RU2359696C2 - Vaccine composition containing transferrin-binding protein and hsf from gram-negative bacteria - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention concerns medicine, namely to creation of immunogene compositions and vaccines for prevention or treatment of the infections caused by Gram-negative bacteria. The immunogene compositions containing a transferrin-binding fiber and Hsf, and a way of their reception are offered. It is shown, that the combination of these two antigens synergically influences on production of antibodies with high activity in the analysis of bactericidal Serum. The composition can be used in vaccines against Gram-negative bacteria, including Neisseria meningitides, Neisseria gonorrhoeae.

EFFECT: creation of immunogene compositions and vaccines for prevention or treatment of the infections caused by Gram-negative bacteria.

56 cl, 10 ex, 1 dwg

Description

Field of Invention

The present invention relates to immunogenic compositions and vaccines based on gram-negative bacteria, their manufacture and use of such compositions in medicine. More specifically, the invention relates to vaccine compositions containing both transferrin-binding protein and Hsf. The presence of both of these antigens leads to higher levels of bacterial antibody production.

State of the art

Gram-negative bacteria are pathogens that cause a number of human pathologies, and there is a need to develop effective vaccines against many of these bacteria. In particular, Bordetella pertussis, Borrelia burgdorferi, Brucella melitensis, Brucella ovis, Chlamydia psittaci, Chlamydia trachomatis, Esherichia coli, Haemophilus influenzae, Legionella pneumophila, Neisseria geroritomaidis, and Yisseria aerinitium bacteria which can be treated with vaccination.

Neisseria gonorrhoeae is the etiological agent of gonorrhea, one of the most commonly reported sexually transmitted diseases in the world with an estimated incidence of 62 million cases per year (Gerbase et al., 1998, Lancet 351; (Suppl.3) 2-4). Clinical manifestations of gonorrhea include inflammation of the mucous membranes of the urogenital tract, rectal lumen, and neonatal eye infections. Ascending gonococcal infections in women can lead to infertility, ectopic pregnancy, chronic pelvic inflammatory disease, and the formation of a tubo-ovarian abscess. Sepsis, arthritis, endocarditis and meningitis are associated with complicated gonorrhea.

A large number of strains of gonococci with antibiotic resistance contribute to increased morbidity and complications associated with gonorrhea. A tempting alternative to antibiotic treatment for gonorrhea would be to prevent it by vaccination. There is currently no vaccine for infections caused by N. gonorrhoeae.

Neisseria meningitidis is an important pathogen, especially in children and young adults. Sepsis and meningitis are the most life-threatening forms of invasive meningococcal disease (IIM). Due to its high prevalence and mortality, this disease has become a global health problem.

Based on antigenic differences in capsular polysaccharides, thirteen N. meningitidis serogroups were identified, the most common of which are A, B, and C, responsible for 90% of cases worldwide. Serogroup B is the most common cause of meningococcal disease in Europe, the United States and some Latin American countries.

Vaccines based on capsular polysaccharides of serogroups A, C, W, and Y have been developed and have been shown to inhibit outbreaks of meningococcal disease (Peltola et al., 1985, Pediatrics 76; 91-96). However, serogroup B is weakly immunogenic and induces only transient antibody production mainly of IgM (immunoglobulin M) isotype (Ala'Aldeen D and Cartwright K 1996, J.Infect. 33; 153-157). Therefore, currently there is no sufficiently effective vaccine against serogroup B meningococci, which are responsible for most diseases in temperate countries. This presents a particular problem due to the fact that the prevalence of serotype B disease is increasing in Europe, Australia and America, mainly in children under 5 years of age. The development of a vaccine against serogroup B meningococci causes particular difficulties, since the polysaccharide capsule is weakly immunogenic due to its immunological proximity to the adhesion molecule of human nerve cells. Accordingly, vaccine production strategies were focused on the surface structures of the outer membrane of meningococci, but the difficulty was a marked variation in these antigens among the strains.

Further developments led to the introduction of vaccines made from vesicles of the outer membranes, which contain a number of proteins that make up the normal contents of the bacterial membrane. One of them is VA-MENGOC-BC®, a Cuban vaccine against N.meningitidis serogroups B and C (Rodriguez et al 1999 Mem Inst. Oswaldo Cruz, Rio de Janeiro 94; 433-440). This vaccine was developed to combat the epidemic of invasive meningococcal disease in Cuba, which was not eliminated by a vaccination program using the capsule-polysaccharide AC vaccine. The predominant serogroups were B and C, and using the VA-MENGOC-BC® vaccine, the outbreak was successfully controlled with an estimated vaccine efficiency of 83% for serogroup B N.meningitidis strains (Sierra et al., 1990 In Neiserria, Walter Gruyter, Berlin, m Atchman et al (eds) p. 129-134, Sierra et al. 1991, NIPH Ann. 14; 195-210). This vaccine was effective against a specific epidemic, but an elicited immune response would not protect against other strains of N.meningitidis.

The following efficacy studies conducted in Latin America during epidemics caused by homologous and heterologous serogroup B meningococcal strains showed some efficacy in older children and adults, but its effectiveness was significantly lower in younger children who are at greatest risk of infection (Milagres et al., 1994, Infect. Immun. 62; 4419-4424). It is unclear how effective the vaccine would be in countries with multiple strains of endemic disease such as the UK. Studies of the immunogenicity of heterologous strains have shown only limited cross-reactive serum bactericidal activity, especially in children (Tappero et al. 1999, JAMA 281; 1520-1527).

A second vaccine based on outer membrane vesicles was developed in Norway using the serotype B isolate, a typical serotype prevailing in Scandinavia (Fredriksen et al., 1991, NIPH Ann, 14; 67-80). This vaccine was tested in clinical trials and was found to have a protective efficacy of 57% after 29 months (Bjune et al., 1991, Lancet, 338; 1093-1096).

However, the use of external membrane vesicles (VNM) in vaccines is associated with some problems. For example, BHMs contain toxic lipopolysaccharides, and they may contain immunodominant antigens that are either strain specific or are variant expressed. A number of methods are described that can be used to overcome some of the problems of vaccines based on external membrane vesicle preparations. WO 01/09350 describes methods that address some of these problems, for example, by reducing the toxicity and modifying the antigens present on the vesicles of the outer membranes.

There are various problems associated with the currently available anti-meningococcal vaccines. Protein-based outer membrane vaccines are usually specific and effective for only a few strains. Polysaccharide vaccines are also partially optimal, as they usually elicit weak and short immune responses, in particular with respect to serogroup B (Lepow et al., 1986; Peltola 1998, Pediatrics 76; 91-96).

Infections caused by Neisseria pose a significant public health problem for which the vaccine can be solved or not, as in the case of N..gonorrhoeae, or, as in the case of N. meningitides, there are vaccines with restrictions on the effectiveness and protective ability for heterologous strains. Obviously, there is a need to develop better quality vaccines against Neisseria infections that will improve the effectiveness of currently available vaccines and make it possible to protect against a wider range of strains.

Description of graphic materials

Drawing - Coomassie-stained gel, showing the levels of expression of Hsf, TbpA and NspA in preparations of the outer membrane vesicles derived from various strains of N.meningitidis. Lane 1 - molecular weight markers; lane 2 - vesicles of the outer membranes obtained from strain H44 / 76, in which the capsular polysaccharides were negatively regulated; lane 3 - vesicles of the outer membranes obtained from strain H44 / 76, in which the capsular polysaccharides and PorA were negatively regulated; lane 4 - vesicles of the outer membranes obtained from strain H44 / 76, in which the capsular polysaccharides and PorA were regulated negatively, and NspA was regulated positively; lane 5 — vesicles of the outer membranes obtained from strain H44 / 76, in which the capsular polysaccharides and PorA were negatively regulated, and Hsf was regulated positively; lane 6 - outer membrane vesicles obtained from strain H44 / 76, in which capsular polysaccharides and PorA were negatively regulated, and TbpA was regulated positively; lane 7 - vesicles of the outer membranes obtained from strain H44 / 76, in which the capsular polysaccharides and PorA were negatively regulated, and TbpA and Hsf were regulated positively; lane 8 — vesicles of the outer membranes obtained from strain H44 / 76, in which the capsular polysaccharides and PorA were negatively regulated, and TbpA and NspA were regulated positively.

DETAILED DESCRIPTION OF THE INVENTION

The present invention discloses a combination of antigens which, when combined in an immunogenic composition or vaccine, can induce bactericidal antibody titers higher than the titers induced by these antigens when administered individually. Preferably, the combination of antigens causes synergistically higher bactericidal antibody titers. Since bactericidal antibodies directly reflect the effectiveness of candidate vaccines, the combination of Tbp and Hsf in vaccines will result in highly effective vaccines. An additional advantage of the invention is that the combination of two antigens, Tbp and Hsf, will also make possible protection against a wide range of strains.

This invention relates to an immunogenic composition comprising a transferrin-binding protein and an Hsf-like protein or antigenic fragments thereof. These proteins are either isolated or preferably purified to at least 30%, 40%, more preferably 50%, 60%, 70%, 80%, 90%, 95% or 99% purity, or enriched in a mixture with other antigens. The transferrin-binding protein and the Hsf-like protein can be isolated or can be derived from the same or from different strains of gram-negative bacteria.

“Isolated” means isolated from the normal environment of a protein by human hands. “Purified” means purified to at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% purity before this antigen is combined with other components of the immunogenic composition according to the invention.

“Derived from” means that the gene encoding the protein is derived from a particular bacterial strain, or that the protein is purified from a particular bacterial strain. Therefore, “derived from” includes recombinant proteins produced in a separate expression system if the gene encoding this protein is derived from the named bacterium.

In a combination, Tbp and Hsf have been shown to interact successfully and preferably synergistically, causing an immune response that is higher in units of bactericidal activity (for example, when measured by analysis of serum bactericidal activity, or ABS) and preferably higher than the additive response caused by these antigens individually more preferably at least 1.1, 1.2, 1.5, two, three, four, five, six, seven, eight, nine times, most preferably at least ten times. A significant advantage of adding both Tbp and Hsf to the vaccine over existing vaccines will be a strong bactericidal immune response and the ability to protect against many strains.

One embodiment of the invention is an immunogenic composition comprising both a transferrin-binding protein and an Hsf-like protein. An immunogenic composition is a composition containing at least one antigen that can elicit an immune response when administered to a host. Tbp and Hsf-like protein can come from any strain of gram-negative bacteria, including Moraxella catarrhalis, Haemophilus influenzae, Bordetella, Neisseria (including Neiserria meningitidis, which can be serogroup A, B, C, W135 or Y, and Neisseria gonorrrhoeae) from gram-negative bacteria described above. This invention encompasses immunogenic compositions in which Tbp and Hsf-like protein are derived from either one or different strains of gram-negative bacteria.

Transferrin binding proteins

Transferrin binding protein (TbpP) is a protein or protein complex on the outer membrane of gram-negative bacteria that binds transferrin. Some proteins in this family form a beta cylinder anchored in the outer membrane. The structure of the transferrin binding protein may contain an intracellular N-terminal domain with a TonB box and a “plug” domain, multiple transmembrane beta chains linked by short intracellular and longer extracellular loops. Other examples are lipoproteins, which interact with the formation of a complex with an integral membrane protein. Examples of this protein family are TbpA and TbpB. The Tbp designation encompasses any of these proteins, either singly or in combination, as well as a complex formed from TbpA and TbpB. Preferably, at least TbpA is present in the immunogenic composition of the invention.

There are two families of TbpB having a high molecular weight and a low molecular weight, respectively. High and low molecular weight forms of TbpB (WO 93/06861; EP 586266) combine with different families of TbpA (WO 93/06861; EP 586266; WO 92/03467; US 5912336), which differ on the basis of homology. Despite the same molecular weight, TbpA are known as high molecular weight and low molecular weight families due to their combination with high or low molecular weight TbpB (Rokbi et al., FEMS Microbiol. Lett. 100; 51, 1993). It is known that TbpA and TbpB are expressed in a number of bacteria, including N.meningitidis (WO 93/06861; EP 586266; WO 92/03467; US 5912336), N.gonorrhoeae (WO 92/03467; US 5912336), H.influenzae ( Gray-Owen et al., Infect. Immun. 1995; 63: 1201-1210, Schryvers J. Med. Microbiol. 1989; 29: 121-130; WO 95/13370; WO 96/40929), A.pleuropneumoniae, M .cararrhalis (Mathers et al., FEMS Immunol. Med. Microbiol. 1997; 19: 231; Chen et al., Vaccine 1999; 18: 109; WO 97/13785; WO 99/52947) and P. haemolytica (Cornelissen et al., Infection and Immunity 68; 4725, 2000). TbpA and TbpB are also referred to as Tbp1 (NMB 0461) and Tbp2 (NMB 0460), respectively (Cornelissen et al., Infection and Immunity 65; 822, 1997).

