higher. For the use of liposomes for nucleic acid delivery, see Hug & Sleight, 1991, Biochim for a review. Biophys. Acta, 1097, 1-17; Straubinger, 1983, Meth. Enzymol., 101, 512-527.

Liposome preparations for use in the present invention include cationic (i.e., positively charged), anionic (i.e., negatively charged) and neutral preparations. Cationic liposomes have been shown to provide intracellular delivery of plasmid DNA (Felgner, 1987, Proc. Nati. Acad. Sci. USA, 84, 7413-7416), mRNA (Malone, 1989, Proc. Natl. Acad. Sci. USA, 86, 6077-6081) and purified transcription factors (Debs, 1990, J. Biol. Chem., 265, 10189-10192), which retain their funcionality.

Cationic liposomes are readily available. For example, N- [1,2,3-dioleyloxy) propyl] -N, N, N-triethylammonium (DOTMA) liposomes are available under the trade name "Lipofectin" from Gibco BRL (Grand Island, NY) (see also Felgner, cit. above). Other commercially available liposomes are transfectants (DDAB / DOPE) and DOTAP / DOPE (Boehringer). Other cationic liposomes can be prepared from readily available materials using techniques well known in the art: see, for example, Szoka, 1978, Proc. Natl. Acad. Sci. USA 75, 4194-4198; and the synthesis of DOTAR (1,2-bis (oleoyloxy) -3- (trimethylammonium) propane) liposomes is described in international patent application WO 90/11092.

Similarly, anionic and neutral liposomes, such as those obtained from Avanti Polar Lipids (Birmingham, AL), or can be readily prepared using readily available materials, are readily available. Such materials include, but are not limited to, phosphatidylcholine, cholesterol, phosphatidylethanolamine, dioleylphosphatidylcholine (DOPC), dioleylphosphatidylglycerol (DOPG), dioleylphosphatidylethanolamine (DOPE). These materials can also be mixed with DOTMA and DOTAP starting materials in suitable ratios. Methods for preparing liposomes using these materials are well known in the art.

Liposomes may include multilayer vesicles (MLV), small unilamellar vesicles (SUV), or large unilamellar vesicles (LUV). Various complexes of liposomes and nucleic acids are formed using methods well known in the art: see, for example, Straubinger, 1983, Meth. Immunol., 101, 512-527; Szoka, 1978, Proc. Natl. Acad. Sci. USA 75, 4194-4198; Papahadjopoulos, 1975, Biochim. Biophys. Acta, 394, 483; Wilson, 1979, Cell, 17, 77; Deamer & Bangham, 1976, Biochim. Biophys. Acta, 443, 629; Ostro, 1977, Biochem. Biophys. Res. Commun., 76, 836; Fraley 1979, Proc. Natl. Acad. Sci. USA, 76, 3348; Enoch & Strittmatter, 1979, Proc. Natl. Acad. Sci. USA, 76, 145; Fraley, 1980, J. Biol. Chem., 255, 10431; Szoka & Papahadjopoulos, 1978, Proc. Natl. Acad. Sci. USA, 75, 145; Schaefer-Ridder, 1982, Science, 215, 166.

E. Lipoproteins

In addition, lipoproteins can be combined with a polynucleotide or polypeptide for delivery. Examples of lipoproteins that can be used are: chylomicrons, HDL, IDL, LDL and VLDL. Mutants, fragments, and chimeras of these proteins may also be used. Modifications of naturally occurring lipoproteins, such as acetylated LDL, may also be used. These lipoproteins can deliver polynucleotides to specific cells expressing lipoprotein receptors. Preferably, if lipoproteins are included in the composition along with the polynucleotide intended for delivery, other ligands that provide targeted delivery to the targets are not included in the composition.

Naturally occurring lipoproteins are composed of lipid and protein constituents. The protein component is known as "apoprotein." At present, apoproteins A, B, C, D, and E have been isolated and identified. At least two of these classes have several proteins, which are indicated by Roman numerals: AI, AII, AIV; CI, CII, CIII.

Lipoprotein may contain more than one apoprotein. For example, naturally occurring chylomicrons include apoproteins A, B, C and E, and over time they lose apoprotein-A and acquire apoproteins C and E. VLDL contains apoproteins A, B, C and E, LDL contains apoprotein-B, and the composition of HDL includes apoproteins A, C and E.

The amino acid composition of these apoproteins is known and described, for example, in Breslow, 1985, Annu. Rev. Biochem., 54, 699; Law, 1986, Adv. Exp. Med. Biol., 151, 162; Chen, 1986, J. Biol. Chem., 261, 12918; Kane, 1980, Proc. Natl. Acad. Sci. USA, 77, 2465; Utermann, 1984, Hum. Genet., 65, 232.

The composition of lipoproteins includes various lipids, including triglycerides, cholesterol (free or in the form of esters) and phospholipids. The composition of lipids varies in naturally occurring lipoproteins. For example, chylomicrons mainly include triglycerides. A more detailed description of the lipid composition of naturally occurring lipoproteins can be found, for example, in the 128th volume of the journal Methods in Enzymology (1986). The lipid composition is selected in such a way as to correspond to the conformation of apoprotein, thereby ensuring binding activity at the corresponding receptor. The composition of lipids can also be selected so as to facilitate hydrophobic interaction and binding to a molecule that binds a polynucleotide.

Naturally occurring lipoproteins can, for example, be isolated from blood serum by ultracentrifugation. Some methods are described in Methods in Enzymology (cited above) and Pitas, 1980, J. Biochem., 255, 5454-5460 and Mahey, 1979, J. Clin. Invest., 64, 743-750. Lipoproteins can also be obtained in vitro or by recombinant techniques by expressing apoprotein genes in a desired host cell: see, for example, Atkinson, 1986, Annu. Rev. Biophys. Chem., 15, 403 and Radding, 1958, Biochim. Biophys. Acta 30, 443. Lipoproteins can also be obtained from commercial distributors such as Biomedical Technologies Inc. (Stoughton, MA, USA). Further description of lipoproteins can be found from Zuckermann et al. In PCT / US 97/14465.

F. Polycationic components

The composition comprising the desired polynucleotide or polypeptide whose delivery is contemplated may include polycationic agents with or without lipoprotein.

Typically, polycationic components are characterized by a common positive charge at physiological pH values and are able to neutralize the electrical charge of nucleic acids, which should facilitate delivery to the desired location. These components can be used both in vitro and ex vivo, and in vivo. Polycationic components can be used for the delivery of nucleic acids intravitally to patients by intramuscular, subcutaneous injection, etc.