When used here, Tbp means a transferrin binding protein from gram-negative bacteria, including Moraxella catarrhalis and Haemophilus influenzae, preferably Neisseria, more preferably N.meningitidis or N..gonorrheoea and most preferably N.meningitidis serotype B. Tbp encompasses both TbpA and TbpB as well as high and low molecular forms of TbpA and TbpB. Tbp encompasses the individual proteins described above and the complexes of these proteins and any other proteins or their complexes capable of binding transferrin.

Although Tbp can refer to both high and low molecular weight forms of TbpA or TbpB, it is preferred that both the high molecular weight and low molecular weight forms of TbpA and / or TbpB are present in the immunogenic composition of the invention. Most preferably, high molecular weight and low molecular weight TbpA are present.

It is contemplated that instead of Tbp proteins or in addition to them, other proteins involved in iron uptake may be included in the immunogenic compositions of the invention. Proteins involved in iron uptake from Moraxella catarrhalis include TbpA, TbpB, Ton-dependent receptor, CpB (Sethi et al., Infect. Immun. 1997; 67: 3666-3671), HasR, OmpB1 and LbpB (Du et al., Infect. Immun. 1998; 66: 3656-3665; Mathers et al., FEMS Immunol. Med. Microbiol. 1997; 19: 231-236; Chen et al., Vaccine 1999; 18: 109-118). Proteins involved in iron absorption from Haemophilus influenzae include TbpB, HasR, TonB-dependent receptor, hemoglobin binding protein, HhuA, HgpA, HgbA, HgbB and HgbC (Cora et al. Infect. Immun. 2000; 68: 4092-4101 ; Maciver et al., Infect Immun. 1996; 64: 3703-3712; Jin et al., Infect. Immun. 1996; 64: 3134-3141; Morton et al., J. Gen. Microbiol. 1990; 136: 927 -933; Schryvers J. Med. Microbiol. 1989; 29: 121-130). Iron-capturing proteins from Neisseria meningitidis include Tbp1 (NMB 0461), Tbp2 (NMB 0460), FbpA (NMB 0634), FbpB, BfrA (NMB 1207), BfrB (NMB 1206), LbpA (NMB 1540), LbpB (NMB 1541) Lipo 28, also known as GNA2132 (NMB 2132), Sibp (NMB 1882), Ton-dependent receptors (NMB 0964 and NMB 0293) and HmbR (Tettelin et al., Science 287; 1809-1815, 2000).

Tbp proteins included in the immunogenic compositions of the invention are proteins having homology with TbpA and TbpB from N. meningitidis as described in WO 93/06861 and EP 586266; preferably having more than 40%, 45%, 50%, 60%, 70%, more preferably more than 80 or 90%, most preferably more than 95%, 96%, 97%, 98%, 99% identity with the amino acid sequence of TbpA and TbpB as described in WO 93/06861 and EP 586266.

Tbp contains several distinct areas. For example, in the case of TbpA from N. meningitidis strain H44 / 76, 186 amino-terminal amino acids form an internal globular domain, 22 beta chains span the membrane, forming a beta-cylindrical structure. They are connected by short intracellular loops and larger extracellular loops. Extracellular loops 2, 3 and 5 have the highest degree of sequence variation, and loop 5 is surface exposed. Loops 5 and 4 are involved in ligand binding and are preferably TbpA fragments for inclusion in the immunogenic compositions of the present invention.

In addition to the genetic upregulation techniques described in the application, transferrin-binding proteins can also be upregulated in gram-negative bacteria when they grow under conditions of iron restriction, as described below. In the immunogenic compositions of the invention in which the transferrin-binding protein is upregulated in the vesicle of the outer membranes, upregulation is preferably achieved by culturing the host strain under conditions of iron restriction. This method also leads to upregulation of variable iron-regulated proteins, in particular FrpB in Neisseria strains, and heme / hemopexin utilizing protein C, HgpA and HgpB in Haemophilus influenzae, which may become immunodominant. Therefore, it is useful to upregulate the expression (and preferably deletion of coding genes) of such proteins as described below to ensure that the immunogenic composition of the invention will elicit an immune response against antigens present in a wide variety of strains.

Hsf-like proteins

Hsf-like proteins are motor transporter proteins having homology with Hsf N.meningitidis with the sequences described in WO 99/31132; preferably they have more than 40%, 50%, 60%, 70%, more preferably more than 80%, most preferably more than 90%, most preferably more than 95%, 96%, 97%, 98%, 99% identity with the Hsf amino acid sequence disclosed in WO 99/31132 (preferably SEQ ID NO 2, 4, 6 or 8). Hsf-like proteins are surface-exposed proteins and are believed to function as adhesins. These proteins form a multidimensional complex and are expressed during infection and colonization.

Hsf-like proteins are found in many gram-negative bacteria, including Neisseria meningitidis, Neisseria gonorrheoea, Haemophilus influenzae, Moraxella catarrhalis and Escherichia coli. Examples of Hsf-like proteins found in Neisseria meningitidis include Hsf (also known as NhhA-NMB 0992) (WO 99/31132), Aida-1-like protein (Peak et al., 2000, FEMS Imm. Med. Vicrobiol. 28; 329), IgA protease, Ssh-2, Nar (WO 99/55873), NadA (J. Exp. Med. 2002 195; 1445), UspA2 and Tsh. Examples of Hsf-like proteins in Moraxella catarrhalis include Hsf, UspA1 (WO 93/03761), UspA2 (WO 93/03761), external membrane esterase and YtfN. Examples of Hsf-like proteins in Haemophilus influenzae include Hia / Hsf (St Geme et al., J. Bacteriol. 2000 182: 6005-6013), Nar, IgA1 protease, HMW1, HMW2 (Barenkamp et al., Infect. Immun. 1992. 60; 1302-1313), YadA, YadAc, and YtfN (Hendrixson et al., Mol. Cell 1998; 2: 941-850; St Geme et al., Mol. Micribiol. 1994; 14: 217-233; Grass and St Geme Infect. Immunol. 201; 69; 307-314; St. Geme and Gutter J. Bacteriology 2000; 182; 6005-6013). Examples of Hsf-like proteins in Escherichia coli include Hsf, Hia and Nar.

Hsf has a structure common to transporter proteins. For example, the Hsf from strain N44 / 76 N.meningitidis consists of a head region at the amine end of the protein (amino acids 52-479), which is exposed on the surface and contains variable regions (amino acids 52-106, 121-124, 191-210 and 230- 234), the cervical region (amino acids 480-509), the hydrophobic alpha-helix region (amino acids 518-529), and the anchor domain in which four transmembrane chains span the outer membrane (amino acids 539-591).

Hsf may refer to full-sized polypeptides, including a signal sequence that consists of amino acids 1-51. The present invention also includes Hsf with a deleted signal sequence, so that the polypeptide consists of a mature form of Hsf. Other preferred forms of Hsf may be truncated to exclude the variable regions of the protein disclosed in WO 01/55182.

Preferred options include the removal of one, two, three, four or five variable regions, as defined in WO 01/55182. In preferred embodiments, residues are removed from the region of the amino acid sequence from 52 to 237, or amino acids from 54 to 237 are removed, more preferably, residues between amino acids from 52 to 133 or amino acids from 55 to 133 are removed. Obviously, truncated variants can be included either from they excluded the signal sequence from Hsf from amino acids 1 to 51. The above sequence and those described below can be truncated or extended by 1, 2, 3, 4, 5, 7, 10, or 15 amino acids on any of N- and In the end or on both of them.

When Hsf is used as a subunit vaccine, it is preferable to use a portion of the soluble "passenger" domain; for example, a complete domain of amino acids 52-479, most preferably a conservative part thereof, for example amino acids 134-479.

Although it is preferable to use full-sized Tbp and / or Hsf-like proteins (in particular TbpA and Hsf) or their naturally occurring variants, or such full-sized sequences in which no more than 60 amino acids from the N- and / or C-terminus are absent, antigenic fragments of Tbp and / or Hsf-like proteins are also included in the immunogenic composition of the invention. These are fragments containing at least 10 amino acids, preferably 20 amino acids, more preferably 30 amino acids, more preferably 40 amino acids, or most preferably 50 amino acids taken directly one after the other from the amino acid sequence of Tbp and Hsf-like protein, preferably TbpA and Hsf. In addition, antigenic fragments mean fragments that are immunologically reactive with antibodies generated against N. meningitidis Tbp or Hsf-like protein, preferably TbpA or Hsf, or with antibodies generated by infection of a mammalian host N.meningitidis. Antigenic fragments also include fragments that elicit an immune response specific against a Tbp or Hsf-like protein, preferably TbpA or Hsf from the gram-negative bacteria from which they originate. Preferably, it protects against infection by the bacterium from which it occurs, preferably from Neisseria infection, more preferably it protects against infection of N. meningitidis, most preferably it protects against infection of N. meningitidis serogroup B.

Preferred TbpA fragments include extracellular TbpA loops. When using the TbpA sequence from N. meningitidis strain H44 / 76, these loops correspond to amino acids 200-202 for loop 1, amino acids 226-303 for loop 2, amino acids 348-395 for loop 3, amino acids 438-471 for loop 4, amino acids 512- 576 for loop 5, amino acids 609-625 for loop 6, amino acids 661-671 for loop 7, amino acids 707-723 for loop 8, amino acids 769-790 for loop 9, amino acids 814-844 for loop 10 and amino acids 872-903 for loops 11. The corresponding sequences, after sequence alignment, in other Tbp proteins also amounted to respectful fragments. Most preferred fragments include amino acid sequences comprising loop 2, loop 3, loop 4 or Tbp loop 5.

Although the preferred fragments of Tbp or TbpA proteins described above relate to N. meningitidis, one skilled in the art can easily find equivalent peptides in Tbp or TbpA proteins from all of the above gram-negative strains that are also fragments of the invention.

Preferred Hsf fragments include the whole Hsf head region, preferably containing amino acids 52-473 of Hsf. Additional preferred Hsf fragments include surface exposed head regions, including amino acids 52-62, 76-93, 116-134, 147-157, 157-175, 199-211, 230-252, 252-270, 284-306, 328 -338, 362-391, 408-418, 430-440 and 469-479. The most preferred fragments are 134-591 for use in the HMF preparation of the invention, and 134-479 for use in the subunit composition of the invention.

Although the preferred fragments of the Hsf-like or Hsf proteins described above relate to N. meningitidis, one skilled in the art can easily find equivalent peptides in the Hsf-like or Hsf proteins from all the above gram-negative strains, which are also fragments according to the invention.

Also included in the invention are fusion proteins from Tbp and Hsf-like protein, preferably TbpA and Hsf. They can combine Tbp and Hsf-like protein, preferably TbpA and Hsf, or fragments thereof, combined in the same polypeptide. Alternatively, the invention also includes individual fusion proteins from Tbp and Hsf-like protein, preferably TbpA and / or Hsf, or fragments thereof, provided that both Tbp and Hsf-like protein, preferably TbpA and Hsf, or fragments thereof are present in compositions of the invention. TbpA or Hsf can, for example, form a fusion protein with β-galactosidase, glutathione-S-transferase, green fluorescent proteins (GFP), tags of epitopes such as FLAG, myc tag, polyhistidine, or surface viruses / bacteria proteins such as influenza virus hemagglutinin, tetanus toxoid, diphtheria toxoid or CRM197.

Isolated transferrin-binding proteins that can be incorporated into immunogenic compositions are well known in the art (WO 0025811). They can be expressed in a host bacterium, extracted using a detergent (e.g. 2% Elugent) and purified by affinity chromatography or using a standard column chromatography procedure well known in the art (Oakhill et al., Biochem J. 2002 364; 613-6 ) Similarly, Hsf can be isolated using techniques well known in the art. Recombinant Hsf can be expressed in E. coli or in other bacterial strains. Protein can be purified using affinity chromatography. This can be a standard procedure if tag is entered in an Hsf sequence.

The terms “comprising”, “comprise” and “comprises” in this description, as the authors of the invention mean, can be replaced by the terms “consisting of”, “consist of” and “consists of”, respectively, in each case.