The following polypeptides are examples of useful polycationic components: polylysine, polyarginine, polyornithine and protamine. Other examples are histones, protamines, serum human albumin, DNA-binding proteins, non-histone chromosomal proteins, capsid proteins of DNA viruses, such as X174 virus; transcription factors also include DNA binding sites and therefore they can also be used as nucleic acid condensing agents. Briefly, the base domain on which DNA binds is part of transcription factors such as C / CEBP, c-jun, c-fos, AP-1, AP-2, AP-3, CPF, Prot-1, Sp- 1, Oct-1, Oct-2, CREP and TFIID.

Organic polycationic components are spermine, spermidine and putrescine.

The dimensions and physical properties of the polycationic component can be extrapolated based on the above list with the aim of constructing other polycationic components of a polypeptide nature or with the aim of creating synthetic polycationic components.

Synthetic polycationic components that are applicable include, for example, DEAE-dextran and polybrene. Lipofectin ™ and lipofectamine ™ are monomers that form polycationic complexes when combined with polynucleotides / polypeptides.

Immunological diagnostic tests

Neusserie antigens of the present invention can be used in immunological tests aimed at determining antibody levels (or, on the other hand, antibodies to neysseria antigens can be used to determine antigen levels). Immunological tests based on well-defined recombinant antigens can be developed to replace invasive diagnostic tests. Antibodies to neisseria proteins can be detected as part of biological samples, such as, for example, blood or blood serum. The development of such immunological tests is the subject of numerous modifications, and a wide range of them is known in the art. The formulations of these immunological tests can be based, for example, on tests with competitive binding or direct reaction, or the principle of "sandwich". For example, solid supports or immunoprecipitation can also be used in these protocols. Many tests use a labeled antibody or polypeptide; labels can be, for example, fluorescent, chemiluminescent, radioactive molecules or dye molecules. Tests are also known that use amplification of signals based on the corresponding probe: examples in this case are tests that use biotin and avidin, as well as enzyme-labeled and indirect immunological tests, such as TIFA (enzyme-linked immunosorbent assay).

Reagent kits suitable for immunodiagnostics, including suitable labeled reagents, are formed by packaging suitable materials, including the compositions of the present invention, in suitable containers, along with other materials and reagents (e.g., suitable buffers, salt solutions, etc.) necessary to conduct a competitive test, as well as the necessary set of informative instructions.

Nucleic Acid Hybridization

The term "hybridization" means the binding of two nucleotide sequences to each other via hydrogen bonds. Typically, one of these sequences is fixed on a solid support, and the other is in solution in a free state. Then provide contact between the two sequences under conditions that favor the formation of hydrogen bonds. Factors that influence the formation of such bonds are: type and amount of solvent; reaction temperature; hybridization duration; stirring the presence of components that would prevent nonspecific binding of the liquid phase sequence to a solid support (Denhardt's solution or BLOTTO reagent); sequence concentration; the use of compounds that are capable of increasing the binding rate of sequences (dextran sulfate or polyethylene glycol); and the stringency of the washing conditions after hybridization (see Sambrook et al., cited above, volume 2, chapter 9, pages 9.47-9.57).

By "stiffness" is meant hybridization conditions that favor the binding of very similar sequences in contrast to different sequences.

For example, the combination of temperature and salt concentration should be selected so that it is 120-200 ° C lower than the calculated dissociation temperature (T _m ) of the analyzed hybrid. The temperature and salt concentration parameters can, as a rule, be determined empirically in preliminary experiments in which samples of genomic DNA immobilized on filters are hybridized with the analyzed sequence and then washed under conditions of varying degrees of hardness (see Sambrook et al., P. 9.50).

The variables that need to be considered when performing, for example, Southern blotting, are: (1) the complexity of the DNA structure to be blotted, and (2) the level of homology of the probe and the detected sequences. The total amount of the studied fragment (s) can vary by an order of magnitude - from 0.1 to 1 μg for a plasmid or phage, or from 10 ^-9 to 10 ^-8 g for a single-copy gene from a complex eukaryotic genome. For less complex polynucleotides, substantially shorter blotting, hybridization, and exposure periods, smaller amounts of starting polynucleotides, and lower specific probe activities can be used. For example, a single-copy yeast gene can be detected with an exposure time of only 1 hour with the initial yeast DNA amount of 1 μg, blotting for 2 hours and hybridization for 4-8 hours with a probe having an activity of 100 million imp./min per 1 μg . For a single-copy mammalian gene, a conservative approach will start with 10 μg of DNA, blotting overnight and hybridization overnight in the presence of 10% dextransulfate using a probe with specific activity of over 100 million cpm per 1 μg, resulting in an exposure time will be approximately 24 hours.

Some factors may affect the melting temperature (T _m ) of the DNA / DNA hybrid formed between the probe and the fragment to be analyzed, and therefore the appropriate conditions for hybridization and washing. In many cases, the probe is not 100% homologous to this fragment. Other commonly taken variables are the length and total content of the GC pair in the hybridizing sequences, as well as the ionic strength and the content of formamide in the hybridization buffer. The influence of all these factors can be taken into account using a single equation:

T _m = 81 + 16.6 · (log ₁₀ Ci) + 0.4 · (% GC pair) -0.6 · (% formamide) -600 / n-1.5 · (% error),

where Ci is the concentration of salts (monovalent ions), and n is the length of this hybrid, expressed as the number of nucleotide pairs (the formula is slightly modified compared to that published by Meinkoth & Wahl, 1984, Anal. Biochem., 138, 267-284).

When constructing a hybridization experiment scheme, in a standard manner, some factors affecting nucleic acid hybridization can be changed. The simplest from the point of view of optimizing the temperature of hybridization and washing and the concentration of salts used during washing. With an increase in the temperature at which the hybridization reaction is carried out (ie, the stiffness of the reaction), it becomes less likely that hybridization will occur between nucleotide chains that are non-homogeneous, leading to a decrease in background “noise”. If the radioactively labeled probe is not completely homologous to the immobilized fragment (the situation is usual in the analysis of gene families and in carrying out experiments on interspecific hybridization), then the hybridization temperature should be reduced — then the background “noise” will increase. The washing temperature similarly affects the intensity of the hybridization band and the background level. The rigidity of the washing conditions also increases with decreasing salt concentrations.

In general, the standard hybridization temperatures in the presence of 50% formamide are 42 ° C for a probe homologous to a target fragment of 95-100%, 37 ° C for a homology of 90-95% and 32 ° C for a homology level of 85-90% . For lower homology levels, the formamide content should be reduced, followed by appropriate temperature adjustments based on the above equation. If the level of homology between the probe and the target fragment is unknown, then the simplest approach is to conduct an experiment initially with non-stringent conditions of hybridization and washing.

If after autoradiographic analysis nonspecific binding bands and the level of background “noise” turn out to be intense, then the filter can be washed at a high level of stiffness and retested. If the time required for exposure makes this approach impractical, then several options for the rigidity of hybridization and / or washing can be tested simultaneously ("in parallel").