Vaccine combinations

This invention relates to combinations of antigens, including Tbp and Hsf-like protein, which effectively provide high bactericidal activity against gram-negative bacteria. The antigenic compositions of the invention may contain antigens in addition to Tbp and Hsf. They may contain other protein antigens from gram-negative bacteria, preferably Neisseria, and more preferably from N.meningitidis.

N.meningitidis

For N. meningitidis, the immunogenic compositions of the invention preferably comprise Hsf and TbpA. Upon receipt of BHM, it is preferable that Hsf and TbpA are up-regulated in the N.meningitidis strain from which the BHM is derived. TbpA may be present in both high and low molecular weight form, and preferably both high molecular weight and low molecular weight forms are present. Hsf is preferably present in BHM in a truncated form integrated into the membrane, preferably from amino acids 134-591. Hsf may also be present as a subunit vaccine, preferably as a passenger domain (amino acids 52-479), most preferably as a truncated passenger domain of amino acids 134-479.

Additional antigens can be added to the above compositions (or positively regulated if present in the BHM), for example NspA (WO 96/29412), Nar (PCT / EP 99/02766), PorA, PorB, OMP85 (also known as D15) (WO 00/23595), PilQ (PCT / EP 99/03603), PldA (PCT / EP 99/06718), FrpB (in WO 96/31618 see SEQ ID NO: 38), FrpA (NMB 0585) or FrpC or a conserved region common to both of at least 30, 50, 100, 150, 500, 750 amino acids (WO 92/01460), LbpA and / or LbpB (PCT / EP 98/05117; Schryvers et al., Med. Microbiol. 1999, 32: 1117), FhaB (WO 98/02547, SEQ ID NO: 38 [nucleotides 3083-9025]), HasR (PCT / EP 99/05989), lipo02 (PCT / EP 99/08315), MltA (WO 99/57280) (NMB0033) and ctrA (PCT / EP 00/00135).

Preferred combinations of antigens in the immunogenic composition of the invention include combinations comprising Tbp and Hsf-like protein and FhaB; Tbp and Hsf-like protein and PilQ; Tbp and Hsf-like protein and NspA; Tbp and Hsf-like protein and FrpC; more preferably containing Tbp and Hsf-like protein and Nar; Tbp and Hsf-like protein and FrpA / C; Tbp and Hsf-like protein and LbpB; Tbp and Hsf-like protein and D15. Most preferably, D15 will be included as part of the outer membrane vesicle preparation.

Antigens Moraxella catarrhalis

One or more of the following Moraxella catarrhalis proteins are preferred for inclusion in the immunogenic composition of the invention (preferably when TbpA and Hsf-like proteins are derived from Moraxella catarrhalis): OMP106 (WO 97/41731 and WO 96/34960), HasR (PCT / EP 99/03824), PilQ (PCT / EP 99/03823), OMP85 (PCT / EP 00/01468), lipo06 (GB 9917977.2), lipo10 (GB 9918208.1), lipo11 (GB 9918302.2), lipo18 (GB 9918038.2), P6 (PCT / EP 99/03038), ompCD, CopB (Helminen ME, et al. (1993) Infect. Immun. 61: 2003-2010), D15 (PCT / EP 99/03822), OmplA1 (PCT / EP 99 / 06781), Hly3 (PCT / EP 99/03257), LbpA and LbpB (WO 98/55606), TbpA and TbpB (WO 97/13785 and WO 97/32980), OmpE, UspA1 and UspA2 (WO 93/03761) and Omp21.

Haemophilus influenzae antigens

One or more of the following Haemophilus influenzae proteins is preferred for inclusion in the immunogenic composition of the invention (preferably when TbpA and Hsf-like proteins are derived from Haemophilus influenzae): D15 (WO 94/12641), P6 (EP 281673), TbpA, TbpB, P2, P5 (WO 94/26304), OMP26 (WO 97/01638), HMW1, HMW2, HMW3, HMW4, Hia, Hsf, Nar, Hin47 and Hif.

Another aspect of the invention are vaccine combinations containing the antigenic composition of the invention in conjunction with other antigens that are successfully used against certain disease states, including conditions associated with viruses or gram-positive bacteria.

In one preferred combination, the antigenic compositions of the invention containing Tbp and an Hsf-like protein are formulated with 1, 2, 3, or preferably all 4 of the following meningococcal capsular polysaccharides or oligosaccharides, which may be pure or protein conjugated : A, C, Y or W-135. Such a vaccine containing TbpA and Hsf from N. Meningitidis can be successfully used as a global meningococcal vaccine. Preferably, conjugated meningococcal capsular polysaccharide C, C and Y or A and C are included.

In yet another preferred embodiment, the antigenic compositions of the invention containing TbpA and Hsf, preferably formulated with 1, 2, 3 or all 4 pure or conjugated meningococcal capsular polysaccharides (or oligosaccharides) A, C, Y or W-135 as described above, is formulated with a conjugated H. influenzae b capsular polysaccharide or oligosaccharide and / or with one or more pure or conjugated pneumococcal capsular polysaccharide or oligosaccharide. Optionally, the vaccine may also contain one or more protein antigens that can protect the host from infection with Streptococcus pneumoniae. Such a vaccine can be successfully used as a vaccine against meningitis / streptococcal pneumonia.

In yet another preferred embodiment, an immunogenic composition of the invention comprising Tbp and an Hsf-like protein is formulated with capsular polysaccharides derived from one or more of the following microorganisms: Neisseria meningitidis, Haemophilus influezae b, Streptococcus pheumonia, group A streptococcus, group B streptococci, Staphylococcus aureus or Staphylococcus epidermidis. In a preferred embodiment, the immunogenic composition comprises capsular polysaccharides derived from one or more serogroups A, C, W-135 and Y of Neisseria meningitidis. Another preferred embodiment includes capsular polysaccharides derived from Streptococcus pneumoniae. Pneumococcal capsular polysaccharide antigens are preferably selected from serotypes 1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F (most preferably of serotypes 1, 3, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F). Another preferred embodiment includes PRF (polyribosyl ribitol phosphate) capsular polysaccharides of Haemophilus influenzae. Another preferred embodiment includes capsular polysaccharides of type 5, type 8 or 336 of Staphylococcus aureus. Another preferred embodiment includes capsular polysaccharides of type I, type II or type III from Staphylococcus epidermidis. Another preferred embodiment includes capsular polysaccharides of type Ia, type Ic, type II or type III of group B streptococci. Another preferred embodiment includes capsular polysaccharides of group A streptococci, preferably additionally containing at least one protein M and more preferably various types of protein M.

Preferred pneumococcal protein antigens are those pneumococcal proteins that are exposed on the outer surface of pneumococci (which can be recognized by the host immune system during at least part of the pneumococcal life cycle), or proteins that are secreted or released by pneumococcus. Most preferably, this protein is a toxin, adhesin, a 2-component signal transducer or lipoprotein from Streptococcus pneumoniae, or fragments thereof. Particularly preferred proteins include, but are not limited to: pneumolysin (preferably detoxified by chemical treatment or mutation) [Mitchell et al. Nucleic Acids Res. 1990 Jul 11; 18 (13): 4010 "Comparison of pheumolysin genes and proteins from Streptococcus pneumoniae types 1 and 2", Mitchell et al. Biochim. Biophys. Acta 1989, Jan 23; 1007 (1): 67-72 "Expression of the pneumolysin gene in Escherichia coli: rapid purification and biological properties", WO 96/05859 (A. Cyanamid), WO 90/06951 (Paton et al), WO 99/03884 ( NAVA)]; PspA and variants thereof with a transmembrane deletion (US 5804193 - Briles et al.); PspC and variants thereof with a transmembrane deletion (WO 97/09994 - Briles et al); PsaA and variants thereof with a transmembrane deletion (Berry & Paton, Infect. Immun. 1996 Dec; 64 (12): 5255-62 "Sequence heterogeneity of PsaA, a 37-kilodalton putative adhesion essential for virulence of Streptococcus pneumoniae"); pneumococcal choline-binding proteins and their variants with a transmembrane deletion; CbpA and variants thereof with a transmembrane deletion (WO 97/41151; WO 99/51266); glyceraldehyde-3-phosphate dehydrogenase (Infect. Immun. 1996, 64: 3544); HSP70 (WO 96/40928); PcpA (Sanchez-Beato et al. FEMS Microbiol. Lett. 1998, 164: 207-14); M-like protein (EP 0837130) and adhesin 18627 (EP 0834568). Further preferred pneumococcal protein antigens are those described in WO 98/18931, in particular those selected in WO 98/18930 and PCT / US 99/30390.

Also, the vaccine may possibly contain antigens that provide protection against one or more diphtheria, tetanus and Bordella pertussis infections. The pertussis component can be killed whole cell B.pertussis (Pw) or the cell-free pertussis component (Ra), which contains at least one antigen (and preferably all three) of the following: PT, FHA and 69 kDa pertactin. Typically, antigens that provide protection against diphtheria and tetanus are diphtheria toxoid and tetanus toxoid. Anatoxins can be chemically inactivated toxins or toxins inactivated by the introduction of point mutations.

The vaccine may also optionally contain one or more antigens that can protect the host against the untypable Haemophilus influenzae, RSV, and / or one or more antigens that can protect the host against the flu virus. Such a vaccine can be successfully used as a global vaccine against otitis media.

Preferred non-typeable H. influenzae protein antigens include Fimbrin protein (US 5,766,608) and fusion products containing its peptides (e.g., LB1 fusion product) (US 5,843,64 - Ohio State Research Foundation), OMP26, P6, Protein D, TbpA, TbpB, Hia , Hmw1, Hmw2, Nar and D15.

Preferred influenza antigens include a whole, live or inactivated virus, a cleaved influenza virus grown in eggs or MDCK cells, or Vero cells, or whole flu virosomes (as described by R. Gluck, Vaccine, 1992, 10, 915-920) or purified or recombinant proteins thereof, such as HA, NP, NA or M proteins, or combinations thereof.

Preferred RSV (respiratory syncytial virus) antigens include glycoprotein F, glycoprotein G, protein HN, protein M, or derivatives thereof.

Obviously, the antigenic compositions of the invention may contain one or more capsular polysaccharides from one species of bacteria.

Antigenic compositions may also contain capsular polysaccharides derived from one or more types of bacteria.

Such capsular polysaccharides may be unconjugated or may be conjugated to a carrier protein such as tetanus toxoid, tetanus toxoid fragment C, diphtheria toxoid, CRM197, pneumolysin, Protein D (US6342224), TbpA or Hsf. One embodiment of the invention includes single capsular polysaccharides conjugated to TbpA and Hsf.

A polysaccharide conjugate can be prepared using any known coupling technique. For example, a polysaccharide may be attached via a thioether bond. This conjugation method is based on the activation of 1-cyano-4-dimethylaminopyridinium polysaccharide with tetrafluoroborate (CDAP) to form a cyanate ester. The polysaccharide so activated can be attached to the amino group on the carrier protein directly or via a spacer group. Preferably, a combination of a cyanate ester with hexanediamine is carried out and the aminoderivatized polysaccharide is conjugated to a carrier protein using heteroligation chemistry, including the formation of a thioether bond. Such conjugates are described in PCT published application WO 93/15760 Uniformed Services University.

Conjugates can also be obtained by direct reductive amination methods, as described in US 4,365,170 (Jennings) and US 4,673,574 (Anderson). Other methods are described in EP-0-161-188, EP-208375 and EP-0-477508.

Another method involves combining a cyanogen bromide activated polysaccharide derivatized with adipic acid hydrazide (ACG) with a carrier protein via carbodiimide condensation (Chu C. et al., Infect. Immunity, 1983, 245-256).

Antigenic compositions containing outer membrane vesicles

A preferred aspect of the present invention is the upregulation, or overexpression, Tbp and Hsf in BHM. Gram-negative bacteria are separated from the environment by two successive layers of membrane structures, the cytoplasmic membrane and the outer membrane. The outer membrane of gram-negative bacteria is dynamic and, depending on environmental conditions, can undergo strong morphological transformations. Among these manifestations, the formation of vesicles of the outer membranes or “vesicles” has been studied and documented in many gram-negative bacteria (Zhou et al., 1998). Among others, a non-exhaustive list of bacterial pathogens that have been reported to form vesicles includes: Bordetella pertussis, Borrelia burgdorferi, Brucella melitensis, Brucella ovis, Chlamydia psittaci, Chlamydia trachomatis, Escherichia coli, HaemophilusheisserieferiaeferiaefheriefherieferiaeriaeriaeriefherieferiaeriaeriaeriefherieferiaeriaeriaeriefherieferiaeriiefherieferiaeriaeriefherieferiaeriiefherieferiaeriiefherieferiaeriiefherieferiaeriiefheriEFeerefefeferievefeferiEFerieferofeissefeyerofeissa , Pseudumonas aeroginosa and Yersinia enterocolitica. Although the biochemical mechanism responsible for the production of BHM / vesicles is not fully understood, such outer membrane vesicles have been extensively studied because they provide a powerful methodology for isolating the outer membrane protein preparations in their native conformation. In this context, the use of external membrane preparations is of particular interest for the development of vaccines against Neisseria, Moraxella catarrhalis, Haemophilus influenzae, Pseudomonas aeruginosa and Chlamydia. In addition, vesicles of the outer membranes combine various protein and non-protein antigens, which are supposed to provide broad protection against intraspecific variants.