Tests with nucleotide probes

In methods such as PCR, branched DNA probe assays, and nucleotide probe blotting techniques in accordance with the present invention, cDNA or mRNA can be detected. The probe is said to “hybridize” to the sequence of the present invention when it forms a duplex or double-stranded complex that is stable enough to be detected.

The corresponding nucleotide probes must hybridize with the nucleotide sequences of the neisseries of the present invention (including both sense and non-sense strands). Although a particular amino acid sequence can encode many different nucleotide sequences, native neisseria sequences are preferred because they are actually present in the cells of these organisms. mRNA represents a coding sequence, therefore, the probe must be complementary to the corresponding coding sequence; single stranded cDNA is complementary to mRNA; therefore, a cDNA probe must be complementary to a non-coding sequence.

It is not necessary that the nucleotide sequence of the probe be identical to the sequence of the neisseria (or its complement) - a certain level of sequence variability and the length of the nucleotide sequence may increase the sensitivity of the test when the nucleotide probe can form a duplex with target nucleotides, which can be detected. Also, the nucleotide probe may include additional nucleotides, which are designed to stabilize the resulting duplex. The additional sequence of the neisseria can also fulfill the auxiliary role of the label, necessary to identify the resulting duplex. For example, a non-complementary nucleotide sequence can be attached to the 5'-end of the probe, while the rest of the sequence of this probe is complementary to the sequence of the neisseria. On the other hand, non-complementary nucleotides or extension sequences can be dispersed along the length of this probe, taking into account that the nucleotide sequence of this probe is characterized by a significant level of complementarity with respect to the neisseria sequence, as a result of which they will hybridize and form a duplex, which can be identified.

The exact length and nucleotide sequence of the probe will depend on hybridization conditions such as temperature, salt conditions and the like. For example, for diagnostic use, depending on the complexity of the nucleotide sequence being analyzed, a nucleotide probe usually consists of at least 10-20 nucleotides, preferably 15-25, and most preferably at least 30 nucleotides, although it may be shorter. Short seeds, as a rule, require lower temperatures for the formation of sufficiently stable hybrid complexes with the matrix.

Probes can be formed using synthesis procedures, for example, using the Matheucci ethereal triester method. (Matteucci et al., 1981, J. Amer. Chem. Soc., 103, 3185) or in accordance with Urdea et al., 1983, Proc. Natl. Acad. Sci. USA, 80, 7461, or using commercially available automated oligonucleotide synthesizers.

The chemical nature of the probe can be selected based on specific preferences. For some uses, DNA or RNA is preferred. For other applications, various modifications can be made: for example, modifications of the axial structure, such as phosphothioates or methylphosphonates, can be used to increase the in vivo half-life, change the level of RNA affinity, increase the resistance to the action of nucleases, etc. (see, for example, Agrawal & Iyer, 1995, Curr. Opinion Biotechnol., 6, 12-19; Agrawal, 1996, TIBTECH, 14, 376-387); analogs such as nucleic acid peptides can also be used (see, for example, Corey, 1997, TIBTECH, 15, 224-229; Buchardt et al., 1993, TIBTECH, 11, 384-386).

Polymerase chain reaction (PCR), on the other hand, is another well-known method for detecting small amounts of target nucleic acids. This test is described by Mullis et al. (Mullis et al., 1987, Meth. Enzymol., 155, 335-350) and in US Pat. Nos. 4,683,195 and 4,683,202. Two seed nucleotides are hybridized with target nucleic acids and used to seed the reaction. Such seeds can include a sequence that does not hybridize with a sequence designed to amplify a target (or its complement) to give stability to the duplex or, for example, to introduce a standard restriction site. Typically, such a sequence should flank the desired sequence of the neisseria.

Thermostable polymerase creates copies of nucleotide target acids from seed using the original target nucleic acid as a template. Once such a polymerase provides a certain threshold number of target nucleic acids, they can be detected using more traditional methods, such as Southern blotting. When using Southern blotting, the labeled probe should hybridize to the neisseria sequence (or its complement).

Also, mRNA or cDNA can be detected using traditional blotting techniques described by Sambrook et al. (cit. above). The mRNA or cDNA formed on the mRNA matrix using the polymerase enzyme can be purified and separated using gel electrophoresis. The nucleic acids in the gel are then blotted on a solid support such as nitrocellulose. The solid support is treated with a labeled probe and then washed to remove any non-hybridized probes. Next, the resulting duplexes are detected, including the labeled probe. Typically, such a probe is labeled using a radioactive component.

Brief Description of the Figures

) обозначает контрольные данные GST; кружок (•) показывает данные, полученные с рекомбинантным белком N.meningitidis. Компьютерный анализ показывает кривую гидрофильности (вверху), кривую антигенного индекса (в середине) и данные анализа AMPHI (внизу).Figure 1-20 shows the biochemical analysis data obtained in the following examples, as well as sequencing data for the ORF coding frame 37, 5, 2, 15, 22, 28, 32, 4, 61, 76, 89, 97, 106, 138, 23, 25, 27, 79, 85 and 132. M1 and M2 correspond to molecular weight markers. The arrows indicate the position of the main recombinant product or (in Western blotting) the position of the main immunoreactive band N.meningitidis. TP is the total protein extract of N.meningitidis; OMV stands for N.meningitidis vesicle outer membrane preparation. In the description of the bactericidal test: the diamond sign (◆) indicates preimmune data; triangle sign (

) denotes GST control data; the circle (•) shows the data obtained with the recombinant protein N.meningitidis. Computer analysis shows a hydrophilicity curve (above), an antigenic index curve (in the middle) and AMPHI analysis data (below).

The AMPHI computer program is used to predict the location of T-cell epitopes (Gao et al., 1989, J. Immunol., 143, 3007; Roberts et al., 1996, AIDS Res. Hum. Retroviruses, 12, 593; Quakyi et al. , 1992, Scand. J. Immunol., Suppl. 11, 9) and it is available from the Protean software package of DNASTAR Inc. (1228 S, Park str., Madison, WI 53715, USA).

Examples

The following examples describe the nucleotide sequences that were identified in N.meningitidis in parallel with the translation products that they supposedly encoded, as well as the same N..gonorrhoeae sequences. He all of the given nucleotide sequences are complete: i.e. they encode less than the full-sized wild-type protein.

The above examples are generally formed in this way:

- the nucleotide sequence that has been identified in N.meningitidis (strain B);

- a product predictably translated from this nucleotide sequence;

- data from a computer analysis of the translation product based on a comparison of available databases;

- indications of the corresponding gene and protein sequences identified in N.meningtidis (strain A) and N..gonorrhoeae;

- a description of the characteristics of the proteins, which indicate that they may have suitable antigenicity;

- results of biochemical analysis (expression, purification, TIFA, FACS, etc.).