The outer membrane vesicles of the invention contain Tbp and Hsf-like protein (preferably TbpA and Hsf) that are upregulated. This can be achieved by having positively regulated Hsf-like protein and Tbp in the vesicles of the outer membranes originating from a single strain of gram-negative bacteria, preferably a strain of Neisseria. The Hsf-like protein and Tbp can also be positively regulated separately in the vesicles of the outer membranes derived from various strains of gram-negative bacteria, preferably Neisseria strains. In a preferred embodiment, the different strains of Neisseria in which Tbp and Hsf-like protein, more preferably TbpA and Hsf, are positively regulated are L2 and L3 or L3 and L2, in accordance with the N. meningitidis immunotype.

You can make vesicle preparations from Neisseria strains by any means well known to those skilled in the art. Preferably, the methods described in EP 301992, US 5,597,572, EP 11243 or US 4,271,147, Frederikson et al. (NIPH Annals [1991], 14: 67-80), Zollinger et al., (J. Clin. Invest. [1979], 63: 836-848), Saunders et al., (Infect. Immun. [1999] 67: 113-119], Drabick et al. (Vaccine [2000], 18: 160-172) or WO 01/09350 (Example 8). In general, BHMs are extracted with a detergent, preferably deoxycholate, and nucleic acids are optionally removed enzymatically The purification is carried out by ultracentrifugation, possibly followed by size exclusion chromatography. If 2 or more different bubbles according to the invention are included, they can be combined in one container to form a polyvalent preparation according to the invention (although the preparation It is also considered to be multivalent if the different vesicles of the invention are separate compositions in separate containers that are administered to the host at the same time [for one visit to the doctor]). HMF preparations are usually sterilized by filtration through a 0.2 μm filter and preferably stored in sucrose solution (for example 3 %), which is known to stabilize vesicle preparations.

The positive regulation of Tbp and Hsf-like protein in the preparations of the outer membrane vesicles can be achieved by inserting an additional copy of the gene into the gram-negative bacterium from which the HMF preparation is derived. Alternatively, the promoter of the gene can be replaced by a stronger promoter in the bacterial strain from which the HMF drug is derived. Such techniques are described in WO 01/09350. Positive regulation of the protein will lead to a higher level of protein present in BHM compared to the level of protein present in BHM, derived from unmodified N.meningitidis (for example, strain H44 / 76). Preferably, the level will be at least 1.2, 1.5, 2, 3, 4, 5, 7, 10, or 20 times higher.

When it is assumed that LPS (lipopolysaccharide) will be an additional antigen in the VNM, a protocol using a low concentration of extracting detergent (for example, deoxycholate, or DOC) can preferably be used in the method for producing VNM so as to maintain high levels of bound LPS while removing particularly toxic, loosely coupled LPS. The concentration of DOC used is preferably 0-0.5% DOC, more preferably 0.02-0.4%, 0.03-0.3%, 0.04-0.2%, 0.05-0.15% , 0.05-0.2% DOC, most preferably approximately or exactly 0.1% DOC.

A “stronger promoter sequence" refers to a regulatory control element that enhances the transcription of a gene encoding an antigen of interest.

"Positive regulation of expression" refers to any means of enhancing the expression of an antigen of interest compared to an unmodified (i.e., naturally occurring) vesicle. Obviously, the magnitude of “positive regulation” will vary depending on the particular antigen of interest, but will not exceed the amount that will destroy the integrity of the bubble membrane. Positive antigen regulation refers to expression that is at least 10% higher than that of an unmodified vesicle. Preferably, it is at least 50% higher. More preferably, it is at least 100% (2 times) higher. Most preferably, it is at least 3, 4, 5, 7, 10, 20 times higher. Preferably, the expression level is evaluated when the vesicles are derived from bacteria grown under conditions of iron restriction (for example, in the presence of an iron chelator).

Alternatively or additionally, up-regulation of expression may relate to the conversion of expression to independent of metabolic or nutritional changes, in particular in the case of TbpA, TbpB, LbpA and LbpB.

For the purposes of clarity, the terms “constructing a bacterial strain to produce less of the indicated antigen” or “negative regulation” refer to any means of reducing the expression of the antigen of interest (or the expression of a functional gene product) relative to the expression of an unmodified (i.e., naturally occurring) vesicle, preferably by deletions, so that this expression is at least 10% lower than that of an unmodified vesicle. Preferably, it is at least 50% lower, and most preferably completely absent. If the negatively regulated protein is an enzyme or a functional protein, then negative regulation can be achieved by introducing one or more mutations leading to a 10%, 20%, 50%, 80%, or preferably 100% decrease in enzymatic or functional activity.

The design steps that are required to modulate the expression of Neisseria proteins can be carried out by various methods known to those skilled in the art. For example, sequences (e.g., promoters or open reading frames) can be inserted, and promoters / genes can be destroyed by transposon insertion. For example, to upregulate gene expression, a strong promoter can be inserted through a transposon up to 2 kb. in the opposite direction from the initiating codon of the gene (more preferably 200-600 bp in the opposite direction, most preferably about 400 bp in the opposite direction). You can also use a point mutation or deletion (in particular for negative regulation of gene expression).

Such methods, however, can be quite unstable or uncertain, and therefore it is preferable that the design step is carried out through an episode of homologous recombination. Preferably, an episode occurs between a sequence (recombinogenic region) of at least 30 nucleotides on a bacterial chromosome and a sequence (second recombinogenic region) of at least 30 nucleotides on a vector transformed in the strain. Preferably, these regions are 40-100 nucleotides, more preferably 100-800 nucleotides, most preferably 500 nucleotides. These recombinogenic regions should be similar enough so that they can hybridize with each other under sufficiently stringent conditions.

The methods used to carry out the genetic modifications described here (such as positive or negative regulation of genes through recombination and the introduction of additional gene sequences into the Neisseria genome) are described in WO 01/09350. Typical strong promoters that can be integrated into Neisseria are porA, porB, lgtF, Opa, p110, Ist, and hpuAB. PorA and PorB are preferred as constitutive strong promoters. It was established that PorB promoter activity is contained in the fragment corresponding to nucleotides from -1 to -250 in the opposite direction from the porB initiation codon.

Positive regulation of the expression of proteins involved in the absorption of iron through growth under conditions of iron restriction

The positive regulation of the transferrin-binding protein in the preparation of the outer membrane vesicles of the invention is preferably achieved by isolating the outer membrane vesicles from the parent strain of gram-negative bacteria grown under conditions of iron restriction. A low concentration of iron in the medium leads to an increase in the expression of proteins involved in the absorption of iron, including TbpA and TbpB. The expression of these proteins is therefore positively regulated without the need for recombinant modification of the involved gene, for example, by inserting a stronger promoter or by inserting an additional copy of the gene. The invention also encompasses the upregulation of transferrin-binding protein by growth on iron-restricted media, when the genes have also been recombinantly modified.

Limitations on iron are achieved by the addition of an iron chelator to the culture medium. Suitable iron chelators include 2,2-dipyridyl, EDDGUK (ethylenediamine di (ortho-hydroxyphenylacetic acid) and Desferal (deferoxamine mesylate, Sigma). Desferal is the preferred iron chelator and is added to the culture medium at a concentration between 10 and 100 μM, preferably 25-75 μM, more preferably 50-70 μM, most preferably 60 μM. The iron content in the medium is mainly due to the components of the yeast extract and soy peptone, and its amount in batches may vary. in fact, different Desferal concentrations may be optimal in order to achieve positive regulation of proteins involved in iron absorption in different batches of the medium. The specialist should be able to easily determine the optimal concentration. In general, an amount of iron chelator sufficient to to positively regulate the expression of the target iron-regulated protein, but not so large as to undesirably affect bacterial growth.

Preferably, the positive regulation of the transferrin binding protein by growth under iron restriction conditions is combined with the recombinant positive regulation of the Hsf-like protein so as to obtain outer membrane vesicles of the invention.

Negative regulation / removal of variable and non-protective immunodominant antigens

Many surface antigens are variable in bacterial strains and therefore protect only from a limited number of closely related strains. An aspect of the present invention encompasses outer membrane vesicles containing TbpA and Hsf-like protein, preferably TbpA and Hsf, in which the expression of other proteins is impaired, or preferably the gene (s) encoding the variable (s) surface (s) protein (s) is deleted (s). Such a deletion leads to the fact that the bacterial strain produces vesicles that, when introduced into the vaccine, have a high potential for cross-reactivity against different strains, due to the stronger influence exerted by conserved proteins (stored on the outer membranes) on the immune system of the vaccinee. Examples of such variable antigens include: for Neisseria, pilin (pilC), which is susceptible to antigenic changes, PorA, Ora, OpC, PilC, PorB, TbpB, FrpB; for H.influezae - P2, P5, pilin, IgA1 protease; and for Moraxella, OMP106.

Other types of genes that can be negatively regulated or “turned off” are genes that can be easily turned on (expressed) or turned off by bacteria in vivo. Since the outer membrane proteins encoded by such genes are not always present on bacteria, the presence of such proteins in vesicle preparations can also impair the effectiveness of vaccines for the reasons stated above. A preferred example of downregulation or deletion is the Ors protein from Neisseria. Anti-Ors immunity induced by the Ors-containing vesicle vaccine will have only limited protective ability, since an infectious organism can easily become Ors ^- . Other examples of such proteins are HgpA and HgpB from H. influenzae.

For example, such variable or non-protective genes can be negatively regulated during expression, or permanently turned off. An advantage of this is the concentration of the immune system on more suitable antigens that are present in small amounts on the outer surface of the vesicles.

Methods for downregulating expression are disclosed in WO 01/09350.

By negative regulation of the immunodominant protein of the outer membrane is meant that the expression levels are lowered and preferably turned off, or that mutations and / or deletions of the surface exposed immunodominant loops make the proteins of the outer membranes less immunodominant. By negative regulation of a protein with an enzymatic function, it is meant that the expression level of this protein is reduced or preferably turned off, or it can be understood that the expression of a functional enzyme is reduced or preferably eliminated.

Preferred meningococcal bacterial strains for use in the manufacture of the immunogenic compositions of the invention are negatively regulated, preferably a deletion of 1, 2 or 3 Poa, OpA and Opc. PorA and Opa are preferably negatively regulated; PorA and OpC; OpA and OpC; PorA and Opa and OpC.

Four different Ora genes are known to exist in the meningococcal genome (Aho et al. 1991 Mol. Microbiol. 5: 1429-37), therefore, when it is indicated that Ora expression is negatively regulated, this means that preferably 1, 2, 3 or (preferably) all 4 genes present in meningococcus are thus negatively regulated. Such negative regulation can be carried out genetically, as described in WO 01/09350, or by searching for easily detectable natural stable meningococcal strains that do not or do not express much at the Ora loci. Such strains can be detected using the technique described in Poolman et al. (1985. J. Med. Micro. 19: 203-209), where the cells that are Ora ^- have a phenotype different from the cells expressing Ora, which can be seen by looking at the appearance of the cells on plates or under a microscope. After detection, it can be shown that the strain is stably Ora ^- by performing Western blotting on the contents of the cells after fermentation to establish the absence of Ora.

When positive regulation of the transferrin-binding protein in the vesicles of the outer membranes is achieved through growth under conditions of iron restriction, variable iron-regulated proteins can also be positively regulated. These include FrpB in Neisseria meningitidis and Neisseria gonorrhoeae (Microbiology 142; 3269-3274, (1996); J.Bacteriol. 181; 2895-2901 (1999)), and heme / hemopexin utilizing protein C (J.Bacteriol. 177; 2644 -2653 (1995)), as well as HgpA, HgpB and HgpC (Infect. Immun. 66; 4733-4741 (1998), Infect. Immun. 67; 2729-2739 (1999), Microbiology 145; 905-914 (1999) ) in Haemophilus influenzae. The inventors have found that it is useful to negatively regulate the expression of at least the variable parts of such proteins when iron restriction is used to positively regulate the expression of transferrin-binding proteins. This is achieved both by using the methods described in WO 01/09350, and by deletion of the variable (s) part (s) of the protein. This ensures that the immune response elicited by the immunogenic composition will be directed against antigens that are present in a wide range of strains. Negative regulation of FrpB is preferably combined with negative regulation of PorA and OpaA; PorA and OpC; OpA and OpC; PorA and OpA and OpC in vesicle immunogenic compositions of the invention derived from gram-negative bacterial strains, preferably Moraxella catarrhalis, Haemophilus influenzae or Neisseria (more preferably N.meningitidis) strains.

In an alternative embodiment of the invention, FrpB is negatively regulated in the outer membrane vesicles that were obtained from gram-negative bacterial strains, preferably Moraxella catarrhalis, Haemophilus influenzae or Neisseria (more preferably N.meningitidis) strains, not necessarily grown under conditions of iron restriction.

LPS detoxification

The HMF in the immunogenic composition of the invention can be detoxified by the LPS detoxification methods described in WO 01/09350. In particular, methods for detoxifying LPS include down regulation, preferably a deletion of htrB and / or msbB enzymes, which are disclosed in WO 01/09350. The deletion mutants of these genes are phenotypically characterized by msbB mutant LPS that has lost one secondary acyl chain compared to the wild type, and htrB mutant LPS that has lost 2 (or both) secondary acyl chains. Such methods are preferably combined with VNM extraction methods using low DOC levels, preferably 0-0.3% DOC, more preferably 0.05-0.2% DOC, most preferably about 0.1% DOC.

Additional methods for detoxifying LPS include adding a non-toxic peptide functional equivalent to polymyxin B [a molecule with high affinity for lipid A] (preferably SAEP 2) to bubble preparations (see WO 93/14115, WO 93/03327, Velucchi et al. (1997), J. Endotoxin Res. 4: 1-12, and EP 976402 further details regarding the non-toxic polypeptide functional equivalents of polymyxin B - in particular the use of the SAEP 2 peptide (KTKCKFLKKC sequences where 2 cysteines form a disulfide bridge)).

Cross Reactive Polysaccharides

Isolation of bacterial vesicles of the outer membranes from encapsulated gram-negative bacteria often results in their isolation in conjunction with the capsular polysaccharide. In some cases, this “polluting” substance may be useful, since the polysaccharide can enhance the immune response elicited by other components of the vesicles. However, in other cases, the presence of a polysaccharide contaminant in bacterial vials may be detrimental to the use of vials in a vaccine. For example, at least in the case of N.meningitidis, it has been shown that the capsular polysaccharide of serogroup B does not induce protective immunity and allows the induction of an undesirable autoimmune response in humans. Therefore, the vesicles of the outer membranes of the invention can be isolated from the bacterial strain to produce vesicles that are altered by design in such a way that they do not contain capsular polysaccharides. Then these bubbles will be suitable for use in humans. A particularly preferred example of such a vesicle preparation is a preparation of N. meningitidis serogroup B lacking a capsular polysaccharide. In general, the isolation of outer membrane vesicles should occur from gram-negative bacterial strains that cannot synthesize capsular polysaccharides, in particular when such a strain is the msbB mutant described above.

This can be achieved through the use of modified vascular producing strains in which the genes necessary for capsular biosynthesis and / or export are damaged. Inactivation of the gene encoding the biosynthesis or export of capsular polysaccharides can be achieved by mutation (point mutation, deletion or insertion) or the control region, or the coding region, or both regions (preferably using the homologous recombination techniques described above), or any other way to reduce the enzymatic functions of such genes. In addition, inactivation of the capsule biosynthesis genes can also be achieved by anti-sense overexpression or transposon mutagenesis. A preferred method is the deletion of some or all of the Neiseria meningitidis capsular polysaccharide (cps) genes necessary for biosynthesis and export of polysaccharides. To this end, the substitution plasmid pMF121 (described by Frosh et al., 1990, Mol. Microbiol. 4: 1215-1218) can be used to carry out a mutation that deletes the cpsCAD (+ gaIE) gene cluster.

When the above immunogenic compositions of the invention are derived from a strain of meningococcus B, it is also preferred that the capsular polysaccharide (which also contains human-like saccharide structures) is also removed. Although many genes can be turned off to achieve this result, the inventors have successfully shown that it is preferable that the strain producing vesicles be modified by genetic engineering to constantly negatively regulate the expression of the functional gene product of the siaD gene (i.e., negative regulation of α-2- 8 polysialyl transferase activity), preferably by turning off the gene, most preferably by deleting the entire promoter and / or open reading frame of the gene or part thereof. Such inactivation is described in WO 01/09350. The siaD (also known as synD) mutation is the most preferred among many mutations that can lead to the removal of a human-like epitope from the capsular polysaccharide, as it is the only mutation that does not affect the biosynthesis of protective VOC epitopes (lipooligosaccharide), thus being useful in a method which ultimately aims to use VOCs as a protective antigen and has minimal effect on bacterial growth. A preferred aspect of the invention is therefore an immunogenic vesicle preparation, as described above, which is derived from lgtE ^- siaD ^- , LgtA ^- siaD ^- or preferably lgtB ^- siaD ^- meningococcal B mutant strain. This strain alone constitutes another aspect of the invention.

Although siaD ^- mutation is preferred for the above reasons, it is possible to use other mutations which switch off neysserialnyh synthesis of capsular polysaccharides (preferably meningococcal B). Thus, the vesicle-producing strain can be modified by genetic engineering to constantly negatively regulate the expression of a functional gene product of one or more of the following genes: ctrA, ctrB, ctrC, ctrD, synA (synX and siaA equivalent), synB (siaB equivalent) or synC (equivalent siaC), preferably by turning off the gene, most preferably by deleting the entire promoter and / or open reading frame of this gene or part thereof. LgtE ^- mutation may be combined with one or more of these mutations. Preferably the lgtB ^- mutation is combined with one or more such mutations. Therefore, an additional aspect of the present invention is an immunogenic vesicle preparation, as described above, which is derived from such a combined mutant strain of meningococcus B. The strain itself is another aspect of the present invention.

Heterogeneity within the oligosaccharide grouping of LPS determines the structural and antigenic diversity of different neisserial strains (Griffiss et al., Inf. Immun. 1987; 55: 1792-1800). This phenomenon was used to separate meningococcal strains into 12 immunotypes (Scholtan et al., J. Med. Microbiol. 1994, 41: 236-243). The immunotypes L3, L7 & L9 are immunologically identical and structurally similar (or even the same) and therefore were designated L3, 7, 9 (or generically as “L3” in this description). Meningococcal LPS L3, 7, 9 (L3), L2 and L5 can be modified by sialylation or by the addition of cytidine-5'-monophosphate-N-acetylneuraminic acid. Although L2, L4 and L6 LPSs are immunologically distinguishable, they are structurally similar, and when L2 is mentioned in the description, it can be replaced with either L4 or L6 within the scope of this invention. See M. P. Jennings et at., Microbiology 1999, 145, 3013-3021 and Mol. Microbiol. 2002, 43: 931-43, an additional illustration of the LPS structure and heterogeneity.

The safety of antibodies developed to L3 or L2 LPS was questioned due to the presence of a structure similar to the lacto-N-neotetraose oligosaccharide group (Galβ1-4GlcNAcβ1-3Galβ1-4Glcβ1-) present in human glycosphingolipids. Even if a large number of people were safely vaccinated with vesicular vaccines extracted with deoxycholate containing residual L3 LPS (G. Bjune et al., Lancet (1991), 338, 1093-1096; GVG. Sierra et al., NIPH ann. (1991), 14, 195-210), a deletion of the terminal portion of the saccharide VOC is useful in preventing any cross-interaction with structures present on the surface of human tissues. In a preferred embodiment, inactivation of the lgtB gene leads to an intermediate LPS structure in which there is no terminal galactose residue and sialic acid (after the mutation, the 4GlcNAcβ1-3Galβ1-4Glcβ1 structure remains in L2 and in L3 VOCs). Such intermediates can be obtained in the L3 and L2 LPS strain. An alternative and less preferred (short) variant of LPS can be obtained by turning off the lgtE gene. Another alternative and less preferred variant of LPS can be obtained by turning off the lgtA gene. If such an lgtA mutation ^is chosen, it ^is preferable to also disable lgtC expression to prevent the formation of a non-immunogenic L1 immunotype.

LgtB ^- mutants are most preferred as the inventors have found that this optimal truncation for resolving the safety problems while remaining LPS protective oligosaccharide epitope may still induce the production of bactericidal antibodies.

Therefore, the immunogenic compositions of the invention, additionally containing L2 or L3 preparations (either purified or in an isolated vesicle) or meningococcal vesicle preparations generally come predominantly from a Neisseria strain (preferably meningococcal), which has been modified by genetic engineering to constantly negatively regulate expression functional gene product lgtB, lgtA or lgtE gene, preferably by turning off the gene, most preferably by deletion of the whole romotora and / or open reading frame of this gene or parts thereof.

A Neisseria locus containing various lgt genes, including lgtB and lgtE, and its sequence are known in the art (see M.P. Jennings et al., Microbiology, 1999, 145, 3013-3021 and references cited therein, and J Exp. Med. 180: 2181-2190 [1994]).

In bubble preparations, in particular in preparations extracted with low concentrations of DOC, LPS can be used as an antigen in the immunogenic composition of the invention. However, it is preferable to negatively regulate / delete / inactivate the enzymatic function of either lgtE, lgtA (in particular in combination with lgtC), or preferably lgtB genes / gene to remove lacto-N-neotetraose structures like human ones. The Neisseria locus (and its sequence) containing lgt genes for LPS biosynthesis of oligosaccharide structures is known in the art (Jennings et al., Microbiology, 1999, 145; 3013-3021 and the references cited therein and J.Exp. Med 180: 2181-2190 [1994]). Negative regulation / deletion of lgtB (or functional gene product) is preferred since it leaves the LPS protective epitope intact.

In vesicular formulations N.meningitidis serogroup B of the invention is preferred negative regulation / deletion of both siaD, and lgtB (although a combination of lgtB ^- with any of ^{ctrA -, ctrB -, ctrC -} , ctrD -, synA - ( equivalent to synX ^- and siaA ^- ), synB ^- (equivalent to siaB ^- ) or synC ^- (equivalent to siaC ^- ) in the vesicle ^- producing strain of meningococcus B can also be used), resulting in a vesicle preparation with optimal safety and preservation of the LPS protective epitope.

An immunogenic composition according to the invention may contain at least one, two, three, four or five different preparations of vesicles of the outer membranes. When two or more BHM preparations are included, at least one antigen of the invention is upregulated in each BHM. Such VNM preparations can be derived from Neisseria strains of one species and serogroup, or preferably from Neisseria strains of another class, serogroup, serotype, subserotype or immunotype. For example, an immunogenic composition may comprise one or more outer membrane vesicle preparations that contain LPS of the L2 immunotype and one or more outer membrane vesicle preparations that contain LPS of the L3 immunotype. The L2 or L3 BHM preparations preferably originate from a stable strain that exhibits minimal phase variation at the locus of the LPS synthesis gene of oligosaccharides.

Preferred Neisseria Bubble Preparations

In addition to Hsf and Tbp, one or more of the following genes (encoding protective antigens) is preferred for positive regulation when it is carried by a Neisseria strain, including gonococcus and meningococcus (in particular N.meningitidis B): NspA (WO 96/29412), Nar (PCT / EP 99/02766), PorA, PorB (NMB 2039), OMP85 (WO 00/23595), PilQ (PCT / EP 99/03603), PldA (PCT / EP 99/06718), FrpB (WO 96 / 31618), FrpA / FrpC (WO 92/01460), LbpA / LbpB (PCT / EP 98/05117), FhaB (WO 98/02547), HasR (PCT / EP 99/05989), lipo02 (PCT / EP 99 / 08315), MltA (WO 99/57280), MafA (NMB 0652), MafB (NMB 0643), Omp26 (NMB 0181), adhesive NMB 0315, adhesive NMB 0995, adhesive NMB 1119, P2086 (NMB 0399), Lipo28 (NMB 2132), NM-ADPRT (NMB 1343), VapD (NMB 1753) and ctrA (PCT / EP 00/00135). They are also preferred as genes that can be heterologously introduced into other gram-negative bacteria.