Examples typically include details describing the level of identity between species and strains. Proteins that are similar in their amino acid sequence are similar in their structure and functions, i.e. sequence identity often indicates unity of origin in phylogenesis. Comparison of the amino acid sequences of proteins with a known function is widely used as a method for evaluating the putative functions of proteins with a new sequence, and it has also been proved to be applicable for the analysis of complete genomes.

Comparison of sequences was performed in the NCBI system (http://www.ncbi.nlm.nih.gov) using the algorithms BLAST, BLAST2, BLASTn, BLASTp, tBLASTn, BLASTx & tBLASTx (also see, for example, Altschul et al., 1997 , "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", Nucl. Acids Res., 25, 2289-3402). Searches were performed using the following polypeptide database programs: non-redundant sequences GenBank + EMBL + DDBJ + PDB and GenBank + broadcasts CDS + PDB + SwissProt + SPupdate + PIR.

To compare the sequences of meningococcus and gonococcus, the tBLASTx algorithm, http://www.genome.ou.edu/gono_blast.html, was used. Also, the FASTA algorithm was used to compare the ORF open coding framework (from the GCG Wisconsin Package, version 9.0).

Labeling with dots in the nucleotide sequences (for example, at position 495 in the sequence SEQ ID NO 11) identifies nucleotides that have been randomly inserted to maintain the coding frame. In the same way, double underlining corresponds to deleted nucleotides. Lower case letters (for example, at position 496 in SEQ ID NO 11) correspond to the uncertainties that arise when comparing independently conducted sequencing reactions (some of the nucleotide sequences in the following examples are the result of a generalization of two or more experiments).

Nucleotide sequences were scanned in all six coding frames to predict the presence of hydrophobic domains, using an algorithm based on statistical approaches Esposti et al., 1990, "Critical evaluation of the hydropathy of membrane proteins", Eur. J. Biochem., 190, 207-219). These domains represent potential transmembrane regions or hydrophobic signal (leader) segments.

Open coding frames were predicted based on fragmented nucleotide sequences, for which the ORFFINDER (NCBI) program was used.

The underlining of amino acid sequences indicates the presence of probable transmembrane domains or signal segments in the ORF coding frame in accordance with the predictions of the PSORT program (http: // www. Psort.nibb.ac.jp). Functional domains were also predicted using the MOTIFS program (GCG Wisconsin & PROSITE).

Various tests have been used to evaluate in vivo immunogenicity of the proteins identified in the examples. For example, proteins can be expressed recombinantly and then used for screening patient serum by immunoblotting. The presence of a positive reaction between the protein and serum of the patient indicates that this patient has previously formed an immune response to the analyzed protein, i.e. this protein is an immunogen. This method can be used to identify immunodominant proteins.

Recombinant protein can also be used in a standard manner to prepare antibodies, for example, in mice. They can be used to directly confirm that the protein is localized on the surface of the cells. A labeled antibody (for example, labeled with a fluorescent label for use in the FACS method) can be incubated with intact bacteria - the presence of the label on the surface of bacterial cells confirms this localization of the corresponding protein.

In particular, the following methods (A to S) were used for expression, purification and biochemical analysis of the proteins of the present invention.

A) Obtaining chromosomal DNA

Meningococci N.meningitidis strain 2996 was grown to an exponential stage of growth in 100 ml of GC culture medium, collected by centrifugation and resuspended in 5 ml of buffer (20% sucrose, 50 mM Tris-HCl, 50 mM EDTA - pH 8). After 10 minutes of incubation on ice, the bacteria were lysed by adding 10 ml of a lyse solution (50 mM NaCl, 2% sodium sarcosyl, 50 μg / ml proteinase-K) and the resulting suspension was incubated at 37 ° C for 2 hours. Twice extraction with phenol was carried out (when equilibrated in pH 8) and a single extraction with a mixture of chloroform and isoamyl alcohol (24: 1). DNA was precipitated by the addition of 0.3 M sodium acetate and 2 volumes of ethyl alcohol and then collected by centrifugation. The resulting clot was washed once with 70% ethanol and redissolved in 4 ml of buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8). The concentration of the obtained DNA preparation was determined by optical density at 260 nm.

B) Construction of the oligonucleotide

Synthetic oligonucleotide primers were constructed based on the coding sequence of each ORF coding frame using (a) the meningococcus B sequence (if available) or (b) the gonococcus / meningococcus A sequence adapted to codon preference for meningococcus (if necessary) . Any putative signal segments were discarded, for which the generated 5'-end amplification seeds corresponded to the area immediately following the putative leader segment.

For most ORF frameworks, direct primers (5 ') included two restriction enzyme recognition sites (BamHI / NdeI, BamHI / NheI or EcoRI / NheI, depending on the intrinsic restriction parameters of a particular gene); back (3 ') seeds included the restriction XhoI site. This procedure was undertaken to direct the cloning of each amplification product (corresponding to each ORF coding frame) in two different expression systems: pGEX-KG (using restriction sites either BamHI / XhoI or EcoRI / XhoI) and pET21b + (using restriction sites or NdeI / XhoI, or NheI / XhoI).

For the ORF frames 5, 15, 17, 19, 20, 22, 27, 28, 65, and 89, two independent amplification reactions were performed to clone each of the ORFs in each of the two expression systems. Two different 5'-end seeds were used for each ORF, while the back-seed was the same 3'-seed as mentioned above:

The coding frame 76 was cloned using the pTRC expression vector and expressed as a chimera with an N-terminus attached to a polyhistidine tail. In this particular case, the intended signal segment was included in the final product. NheI / BamHI restriction sites were introduced using the following seeds:

As well as sequences recognized by restrictase enzymes are included, nucleotides that hybridize with the sequence to be amplified are also included in the seeds. The number of hybridizing nucleotides depended on the melting temperature of the complete seed and was determined for each seed using the following formulas:

T _m = 4 · (G + C) +2 (A + T) (tail segment excluded),

T _m = 64.9 + 0.41 · (% GC) -600 / N (full seed).

The average melting temperature of the selected oligonucleotides was 65-70 ° C for complete oligonucleotides and 50-55 ° C for individual hybridization sites.

Table 1 shows the forward and backward seeds used for each amplification reaction. In some cases, it should be noted that the sequence of a specific seed does not completely match the sequence of the coding frame. When the initial amplification reactions were carried out, the full 5 ′ and / or 3 ′ sequences were not known for some meningococcal ORFs, although the corresponding sequences were identified in gonococcus. For amplification of the sequence of gonococci could, therefore, be used as the basis for the design of seeds, carrying out changes in the sequence, based on the preferences of the codons. In particular, such codon changes were carried out: ATA → ATT; TCG → TST; CAG → CAA; AAG → AAA; GAG → GAA; CGA → CGC; CGG → CGC; GGG → GGC. Such changes are reflected in table. 1 by highlighting nucleotides in italics. It should be understood that once a complete sequence has been identified, this described approach becomes further unnecessary.