One or more of the following genes are preferred for down regulation: PorA, PorB, PilC, LbpA, LbpB, Opa, Opc, htrB, msbB and lpxK.

One or more of the following genes are preferred for upstream regulation: pmrA, pmrB, pmrE, and pmrF.

Preferred repressive regulation sequences to be modified are: the region of the fur operator (in particular one of the TbpB or LbpB genes, or both); and the DtxR region of the operator.

One or more of the following genes are preferred for downregulation: galE, siaA, siaB, siaC, siaD, ctrA, ctrB, ctrC and ctrD.

The immunogenic compositions of the invention may also contain BHM / vesicular preparations derived from gram-negative bacteria, including Pseudomonas aeruginosa, Moraxella catarrhalis and Haemophilus influenzae b.

Preferred Pseudomonas aeruginosa Bubble Preparations

In addition to Hsf and Tbp, one or more of the following genes (encoding protective antigens) are preferred for positive regulation: PcrV, OprF, OprI. They are also preferred as genes that can be heterologically introduced into other gram-negative bacteria.

Preferred Bubble Preparations Moraxella catarrhalis

In addition to Hsf and Tbp, one or more of the following genes (encoding protective antigens) are preferred for positive regulation: OMP106 (WO 97/41731 & WO 96/34960), HasR (PCT / EP 99/03824), PilQ (PCT / EP 99/03823), OMP85 (PCT / EP 00/01468), lipo06 (GB 9917977.2), lipo10 (GB 9918208.1), lipo11 (GB 9918302.2), lipo18 (GB 9918038.2), P6 (PCT / EP 99/03038), ompCD CopB (Helminen ME, et al (1993) Infect. Immun. 61: 2003-2010), D15 (PCT / EP 99/03822), OmplA1 (PCT / EP 99/06781), Hly3 (PCT / EP 99/03257 ), LbpA and LbpB (WO 98/55606), TbpA and TbpB (WO 97/13785, WO 95/13370 & WO 97/32980), OmpE, UspA1 and UspA2 (WO 93/03761) and Omp21. They are also preferred as genes that can be heterologically introduced into other gram-negative bacteria.

One or more of the following genes are preferred for downregulation: CopB, OMP106, OmpB1, LbpA, and LbpB.

One or more of the following genes are preferred for downregulation: htrB, msbB, and lpxK.

One or more of the following genes are preferred for positive regulation: pmrA, pmrB, pmrE, pmrF.

Preferred Bubble Preparations Haemophilus influenzae

In addition to Hsf and Tbp, one or more of the following genes (encoding protective antigens) are preferred for positive regulation: D15 (WO 94/12641, WO 95/12641), P6 (EP 281673), P2, P5 (WO 94/26304) , OMR26 (WO 97/01638), HMW1, HMW2, HMW3, HMW4, Hia, Nar, Hin47 and Hif (all genes in this operon must be positively regulated for positive regulation of pilin). They are also preferred as genes that can be heterologously introduced into other gram-negative bacteria.

One or more of the following genes are preferred for downregulation: P2, P5, Hif, IgA1 protease, HgpA, HgpB, HMW1, HMW2, Hxu, htrB, msbB and lpxK.

One or more of the following genes is preferred for upstream regulation: pmrA, prmB, pmrE, and prmF.

Preferably, the immunogenic compositions or vaccines of the invention do not consist of and / or do not contain the specific combinations of SEQ IDs listed in the table from page 3, line 18, to page 52, line 2, in WO 00/71725, and / or any other particular combination described in examples 1-11 in WO 00/71725.

In addition, preferably or alternatively, any specific combinations disclosed in WO 01/52885 are not claimed in the present invention.

Vaccine preparations

A preferred embodiment of the invention is the manufacture of a preparation of the immunogenic composition of the invention in the form of a vaccine, which may also contain a pharmaceutically acceptable excipient or carrier.

The manufacture of outer membrane vesicle preparations from any of the above modified strains can be accomplished by any means well known to those skilled in the art. Preferably, the methods disclosed in EP 301992, US 5597572, EP11243 or US 4271147 are used. Most preferably, the method described in WO 01/09350 is used.

Vaccine preparation is generally described in Vaccine Design ("The subunit and adjuvant approach" (eds Powell M.F. & Newman M.J.) (1995) Plenum Press New York).

The antigenic compositions of the present invention in the vaccine preparation of the invention may be adjuvant. Suitable adjuvants include an aluminum salt, such as an aluminum hydroxide gel (alum) or aluminum phosphate, but may also be a calcium salt (in particular calcium carbonate), iron or zinc, or may be an insoluble suspension of acylated tyrosine or acylated sugars, cationic or anionic derivatives of polysaccharides or polyphosphazenes.

Suitable Th1 adjuvant systems that can be used include monophosphoryl lipid A, in particular 3-de-O-acylated monophosphoryl lipid A, and a combination of monophosphoryl lipid A, preferably 3-de-O-acylated monophosphoryl lipid A (3D-MPL) with an aluminum salt. The enhanced system includes a combination of monophosphoryl lipid A and a saponin derivative, in particular a combination of QS21 and 3D-MPL, as described in WO 94/00153, or a less reactive composition, where QS21 is quenched with cholesterol, as described in WO 96/33739. A particularly strong adjuvant preparation, including QS21, 3D-MPL and tocopherol in an oil-in-water emulsion, is described in WO 95/17210 and is the preferred preparation.

The vaccine may contain saponin, more preferably QS21. It may also contain an oil-in-water emulsion and tocopherol. Unmethylated CpG containing oligonucleotides (WO 96/02555) is also a preferred TH1 response inducer and is suitable for use in the present invention.

The vaccine preparation of the present invention can be used to protect or treat a mammal susceptible to infection by administering the indicated vaccine systemically or through the mucous membrane. Such administrations may include injection by intramuscular, intraperitoneal, intradermal or subcutaneous route; or mucosal administration to the mouth / digestive, respiratory, urogenital tract. Thus, one aspect of the present invention is a method of immunizing a human host against a disease caused by infection with gram-negative bacteria, comprising administering to this host an immunoprotective dose of a vial preparation of the present invention.

The amount of antigen in each dose of the vaccine is selected as the amount that induces an immunoprotective response without significant adverse side effects in typical vaccines. This amount will vary depending on the specific immunogen used and how it will be presented. In the General case, it is assumed that each dose will contain 1-100 μg of protein antigen, preferably 5-50 μg and most typically in the range of 5-25 μg.

The optimal amount for a particular vaccine can be determined through standard studies, including observation of appropriate immune responses in subjects. After the initial vaccination, subjects can receive one or more re-immunizations with adequate time separation.

Polynucleotides of the Invention

A “polynucleotide" generally refers to a polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA, or modified RNA or DNA. "Polynucleotides" include, but are not limited to, single and double stranded DNAs, DNAs that are a mixture of single and double stranded regions, single and double stranded RNAs and RNAs, which are a mixture of single and double stranded regions, hybrid molecules containing DNA and RNA, which can be single-stranded or, more typically, double-stranded or a mixture of single and double-stranded areas. In addition, “polynucleotide” refers to trilinear regions containing RNA or DNA, or both RNA and DNA. The term "polynucleotide" also includes DNA or RNA containing one or more modified bases, and DNA or RNA whose skeletons are modified for stability or for other reasons. “Modified” bases include, for example, tritylated bases and unusual bases such as inosine. Many modifications have been made to DNA and RNA; thus, a "polynucleotide" encompasses chemically, enzymatically or metabolically modified forms of polynucleotides, both commonly found in nature, and chemical forms of DNA and RNA characteristic of viruses and cells. A "polynucleotide" also encompasses relatively short polynucleotides, often referred to as oligonucleotides.

Another aspect of the invention relates to an immunological / vaccine preparation that contains one or more polynucleotides encoding Tbp and Hsf-like protein, in particular to those that correspond to the protein combinations of the invention. Such techniques are known in the art, see, for example, Wolff et al., Science (1990) 247: 1465-8.

The expression of Tbp and Hsf-like protein, preferably TbpA and Hsf, in such a polynucleotide will be controlled by a eukaryotic promoter capable of driving expression in a mammalian cell. The polynucleotide may further comprise a sequence encoding other antigens. Examples of such eukaryotic promoters include promoters from viruses using mammalian cells as a host, including adenovirus promoters, retroviral promoters. Alternatively, mammalian promoters can be used to regulate TbpA and Hsf-like protein.

Antibodies and Passive Immunization

Another aspect of the present invention is the use of an immunogenic composition comprising TbpA and an Hsf-like protein for the synthesis of immunoglobulin, which can be used to treat or prevent infection with gram-negative bacteria, or preferably Neisseria, more preferably Neisseria meningitidis and most preferably Neisseria meningitidis serogroup B.

An inoculum for producing polyclonal antibodies is usually prepared by dispersing the antigenic composition in a physiologically acceptable diluent, such as physiological saline or other adjuvants suitable for use in humans, to form an aqueous composition. An immunostimulating amount of the inoculum is administered to the mammal, and then the inoculated mammal is maintained for a time sufficient for the antigenic composition to induce protective antibodies.

These antibodies can be isolated to the desired extent by well-known methods, such as affinity chromatography.

Antibodies may include immune serum preparations from a number of commonly used animals, such as goats, primates, donkeys, pigs, horses, guinea pigs, rats or humans. Blood was collected from animals and serum was recovered.

The immunoglobulin produced in accordance with the present invention may include whole antibodies, antibody fragments or subfragments. Antibodies can be whole immunoglobulins of any class, for example IgG, IgM, IgA, IgD or IgE, chimeric antibodies or hybrid antibodies with double specificity for Tbp and Hsf. They can also be fragments, for example F (ab ') 2, Fab', Fab, Fv and the like, including hybrid fragments. Immunoglobulin also includes natural, synthetic, or genetically engineered proteins that act as an antibody that binds to specific antigens to form a complex.

The vaccine of the present invention can be administered to the recipient, who then acts as a source of immunoglobulin produced in response to stimulating infection from a particular vaccine. A subject treated in this manner will produce plasma from which hyperimmunoglobulin will be obtained by plasma fractionation technique. Hyperimmunoglobulin will be administered to another subject in order to transmit resistance to neisserial infection, or to treat it. The hyperimmunoglobulin of the invention is particularly useful for treating or preventing a neisserial disease in children, immunologically deficient individuals, or when treatment is required and there is no time for an individual to produce antibodies in response to vaccination. An additional aspect of the invention is a pharmaceutical composition comprising monoclonal antibodies reactive against TbpA and Hsf, which can be used to treat or prevent infection with gram-negative bacteria, or preferably Neisseria, more preferably Neisseria meningitides, and most preferably Neisseria meningitidis serogroup B.

Such pharmaceutical compositions contain monoclonal antibodies, which can be whole immunoglobulins of any class, for example, IgG, IgM, IgA, IgD or IgE, chimeric antibodies, or fusion antibodies with double specificity for Tbp and Hsf-like protein. They can also be fragments, for example F (ab ') 2, Fab', Fab, Fv and the like, including hybrid fragments.

Methods for producing monoclonal antibodies are well known in the art and may include fusion of splenocytes with myeloma cells (Kohler and Milstein, 1975, Nature 256; 495; Antibodies - a laboratory manual, Harlow and Lane 1988). Alternatively, monoclonal Fv fragments can be obtained by screening an appropriate phage display library (Vaughan, T.J. et al., 1998, Nature Biotechnology, 16; 535). Monoclonal antibodies can also be humanized or partially humanized using techniques well known in the art.

Serum Bactericidal Analysis

Serum bactericidal analysis is the preferred method for evaluating the synergistic relationships between antigens when combined in an immunogenic composition.

Such a synergistic response can be characterized by SBA (serum bactericidal activity) caused by a combination of antigens that is at least 50%, twice, three times, preferably four times, five times, six times, seven times, eight times, nine times and most preferably ten times higher than the SBA caused by each antigen separately. Preferably, SBA is measured against the homologous strain from which these antigens are derived, and preferably also against a number of heterologous strains. (See a typical series below, for example, BZ10 (B: 2b: P1.2) belonging to the A-4 cluster; B16B6 (B: 2a: P1.2) belonging to the ET-37 complex; and H44 / 76 (B: 15: P1.7,16)). SBA is the most widely accepted immunological marker for evaluating the effectiveness of a meningococcal vaccine (Perkins et al., J.Infect. Dis. 1998, 177: 683-691). Satisfactory SBA can be detected by any known method. SBA can be performed using sera obtained from animal models (see examples 6-9) or from humans.