Oligonucleotides were synthesized using a Perkin-Elmer Model 394 DNA / RNA synthesizer, eluted from 2 ml NH ₄ OH columns, and released from protection by 5-hour incubation at 56 ° C. These oligonucleotides were precipitated by the addition of 0.3 M sodium acetate and 2 volumes of ethanol. Samples were then centrifuged and clots resuspended in either 100 μl or 1 ml of water. The optical density OD _{260 was} determined using a Laiabda-Bio spectrophotometer (Perkin-Elmer), thereby evaluating the concentration, which was then adjusted to 2-10 pmol / μl.

C) Amplification

The standard PCR protocol was as follows: 50-200 ng of genomic DNA was used as a template in the presence of 20-40 μM of each oligonucleotide, 400-800 μM solution of a mixture of deoxyribonucleotides (dNTP), 1 × PCR buffer (including 1.5 mm MgCl ₂ ) , 2.5 units TaqI DNA polymerase (using an AmpliTaQ Perkin-Elmer amplifier, Gibco Platinum; Pwo DNA polymerase or Tahara Shuzo Taq polymerase).

In some cases, PCR parameters were optimized by adding 10 μl DMSO or 50 μl 2 M betaine.

After a hot start (adding polymerase to a mixture incubated for 3 minutes at 95 ° C), each sample underwent 2-stage amplification: the first 5 cycles were performed using the hybridization temperature determined for oligonucleotides with a restriction tail excluded from the calculation sites, followed by 30 cycles carried out at a hybridization temperature determined for full-sized oligonucleotides. All cycles were completed with the final 10-minute stage of chain completion at 72 ° C.

Standard cycles were as follows:

The duration of the completion phase varied depending on the length of the ORF coding frame, the amplification of which is carried out.

Amplification reactions were carried out using the gene-amplifier models 9600 or 2400 (Perkin-Elmer). To verify the results, a tenth of the amplification volume was loaded into a 1-1.5% agarose gel and the size of each of the amplification products was compared with the molecular weight of the DNA marker.

The amplified DNA was either loaded directly into a 1% gel, or first precipitated with ethanol and resuspended in a suitable volume, which should be loaded into a 1% gel. The DNA fragment corresponding to the correct band size was then eluted and gel purified using the Qiagen Gel Extraction Kit in accordance with the manufacturer's instructions. The final volume of the DNA fragment was 30 μl or 50 μl in water or 10 mm Tris, pH 8.5.

D) Cleavage of PCR fragments

The purified DNA corresponding to the amplified fragment was divided into two aliquots and subjected to double digestion with restriction enzymes:

- NdeI / XhoI or NheI / XhoI for cloning into pET21b + and further expression of the protein in the form of the C-terminal part of the chimera, including polyhistidine tail;

- BamHI / XhoI or EcoRI / XhoI for cloning into pGEX-KG and further expression of the protein as the N-terminal portion of the GST chimera;

- in the variant with the coding frame ORF76 NheI / BamHI for cloning into the vector pTRC-HisA and further expression of the protein in the form of the N-terminal part of the chimera, including polyhistidine tail;

- EcoRI / PstI, EcoRI / SalI, SalI / PstI for cloning into pGex-His and further expression of the protein in the form of the N-terminal part of the chimera, including polyhistidine tail.

Each purified DNA fragment was incubated (at 37 ° C for 3 hours or overnight) with 20 units. each of the restriction enzymes (manufactured by New England Biolabs) at a final volume of either 30 μl or 40 μl in the presence of a suitable buffer. The digested product was then purified using the QIAquick PCR purification kit in accordance with the manufacturer's instructions and eluted into a final volume of 30 μl or 50 μl either in water or in 10 mM Tris-Hcl (pH 8.5). The final DNA concentration was determined by electrophoresis on a 1% agarose gel in the presence of a titrated molecular weight marker.

E) Processing of cloning vectors (pET22B, pGEX-KG, pTRC-HisA, pGex-His)

Double restriction was subjected to 10 μg of the plasmid, using 50 units. each restriction enzyme in 200 μl of reaction volume in the presence of a suitable buffer overnight during incubation at 37 ° C. After the treated volume was fully loaded into a 1% agarose gel, the band corresponding to the restriction enzyme digested vector was gel purified using the QIAquick Gel Extraction kit (Qiagen) and the DNA was eluted in 50 μl in 10 mM Tris-Hcl (pH 8.5 ) DNA concentration was evaluated by measuring the OD _{260 of} each sample and adjusted to 50 μg / ml. 1 μl of each plasmid was used in each cloning procedure.

The pGEX-His vector is a modified pGEX-2T vector carrying a region that encodes six histidine residues located above the thrombin cleavage site and including multiple cloning sites from the pTRC99 vector (Pharmacia).

F) Cloning

Fragments corresponding to each coding frame, pretreated with restriction enzymes and purified, were ligated into both pET22b and pGEX-KG. At a final volume of 20 μl, the molar ratio of 3: 1 of the fragment and vector was ligated using 0.5 μl of NEB T4 phage DNA ligase (400 units / μl) in the presence of a buffer proposed by the manufacturer. The reaction mixture was incubated at room temperature for 3 hours. In some experiments, ligation was performed using the Rapid Ligation kit (Boehringer) in accordance with the manufacturer's instructions.

In order to introduce a recombinant plasmid into a suitable strain, 100 μl of competent E. coli cells (strain DH5) were incubated with ligase reaction for 40 minutes on ice, then at 37 ° C for 3 minutes, then, after adding 800 μl of LB broth, again incubated at 37 ° C for 20 minutes. The cells were then centrifuged at maximum rotation speed of the Eppendorf microcentrifuge and resuspended in approximately 200 μl of the supernatant fraction. The resulting suspension was then plated on LB medium containing ampicillin (100 mg / ml).

Recombinant colonies were screened by growing five randomly selected colonies overnight at 37 ° C in either 2 ml (pGEX or pTC clones) or 5 ml (pET clones) of LB broth + 100 μg / ml ampicillin. Cells were then pelletized and DNA was extracted using the QIAprep Spin Miniprep kit (Qiagen) according to the manufacturer's instructions to a final volume of 30 μl. 5 μl of each individual minidrug (approximately 1 g) was treated with restriction enzymes of either NdeI / XhoI or BamHI / XhoI and the entire treated volume was loaded into a 1-1.5% agarose gel (depending on the expected insert size) along with a molecular marker masses (1Kb DNA Ladder, Gibco). Positive clones were screened for the correct size of the desired insert.

To clone the ORF coding frames 110, 111, 113, 115, 119, 122, 125, and 130, the restriction enzyme-treated PCR product was ligated into a restriction-digested vector using EcoRI / PstI cloning sites, in the case of ORF frames 115 and 127 - EcoRI / SalI sites, and in the case of frame 122, SalI / PstI sites. After cloning, recombinant plasmids were introduced into E. coli cells of strain W3110. Individual clones were cultured overnight at 37 ° C in L-broth with the addition of 50 μl / ml ampicillin.

G) Expression

Each ORF coding frame cloned into an expression vector was used to transform a strain suitable for expression of a recombinant protein product. 1 μl of each construct was used to transform 30 μl of E. coli strain BL21 (vector pGEX), E. coli strain TOP10 (vector pTRC) or E. coli strain BL21-DE3 (vector pET) as described above. In the case of the pGEX-His vector, the same E. coli E. coli strain (W3110) was used for the initial cloning and expression. Separate recombinant colonies were seeded in 2 ml of LB culture broth with ampicillin (100 μg / ml), incubated overnight at 37 ° C, then diluted 1:30 in 20 ml of LB with ampicillin (100 μg / ml) in 100- ml flasks, while checking the optical density of OD ₆₀₀ , which should be 0.1-0.15. These flasks were incubated at 30 ° C in a rotary shaker with a water bath until the optical density parameter indicated the culture's exit to an exponential growth stage suitable for expression induction (0.4-0.8 OD for pET and pTRC vectors; 0 , 8-1 OD for pGEX and pGEX-His vectors). For the pET, pTRC and pGEX-His vectors, protein expression was induced by the addition of 1 mM IPTG, while in the case of the pGEX vector, the final concentration of IPTG was 0.2 mM. After 3 hours of incubation at 30 ° C, the final concentration of the sample was checked by OD. In order to control expression, 1 ml of each sample was removed, centrifuged in a microcentrifuge, the resulting clot was resuspended in phosphate-buffered saline (PBS) and analyzed by SDS-electrophoresis in 12% PAG with Coomassie blue staining. The full volume of the sample was centrifuged at 6000 g and the resulting clot was resuspended in PBS for further use.

H) Large-scale purification of chimeric GST proteins

A single colony was cultured overnight at 37 ° C in LB medium with ampicillin on an agar plate. Bacteria were plated in 20 ml of LD with ampicillin in a liquid culture in a shaker containing a water bath, and cultured overnight. Bacteria were diluted 1:30 into a volume of 600 ml (fresh culture medium) and allowed to grow at the optimum temperature (20-37 ° C) until an optical density of OD ₅₅₀ 0.8-1 was reached. Protein expression was induced by the addition of 0.2 mM IPTG followed by 3-hour incubation. The culture was centrifuged at 8000 rpm at 4 ° C. The supernatant was removed and the clot of bacterial cells resuspended in 7.5 ml of cold phosphate-saline buffer. Cells were sonicated on ice for 30 seconds at 40 W using a B-15 apparatus (Branson), frozen and melted twice and centrifuged again. The supernatant was collected and mixed in 150 μl of glutathione-sepharose-4B resin (Pharmacia) (previously washed in PBS) and incubated at room temperature for 30 minutes. The sample was centrifuged at 700 g for 5 minutes at 4 ° C. The resin was washed 2 times in 10 ml of cold FSB for 10 minutes, resuspended in 1 ml of cold FSB and loaded onto a free column. The resin was washed twice in 2 ml of cold FSB until the flow material reached an optical density of OD ₂₈₀ 0.02-0.06. The chimeric GST protein was eluted by adding 700 μl of cold glutathione elution buffer (10 mM reduced glutathione, 50 mM Tris-Hcl) and fractions were collected until OD ₂₈₀ reached 0.1. 21 μl of each fraction was loaded into a 12% SDS gel using either molecular standards for SDS-PAGE from Biorad (M1) (200, 116.25, 97.4, 66.2, 45, 31, 21.5 , 14.4, 6.5 kD), or a set of standards from Amersham (M2) (220, 66, 46, 30, 21.5, 14.3 kD). While the molecular weight of the GST is 26 kD, this value should be added to the molecular weight determined for each chimeric GST-including protein.

I) Solubility analysis of His-chimeras (coding frames 111-129)

To analyze the solubility of His-chimeric expression products, clots of 3 ml cultures were resuspended in Ml buffer (500 μl PBS - pH 7.2). 25 μl of lysozyme was added and bacteria were incubated for 15 minutes at 4 ° C. The resulting clots were irradiated with ultrasound for 30 seconds at a power of 40 W using a Branson B-15 ultrasonic apparatus, frozen and melted twice, and then again separated into a clot and supernatant by centrifugation. The supernatant was collected and the remaining clot was resuspended in M2 buffer (8 M urea, 0.5 M NaCl, 20 mm imidazole and 0.1 M NaH ₂ PO ₄ ) and incubated for 3-4 hours at 4 ° C. After centrifugation, the supernatant was collected and the clot was resuspended in M3 buffer (6 M guanidine-HCl, 0.5 M NaCl, 20 mM imidazole and 0.1 M NaH ₂ PO ₄ ) overnight at 4 ° C. The supernatant fractions at each of these steps were analyzed by electrophoresis in SDS-PAGE.

Proteins expressed by frames ORF 113, 119 and 120 have been found to be soluble in phosphate buffered saline, while products from frames ORF 111, 122, 126 and 129 require urea to dissolve, and products from frames ORF 125 and 127-guanidinochloride .

J) Large-scale purification of His-chimeras

A single colony was cultured overnight at 37 ° C in LB medium with ampicillin on an agar plate. Bacteria were sown in liquid culture of 20 ml in LB medium with ampicillin and incubated overnight in a shaker with a water bath. Bacteria were diluted 1:30 in 600 ml of fresh medium and allowed to grow at the optimum temperature (20-37 ° C) to a density of QD _{550 of} 0.6-0.8. Protein expression was induced by the addition of 1 mM IPTG and the culture was further incubated for three hours. The culture was then centrifuged at 8000 rpm at 4 ° C, the supernatant was removed and the bacterial clot was resuspended in 7.5 ml of either (1) cold buffer A (300 mm NaCl, 50 mm phosphate buffer, 10 mm imidazole - pH 8) for soluble proteins, or (2) buffer B (8 M urea, 10 mm Tris-HCl, 100 mm phosphate buffer - pH 8.8) for insoluble proteins.

Cells were destroyed by ultrasound on ice for 30 seconds at a power of 40 W using a Branson B-15 ultrasonic apparatus, frozen twice, melted and centrifuged again.

In the case of insoluble proteins, the supernatant was stored at -20 ° C, while the clots were resuspended in 2 ml of buffer C (6 M guanidine hydrochloride, 100 mM phosphate buffer, 10 mM Tris-Hcl pH 7.5) and processed in a homogenizer for 10 cycles. The resulting product was centrifuged at 13,000 rpm for 40 minutes.