Another preferred method for conducting SBA with human sera is the following method. A blood sample is taken before the first vaccination, two months after the second vaccination and one month after the third vaccination (in this case, three vaccinations in one year are the regimen of typical primary vaccination of people, when introduced, for example, at 0, 2 and 4 months, or 0 , 1 and 6 months). Such initial vaccination regimens can be given to children under the age of 1 year (for example, simultaneously with Hib vaccinations), or for children aged 2-4 years old or teenagers with the same primary vaccination regimen, can also be vaccinated for SBA testing. 6-12 months after the initial vaccination and one month after the repeated dose, if applicable, an additional blood sample can be taken.

SBA will be satisfactory for antigen or vesicle preparation with homologous bactericidal activity if, one month after the third dose of the vaccine (in the initial vaccination regimen) (in children aged 2-4 years or adolescents, but preferably in children of the first year of life), the percentage of subjects with a four-fold increase in units of the SBA titer (antibody dilution) (compared to the titer before vaccination) against the strain of meningococcus from which the antigens of this invention were obtained, above 30%, preferably above 40%, more preferred substantially higher than 50% and most preferably above 60% of the total number of subjects.

Of course, an antigenic or vesicle preparation with heterologous bactericidal activity can also be part of a vesicle preparation with homologous bactericidal activity if it can also cause satisfactory SBA against the meningococcal strain from which it is derived.

SBA will be satisfactory for an antigenic or vesicle preparation with heterologous bactericidal activity if, one month after the third dose of the vaccine (in the initial vaccination regimen) (in children aged 2-4 years or adolescents, but preferably in children of the first year of life), the percentage of fourfold increase in units of titer SBA (antibody dilution) (compared to titer before vaccination) against three heterologous meningococcal strains above 20%, preferably above 30%, more preferably above 35% and most respectfully above 40% of the total number of subjects. Such a test is a good indicator of whether an antigenic or vesicle preparation with heterologous bactericidal activity can induce cross-bactericidal antibodies against various strains of meningococci. The three heterologous strains should preferably have a different (ET) complex of electrophoretic type or multilocus sequence typing (MLTP) character (see Maiden et al. PNAS USA 1998, 95: 3140-5) to each other and preferably to the strain from which this antigenic or a vesicle preparation with heterologous bactericidal activity obtained or occurred. A person skilled in the art will be able to easily identify three strains with different ET complexes that reflect the genetic diversity observed among meningococci, in particular among type B meningococci strains, which are recognized as the cause of significant disease burden and / or which represent hypervirulent lines recognized by MenB (see Maiden et al. , above). For example, the following three strains can be used: BZ10 (B: 2b: P1.2), belonging to the A-4 cluster; B16B6 (B: 2a: p1.2), belonging to the ET-37 complex; and H44 / 76 (B: 15: P1.7,16), belonging to the ET-5 complex, or any other strains belonging to the same ET / cluster. Such strains can be used to test antigenic or vesicle preparation with heterologous bactericidal activity, obtained or derived from, for example, meningococcal strain CU385 (B: 4: P1.15), which belongs to the ET-5 complex. Another example of a strain that can be used is a strain from line 3 of the epidemic clone (eg, NZ124 [B: 4: P1.7,4]. Another ET-37 strain is NGP165 (B: 2a: P1.2).

Methods for measuring SBA are known in the art. For example, a method that can be used is described in WO 99/09176 in Example 10C. In general, a culture of the test strain is grown (preferably under iron depletion conditions by adding an iron chelator such as EDDUK (ethylenediamine diorthohydroxyphenylacetic acid) to the growth medium) in a logarithmic growth phase. It can be suspended in a medium with BSA (bovine serum albumin) (such as Hanks’s medium with 0.3% BSA) to give a working cell suspension brought to approximately 20,000 CFU (colony forming units) / ml. A series of reaction mixtures can be obtained by mixing a series of two-fold dilutions of test sera (preferably inactivated by heating at 56 ° C for 60 min) [for example, in a volume of 50 μl / well] and a test suspension of a meningococcal strain of 20,000 CFU / ml [for example, in a volume of 25 μl / well]. The reaction vessel should be incubated (e.g., 37 ° C for 15 minutes) and shaken (e.g., at 210 rpm). The final reaction mixture [for example, in a volume of 100 μl] additionally contains a source of complement [such as 25% of the final volume of previously tested rabbit serum], and incubated as described above [for example, 37 ° C. for 60 minutes]. For this analysis, a sterile polystyrene 96-well microtiter plate with a U-shaped bottom can be used. An aliquot [for example, 10 μl] can be taken from each well using a multichannel pipette, and dropped onto Mueller-Hinton agar plates (preferably containing 1% Isovilatex and 1% heat-inactivated horse serum) and incubated (for example, for 18 hours at 37 ° C in 5% CO ₂ ). Preferably, individual colonies can be up to 80 CFU per aliquot. The following three test samples can be used as controls: buffer + bacteria + complement; buffer + bacteria + inactivated complement; serum + bacteria + inactivated complement. SBA titers can be directly calculated using a program that, through regression calculations, processes the data to produce a dilution value that corresponds to 50% cell mortality.

All references or patent applications cited within this patent application are hereby incorporated by reference.

Method for industrial application of the invention

The examples below are carried out using standard techniques that are well known and generally accepted by those skilled in the art, with the exception of those that are described in detail. These examples are illustrative but do not limit the invention.

Example 1: Methods of constructing strains of Neisseria meningitidis serogroup B used in the preparation of outer membrane vesicles

WO 01/09350 provides detailed methods for the preparation of outer membrane vesicles and the treatment of bacterial strains from which external membrane vesicles originate. When the outer membrane vesicles are designed to hold lipoproteins such as TbpB and / or lipopolysaccharides, isolation methods with or without low levels of deoxycholate are preferred.

Example 2: Positive regulation of Hsf protein antigen in a recombinant strain of Neisseria meningitidis serogroup B, which lacks functional cps genes but expresses PorA

As described in the examples in WO 01/09350, in some countries the presence of PorA in outer membrane vesicles may be useful and may increase the efficacy of recombinant improved vesicle vaccines. In the following examples, the authors used a modified rSMK (+) vector to upregulate Hsf expression of a protein antigen in a strain that lacks functional cps genes but expresses PorA. The starting pCMK (+) vector contains a chimeric porA / lacO promoter repressed in an E. coli host expressing lacl ^q but transcriptionally active in Neisseria meningitidis. In the modified rSMK (+) native porA promoter is used to start transcription of the hsf gene. The gene encoding Hsf was amplified by PCR (polymerase chain reaction) using the HSF 01-NdeI and HSF 02-NheI oligonucleotide primers shown in the table below. Due to the HSF 01-NdeI primer sequence, the expressed Hsf protein will contain two methionine residues at the 5 ′ end. The conditions used for PCR amplification were those described by the manufacturer (HiFi DNA polymerase, Boehringer Mannheim, GmbH). Thermal citation was as follows: 25 times (94 ° C 1 min, 48 ° C 1 min, 72 ° C 3 min) and 1 time (72 ° C 10 min, 4 ° C until return). The corresponding amplicon was sequentially cloned into the corresponding restriction sites of the pCMK (+) delivery vector. In this recombinant plasmid constructed as pCMK (+) - Hsf, the inventors removed the lacO present in the chimeric porA / lacO promoter by means of a recombinant PCR strategy. rSMK (+) - Hsf plasmid was used as a template for PCR amplification of 2 separate DNA fragments:

- fragment 1 contains a porA 5 ′ recombinogenic region, a kanamycin resistance gene, and a porA promoter. Used oligonucleotide primers RP1 (SacII) and RP2 are presented in the table below. RP1 primer is homologous to the sequence directly in the opposite direction from the lac operator;

- fragment 2 contains the Shine-Dalgarno sequence of the porA gene, the hsf gene, and the porA 3 ′ recombinogenic region. Used oligonucleotide primers, RP3 and RP4 (Apal) are presented in the table below. The RP3 primer is homologous to the sequence directly in the forward direction from the lac operator. The 3'-end of fragment 1 and the 5'-end of fragment 2 has 48 overlapping bases. 500 ng from each batch PCR (1 and 2) was used for the final PCR reaction using primers RP1 and RP4. The last amplicon obtained was subcloned into the pSL1180 vector, which was digested with SacII and ApaI enzymes. The modified plasmid pCMK (+) - Hsf was purified on a large scale using the QIAGEN maxiprep kit, and 2 μg of this substance was used to transform the serogroup B Neisseria meningitidis strain without functional cps genes. To preserve porA expression using a combination of PCR and screening techniques by Western blotting, the integration resulting from a single crossing-over was selected. Kanamycin-resistant clones evaluated positively by porA-specific PCR and Western blotting were stored at -70 ° C as stock solutions in glycerol and used for further studies. Bacteria (corresponding to approximately 5 × 10 ⁸ bacteria) were resuspended in 50 μl SDS-PAGE buffer, frozen (-20 ° C) / boiled (100 ° C) three times and then separated by SDS-PAGE (sodium polyacrylamide gel dodecyl sulfate) electrophoresis in a 12.5% gel. Hsf expression was studied on whole-cell bacterial lysates (CCLs) obtained from NmB [Cps-, PorA +] or NmB [Cps-, PorA +, Hsf +]. Coomassie staining revealed a significant increase in Hsf expression (relative to endogenous Hsf level). This result confirms that the modified pCMK (+) - Hsf vector is functional and can be successfully used to positively regulate the expression of outer membrane proteins without stopping the production of the main PorA protein of the outer membrane antigen.

The oligonucleotides used in this study

Oligonucleotides Sequence Note (i) Hsf 01-nde 5 '- GGA ATT CCA TAT GAT GAA CAA AAT ATA CCG C - 3' NdeI cloning site Hsf 02-nhe 5 '- GTA GCT AGC TAG CTT ACC ACT GAT AAC CGA C - 3' Nhe I cloning site Gfp-mut-asn 5 '- AAC TGC AGA ATT AAT ATG AAA GGA GAA GAA CTT TTC - 3' AsnI NdeI compatible cloning site Gfp-spe 5 '- GAC ATA STA GTT TAT TTG TAG AGC TCA TCC ATG - 3' Spe I NheI compatible cloning site RP1 (SacII) 5 '- TCC CCG CGG GCC GTC TGA ATA CAT CCC GTC-3' SacII cloning site RP2 5 '- CAT ATG GGC TTC CTT TTG TAA ATT TGA GGG CAA ACA CCC GAT ACG TCT TCA - 3' RP3 5 '- AGA CGT ATC GGG TGT TTG CCC TCA AAT TTA CAA AAG GAA GCC CAT ATG - 3' RP4 (ApaI) 5'- GGG TAT TCC GGG CCC TTC AGA CGG CGC AGC AGG - 3 ' APAI cloning site

Example 3: Positive regulation of TbpA gene of N.meningitidis serogroup B through substitution of the promoter

The aim of the experiment was to replace the endogenous promoter region of the TbpA gene with a strong porA promoter to upregulate TbpA antigen production. To this end, a plasmid was constructed to replace the promoter using the E. coli cloning methodology. A DNA region (731 bp), located in the opposite direction from the TbpA coding sequence, was found in the private Incyte PathoSeq database of Neisseria meningitidis strain ATCC 13090. This DNA contains a sequence encoding TbpB antigen. These genes are organized into an operon. The TbpB gene is deleted and replaced with the CmR / porA promoter cassette. To this end, a 3218 bp DNA fragment corresponding to a 5'-flanking region of 509 bp in length tbpB gene, tbpB coding sequence with a length of 2139 bp, intergenic sequence with a length of 87 bp and 483, the first nucleotides of the TbpA coding sequence were amplified by PCR from genomic DNA of serogroup B Neisseria meningitidis using BAD16 oligonucleotides (5 '- GGC STA GCT AGC CGT CTG AAG CGA TTA GAG TTT CAA AAT TTA TTC - 3') and BAD17 (5 GGC C AA GCT T CA GAC GGC GTT CGA CCG AGT TTG AGC CTT TGC - 3 ') containing capture sequences and NheI and HindIII restriction sites (underlined). This PCR fragment was purified using a High Pure Kit (Boerhinger Mannheim, Germany) and directly cloned into the pGemT vector (Promega, USA). This plasmid was subjected to cyclic PCR mutagenesis (Jones & Winistofer (1992)) in order to 1) introduce suitable restriction sites to clone the CmR / PorA promoter cassette, and 2) to delete a 209 bp sequence. 5'-flanking sequence of tbpB and tbpB coding sequence. Cyclic PCR was performed using BAD 18 (5 '- TCC CCC GGG A AG ATC T GG ACG AAA AAT CTC AAG AAA CCG - 3') & BAD 19 (5 '- GGA AGA TCT CCG CTC GAG CAA ATT TAC AAA AGG AAG CCG ATA TGC AAC AGC AAC ATT TGT TCC G - 3 ') oligonucleotides containing suitable restriction sites XmaI, BglII and XhoI (underlined). The CmR / PorA promoter cassette was amplified from the pUC D15 / Omp85 plasmid previously described using primers BAD21 (5 '- GGA AGA TCT CCG CTC GAG ACA TCG GGC AAA CAC CCG-3') & BAD20 (5'-TCC CCC GGG AGA TCT C AC TAG T AT TAC CCT GTT ATC CC-3 ') containing suitable restriction sites XmaI, SpeI, BglII and Xhol (underlined). This PCR fragment was cloned into a cyclic PCR plasmid. This plasmid is used to transform strains of Neisseria meningitidis serogroup B [cps-] and [cps-porA-]. Integration by double crossing over in the region located in the opposite direction from TbpA directs the porA insertion of the promoter directly in the opposite direction from TbpA ATG.