The supernatant fractions were collected and mixed with 150 μl of Nickel-containing (Ni ²⁺ ) resin (Pharmacia) (pre-washed with buffer A or buffer B) and incubated at room temperature with thorough stirring for 30 minutes. The sample was centrifuged at 700 g for 5 minutes at 4 ° C. The resin was washed twice in 10 ml of buffer A or B for 10 minutes, resuspended in 1 ml of buffer A or B and loaded onto a free column. The resin was washed either (1) in 2 ml of cold buffer A at 4 ° C, or (2) at room temperature in 2 ml of buffer B until the flowing material reaches an optical density of OD ₂₈₀ 0.02-0.06.

The resin was washed either in (1) 2 ml cold buffer with 20 mM imidazole (300 mM NaCl, 50 mM phosphate buffer, 20 mM imidazole pH 8), or in (2) D buffer (8 M urea, 10 mM Tris-HCl , 100 mM phosphate buffer - pH 6.3) until the flowing material reaches an optical density of OD ₂₈₀ 0.02-0.06. The chimeric His protein was eluted by adding 700 μl of either (1) cold elution buffer A (300 mM NaCl, 50 mM phosphate buffer, 250 mM imidazole — pH 8), or (2) elution buffer B (8 M urea, 10 mM Tris- HCl, 100 mM phosphate buffer, pH 4.5) and the resulting fractions were taken to a density of OD ₂₈₀ = 0.1. 21 μl of each fraction was loaded into a 12% SDS gel.

K) Renaturation of His-chimeric proteins

10% glycerol was added to the denatured proteins. The proteins were then diluted to a concentration of 20 μg / ml using dialysis buffer I (10% glycerol, 0.5 M arginine, 50 mM phosphate buffer, 5 mM reduced glutathione, 0.5 mM oxidized glutathione, 2 M urea - pH 8.8 ) and dialyzed against the same buffer at 4 ° C for 12-14 hours. Then this protein was dialyzed against dialysis buffer II (10% glycerol, 0.5 M arginine, 50 mM phosphate buffer, 5 mM reduced glutathione, 0.5 mM oxidized glutathione - pH 8.8) for 12-14 hours at 4 ° FROM. Protein concentration was determined using the following formula:

Protein (mg / ml) = (1.55 × OD ₂₈₀ ) - (0.76 × OD ₂₆₀ ).

L) Large-scale purification of His chimera (coding frame ORF 111-129)

500 ml of bacterial cultures were incubated and chimeric proteins were obtained soluble in M1, M2 or M3 buffers, for which the protocols described above were used. The crude bacterial extract was loaded onto a super-fast Ni-NTA (Quiagen) column, equilibrated with M1, M2 or M3 buffer, depending on which of these buffers the chimeric protein was dissolved. Unbound material was eluted by washing the column with the same buffer. The particular protein was eluted using an appropriate buffer containing 500 mM imidazole and dialyzed against the corresponding buffer in which imidazole was absent. After each duct, columns were restored by washing with at least two column volumes of 0.5 M sodium hydroxide and re-equilibrating before next use.

M) Immunization of mice

For immunization of mice by intraperitoneal injection, 20 μg of each of the purified proteins was used. In cases of the ORF coding frame 2, 4, 15, 22, 27, 28, 37, 76, 89, and 97, when immunizing BALB / c mice on days 1, 21, and 42, hydroxide was used as adjuvant aluminum, and the immune response was monitored in samples on day 56. In the case of the ORF coding frames, 44, 106 and 132 mice of the CD1 line were immunized in the same protocol. In the case of the ORF coding frames, 25 and 40 CD1 mice were immunized in the same protocol, replacing aluminum hydroxide with Freund's adjuvant as an adjuvant; also the difference was that the immune response was monitored on the 42nd, and not on the 56th day. Similarly, in the cases of the ORF coding frame 23, 32, 38, and 79, CD1 mice were immunized with Freund's adjuvant, but the immune response was determined on day 49.

N) TIFA test (serum analysis)

The decapsulated MenB M7 strain was plated on chocolate agar plates and incubated overnight at 37 ° C. Bacterial colonies were selected from agar plates using a sterile brush and plated in 7 ml of Mueller-Hinton (Difco) culture broth containing 0.25 glucose.

Bacterial growth was monitored every 30 minutes with an OD of ₆₂₀ . Bacteria were allowed to grow until an optical density of OD 0.3-0.4 was reached. The cultures were centrifuged for 10 minutes at 10,000 rpm. The supernatant was removed and the bacteria washed once with phosphate-buffered saline, resuspended in PBS containing 0.025% formaldehyde, and incubated for 2 hours at room temperature and then, stirring, overnight at 4 ° C. 100 μl of bacterial cells were added to each well of a 96-well graining plate and incubated overnight at 4 ° C. Then, the wells were washed three times with phosphate wash buffer (0.1% Tween-20 in phosphate-buffered saline). 200 μl of saturating buffer (2.7% polyvinylpyrrolidone-10 in water) was added to each well, and the plates were incubated for 2 hours at 37 ° C. Wells were washed three times in the FSB. 200 μl of diluted serum was added to each well (dilution buffer: 1% bovine serum albumin, 0.1% tween-20, 0.1% NaN in PBS) and the plates were incubated for 90 minutes at 37 ° C. Then the wells were washed three times in the FSB. 100 μl of horseradish peroxidase conjugated rabbit anti-mouse serum (Dako) diluted 1: 2000 in dilution buffer was added to each well, and the plates were incubated for 90 minutes at 37 ° C. 100 μl of horseradish peroxidase substrate buffer (25 ml of citrate buffer, pH 5; 10 mg of O-phenyldiamine and 10 μl of water) was added to each well and the plates were left at room temperature for 20 minutes. 100 μl of sulfuric acid was added to each well and OD _{490 was} determined. The TIFA test turned out to be positive when the value of OD ₄₉₀ was 2.5 times higher than the corresponding parameter of pre-immune serum.

O) FACScan bacterial binding test procedure

The decapsulated MenB M7 strain was plated on chocolate agar plates and incubated overnight at 37 ° C. Bacterial colonies were collected from agar plates using a sterile brush and plated in 4 tubes each containing 8 ml of Mueller-Hinton (Difco) culture broth containing 0.25% glucose. Bacterial growth was monitored every 30 minutes according to OD ₆₂₀ . Bacteria were allowed to grow until the optical density reached 0.35-0.5. The cultures were centrifuged for 10 minutes at 4000 rpm. The supernatant was removed and the clot resuspended in blocking buffer (1% bovine serum albumin, 0.4% NaN ₃ ) and centrifuged for 5 minutes at 4000 rpm. Cells were resuspended in blocking buffer until an OD ₆₂₀ of 0.07 was reached. 100 μl of bacterial cells were added to each well of a Costar 96-well plate. 100 μl of diluted (1: 200) serum (in blocking buffer) was added to each well and the plates were incubated for 2 hours at 4 ° C. The cells were centrifuged for 5 minutes at 4000 rpm, the supernatant was aspirated and the cells were washed by adding 200 μl of blocking buffer to each well. 100 μl of goat anti-mouse F (ab) ₂ antibody conjugated with R-phycoerythrin and diluted 1: 100 was added to each well, and the plates were incubated for 1 hour at 4 ° C. Cells were pelleted by centrifugation at 4000 rpm for 5 minutes and washed by adding 200 μl of blocking buffer to each well. The supernatant was aspirated and the cells resuspended in 200 μl PBS per well + 0.25% formaldehyde. Samples were transferred to FACS tubes (sorted cells with excitation fluorescence) and read them. The conditions for the FACS scan were as follows: FL1 - on; FL2 and FL3 - off; FSC-H - threshold value of 92; FSC РМТ - voltage Е-02; SSC РМТ - 474; increase in amplitude - 7.1; FL-2 RMT - 539; the compensation value is 0.

P) Obtaining OMV

Bacteria were cultured overnight on five GC plates, looped and resuspended in 10 ml of 20 mM Tris-Hcl. Heat inactivation was carried out for half an hour at 56 ° C and the bacterial cells were destroyed by ultrasound for 10 minutes on ice (50% of the load in the irradiation cycle, 50% of the output power). Undamaged cells were removed by centrifugation at 5000 g for 10 minutes and the complete fraction of cell membranes was isolated by centrifugation at 50,000 g for 75 minutes at 4 ° C. To extract the cytoplasmic membrane proteins from the crude fraction of the outer membranes, the whole fraction was resuspended in 2% sarcosyl (Sigma) and incubated at room temperature for 20 minutes. The suspension was centrifuged at 10,000 g for 10 minutes to remove aggregates, and the supernatant was further ultracentrifuged at 50,000 g for 75 minutes to obtain a clot of external membranes. External membranes were resuspended in 10 mM Tris-Hcl (pH 8) and protein concentration was determined in the Bio-Rad Protein assay using bovine serum albumin as standard.

Q) Preparation of complete extracts

Bacteria were cultured overnight on a GC plate, harvested by loop, and resuspended in 1 ml of 20 mM Tris-Hcl. Inactivation by heating was carried out for 30 minutes at 56 ° C.

R) Western blotting

Purified proteins (500 ng per lane), external membrane vesicles (5 μg) and total cell extracts (25 μg) derived from cells of strain MenB-2996 were loaded into a 15% polyacrylamide gel for SDS electrophoresis and transferred onto a nitrocellulose membrane . The transfer was carried out for 2 hours at 150 mA at 4 ° C in a special buffer (0.3% basic Tris, 1.44% glycine, 20% methanol). The membranes were saturated by incubation overnight at 4 ° C in saturating buffer (10% skim milk, 0.1% Triton X-100 in PBS). The membranes were washed twice in wash buffer (3% skim milk, 0.1% Triton X-100 in PBS) and incubated for 2 hours at 37 ° C with mouse serum diluted to 1: 200 with wash buffer. Membranes were washed twice and incubated for 90 minutes with horseradish peroxidase labeled anti-mouse immunoglobulin diluted to 1: 2000. Membranes were washed twice with 0.1% Triton X-100 in PBS and processed using a substrate from the Opti-4CN Substrate Kit (Bio-Rad). The reaction was stopped by adding water.

S) bactericidal test

Cells of strain MC58 were cultured overnight at 37 ° C on chocolate agar plates. 5-7 colonies were selected and used for plating in Mueller-Hinton culture broth (7 ml). Suspensions were incubated at 37 ° C in a nutrient and allowed to cultivate until reaching OD ₆₂₀ of 0.5-0.8. Each culture was aliquoted in sterile 1.5 ml Eppendorf tubes and centrifuged for 20 minutes at maximum microcentrifuge speed. The resulting clot was washed once in a Gaia buffer (Gibco) and resuspended in the same buffer to an OD ₆₂₀ value of 0.5, diluted in Gaia buffer to 1: 20,000 and stored at 25 ° C.

To each well of a 96-well tissue culture plate, 50 μl of Gaia buffer in + 1% bovine serum albumin was added. 25 μl of diluted mouse serum (1: 100 Gaia buffer + 0.2% bovine serum albumin) was added to each well and the plate was incubated at 4 ° C. 25 μl of the bacterial cell suspension described above was added to each well. 25 μl of either heat inactivated (up to 56 ° C in a water bath for 30 minutes) or normal rabbit complement was added to each well. Immediately following the addition of complement, rabbits were 22 μl of each sample (wells) plated on Meuller-Hinton agar plates (time 0). 96-well plates were incubated for 1 hour at 37 ° C. under rotation and then 22 μl of each sample (wells) were plated on Mueller-Hinton agar plates (time 1). After incubation overnight, colonies corresponding to time spans 0 and 1 (hours) were counted.

Table 2 summarizes the data on cloning, expression and purification.

Examples 1-104 are given at the end of the text.

Claims (12)

1. A protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 458, 460, 462, 464 and 466, corresponding to the antigenic protein Neisseria meningitidis and having immunogenic properties. 2. A protein characterized by a 50% or higher level of sequence identity to the protein described in claim 1, having its antigenic properties. 3. A protein having antigenic properties, including a fragment of the protein described in claim 1. 4. The antibody obtained using the protein according to any one of claims 1 to 3, which binds to a protein according to any one of claims 1 to 3. 5. The nucleotide sequence that encodes a protein according to any one of claims 1 to 3. 6. The nucleotide sequence according to claim 5, selected from the group consisting of SEQ ID NO: 457, 459, 461, 463 and 465. 7. The nucleotide sequence comprising a fragment of a nucleotide sequence selected from the group of SEQ ID NO: 457, 459, 461, 463 or 465, encoding a fragment of a Neisseria meningitidis protein having antigenic properties. 8. The nucleotide sequence complementary to the nucleotide sequence according to any one of claims 5 to 7. 9. The nucleotide sequence used as a probe, capable of hybridizing with the nucleotide sequence according to any one of claims 5 to 8 under stringent hybridization conditions, such as 65 ° C, in a solution of 0.1 × SSC, 0.5% SDS. 10. A pharmaceutical composition for treating or preventing an infection caused by Neisseria meningitidis, comprising as an active ingredient an effective amount of a substance selected from the group consisting of (a) a protein according to any one of claims 1 to 3; (b) an antibody according to claim 4 and (c) a nucleotide sequence according to any one of claims 5 to 9, and a pharmaceutically acceptable carrier. 11. The pharmaceutical composition according to claim 10, characterized in that it is a vaccine or diagnostic. 12. The pharmaceutical composition of claim 10, characterized in that it is used for the manufacture of a medicament for the treatment or prophylaxis of an infection caused by Neisseria meningitidis.

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