Example 4: Construction of a strain of N.meningitidis serogroup B with positive regulation of the expression of two antigens: TbpA and Hsf

The aim of this experiment was to upregulate TbpA and Hsf expression simultaneously in the same N.meningitidis serogroup B strain. TbpA production was positively regulated by replacing the region of its endogenous promoter with a strong porA promoter (promoter substitution). In this context, the tbpB gene located in the opposite direction from TbpA is deleted, and the TbpB protein is no longer present in the outer membrane. Hsf expression was positively regulated by insertion (homologous recombination) of a second copy of the corresponding gene at the porA locus (gene delivery). Both strains are described in a separate patent, referred to as WO 01/09350. Selective markers used in both strategies (Cm ^R or Kan ^R ) allow the combination of both integrations on the same chromosome.

Total genomic DNA was extracted from the recombinant Nm.B cps-TbpA + / PorA + strain following the Qiagen Genomic tip 500-G protocol. Ten micrograms of DNA was subjected to restriction with the o / n DraIII restriction enzyme and used to transform Neisseria meningitidis serogroup B according to the classical transformation protocol. The cells used for transformation were either recombinant NmB cps- / Hsf + / PorA + (homologous recombination by 1 crossing over at the porA locus) or recombinant NmB cps- / Hsf + / PorA- (allelic exchange / homologous recombination by 2 crossAcrosses ) They were placed overnight on GC agar (gonococcal agar) containing 200 μg / ml kanamycin, diluted to OD ₆₅₀ = 0.1 in GC liquid medium with 10 mM MgCl ₂ and incubated for 6 hours at 37 ° C with vigorous stirring with 10 μg restricted with DraIII genomic DNA. Recombinant Neisseria meningitidis obtained by double crossing over in one act (PCR screening) was selected on GC medium containing 200 μg / ml kanamycin and 5 μg / ml chloramphenicol, and analyzed for TbpA and Hsf expression in OMV preparations. As shown in the drawing, the production of both TbpA and Hsf was significantly increased in BHM obtained from the TbpA / Hsf recombinant NmB strain, compared to BHM obtained from control NmB cps strains. The level of overexpression of each protein in a double recombinant is comparable to the expression level obtained in the corresponding single recombinants. The overexpression level of TbpA and Hsf was comparable in porA + and PorA strains (data not shown). Together, these data demonstrate that: 1) the expression of TbpA and Hsf can be combined and concomitantly positively regulated in N. Meningitidis, and 2) recombinant vesicles enriched in TbpA and Hsf can be obtained and used for immunization.

Analysis of the content of Hsf and TbpA vesicles of the outer membranes

SDS-PAGE, Coomassie Blue

15 μg of protein in preparations based on external membrane vesicles with positive regulation of Hsf or TbpA or both Hsf and TbpA was diluted in sample buffer containing β-mercaptoethanol and heated at 95 ° C for 10 minutes. These samples were then subjected to SDS-PAGE polyacrylamide gel electrophoresis (Novex 4-20% Tris-Glycine 1.5 mm 2Dwell SDS Page), stained with Coomassie blue for one hour, and decolorized by several washes with a bleaching agent. The results are presented in the drawing and show that the level of Hsf and TbpA is significantly higher in preparations of the outer membrane vesicles obtained from N. meningitidis, where their expression level was increased.

Example 5: Immunogenicity of HMF with Positive Regulation of Hsf and / or TbpA

Groups of 20 mice were immunized three times with BHM intramuscularly on days 0, 21 and 28. Each inoculation was performed with 5 μg (protein content) of BHM prepared on AlPO ₄ with MPL (monophosphoryl lipid). VNMs were obtained from N.meningitidis strain H44 / 76, designed so that capsular polysaccharides and PorA are negatively regulated. Compared with VNM, in which Hsf, TbpA, both Hsf and TbpA, or none of them were regulated positively. On the 41st day, blood samples were taken for ELISA analysis (enzyme-linked immunosorbent assay) or serum bactericidal analysis.

ELISA for the detection of antibodies against Hsf

96-well microplates (Nunc, Maxisorb) were coated overnight at 4 ° C. with 100 μl of 1 μg / ml specific antigen in PBS (phosphate buffered saline). After washing with NaCl 150 mM Tween-20 0.05%, the plates were saturated with 100 μl PBS-BSA (bovine serum albumin) 1%, with shaking at room temperature for 30 minutes. Between each stage (carried out by shaking at room temperature for 30 minutes and with PBS-BSA 0.2% as a dilution buffer), excess reagents were removed by washing with NaCl-tween 20. One hundred microliters of diluted serum samples were added to the microwell. Bound antibodies were recognized by biotinylated anti-mouse Ig (Prosan) (1/2000). The antigen / antibody complex was detected by incubation with streptavidin-biotinylated peroxidase (Amersham) conjugate (1/4000). Orthophenylenediamine / H ₂ O ₂ (4 mg / 10 ml citrate buffer 0.1 M pH 4.5 + 5 μl H ₂ O ₂ ) was used for detection in the analysis. The plates were incubated for 15 minutes at room temperature in the dark, then the reaction was stopped by adding 50 μl of 1 N. HCl. Read absorbance at 490 nm.

Midpoint titration (on pooled serum) g1, TbpA-HSF vesicles, IM 15471 g2, TbpA vesicles, IM 15.41 g3, bubbles HSF, IM 14508 g4, vesicles CPS (-) PorA (-), IM - g5, MPL / AlPO4, IM -

The results presented in the table above show that high and equivalent anti-Hsf antibody titers were achieved by immunizing BHM with up-regulation of Hsf or both Hsf and TbpA. Virtually no anti-Hsf antibodies were detected in the sera after inoculation with one adjuvant or BHM, in which neither Hsf nor TbpA were positively regulated, or BHM, in which only TbpA was positively regulated.

Example 6: Serum bactericidal activity of antisera obtained against VNM with positive regulation of Hsf and / or TbpA

The serum bactericidal activity of antisera from mice inoculated with VNM with positive regulation of Hsf, TbpA, both Hsf and TbpA, or without positive regulation, was compared in the analyzes using either a homologous strain H44 / 76 or a heterologous strain Cu385. As shown, the analysis of the bactericidal properties of whey shows a good correlation with protection and, therefore, is a good indicator of how effectively the test composition elicits a protective immune response.

Strains of wild-type Neisseria meningitidis serogroup B (H44 / 76 strain = B: 15 P1. 7.16 L3, 7, 9 and CU385 strain = B: 4 P1. 19.15 L3, 7, 9) were cultured overnight on plates Petri dish with MN + 1% Polyvitex + 1% horse serum at 37 ° С + 5% CO ₂ . They were subcultured for 3 hours in a TSB liquid medium enriched in 50 μM Desferal (Desferal, iron chelator) at 37 ° C. with shaking until an optical density (OD) of approximately 0.5 at 470 nm was reached.

The pooled or single serum was inactivated for 40 min at 56 ° C. Serum samples were diluted 1/100 in HCPX (Hanks balanced salt solution) -BSA 0.3% and then sequentially diluted twice (8 dilutions) in a volume of 50 μl in round bottom microplates.

Bacteria with appropriate OD were diluted with CCPX-BSA 0.3% to obtain 1.3 · 10 ⁴ CFU per ml. 37.5 μl of this dilution was added to serum dilutions and the microplates were incubated for 15 minutes at 37 ° C. with shaking. Then, 12.5 μl rabbit complement was added to each well. After 1 hour of incubation at 37 ° C and with shaking, the microplates were placed on ice to stop the destruction.

Using the tilt method, 20 μl from each well was transferred to Petri dishes with MH + 1% Polyvitex + 1% horse serum and incubated overnight at 37 ° C + CO ₂ . CFU was counted and the percentage of killed cells was calculated. The titer of serum bactericidal activity is the last dilution, causing not less than 50% destruction.

H44 / 76 CU385 VNM GMT % of respondents GMT % of respondents CPS (-) PorA (-) 93 thirty% 58 5% CPS (-) PorA (-) Hsf 158 40% 108 twenty% CPS (-) PorA (-) TbpA 327 60% 147 thirty% CPS (-) PorA (-) Hsf - TbpA 3355 one hundred% 1174 80%

Results similar to those shown in the table above were obtained in two other similar experiments.

Significant increases in bactericidal titers (GMT) against the homologous strain and the heterologous strain were observed after HVM vaccination, when both Hsf and TbpA were regulated positively. By comparison, bactericidal GMTs measured in mice vaccinated with Hsf or TbpA positively regulated by HMN were similar to those obtained in mice vaccinated with control HMF.

The advantage of double positive regulation was also clearly observed in the percentage of mice producing a significant level of bactericidal antibodies (titers greater than 1/100), in particular in experiments using a heterologous strain.

Example 7: Effect of Mixing Anti-Hsf and Anti-TbpA Serums on Bactericidal Activity

Groups of 20 mice were immunized three times with BHM intramuscularly on days 0, 21 and 28. Each inoculation was performed with 5 μg (protein content) of BHM prepared on AlPO ₄ with MPL. VNMs were derived from strain N.meningitidis H44 / 76, designed so that capsular polysaccharides and PorA were negatively regulated. One group of mice was immunized with control BHM, in which there was no positive regulation of proteins. In the second group, Hsf expression was positively regulated, in the third group, TbpA expression was positively regulated, and in the fourth group, both Hsf and TbpA expression were positively regulated.

Sera were pooled either using the serum of mice in the same group, or by mixing sera isolated from the group in which one Hsf or one TbpA was up-regulated. Serum bactericidal activity was measured for each pooled serum, the results are shown in the table below.

SBA performed on pooled sera of mice immunized with: SBA titer TbpA-Hsf vesicles 774 TbpA vesicles 200 Hsf bubbles fifty CPS (-) PorA (-) vesicles fifty Mixture of anti-TbpA + anti-Hsf sera 1162

The results in the above table show that the consequence of mixing anti-Hsf and anti-TbpA antisera was serum bactericidal activity, significantly higher than that achieved by any antiserum separately. It seems that a synergistic effect is achieved due to the presence of antibodies against both Hsf and TbpA.

Example 8: Truncated Hsf Proteins Can Synergistically Combine with TbpA

A series of truncated Hsf constructs were obtained using standard molecular biology techniques. These include a construct that encodes amino acids 1-54 including the Hsf signal sequence, and amino acids 134-592 Hsf (Tr1Hsf). The second truncated Hsf contained amino acids 1-53 of the Hsf signal sequence and the following amino acids 238-592 Hsf (Tr2Hsf). These two truncated Hsf constructs and full-sized Hsf were introduced into N. meningitidis strain MC siaD-, Opc-, PorA- so that their expression was positively regulated, and outer membrane vesicles were obtained using the methods described above.

Outer membrane vesicle preparations were adsorbed onto Al (OH) ₃ and injected into mice on days 0, 21 and 28. On day 42, blood was collected from mice and serum was prepared. These sera were mixed with the sera of mice vaccinated with VNM with positively regulated TbpA, and serum bactericidal analyzes were performed as described above.

Serum bactericidal titers Group H44 / 76 CU385 MC58 PorA + siaD + 25600 25600 MC58 PorA-siaD-Hsf 1530 800 MC58 PorA-siaD-Tr1Hsf 1015 1360 MC58 PorA-siaD-Tr2Hsf fifty fifty Negative control fifty fifty TbpA + MC58 PorA + siaD + 25600 24182 TbpA + MC58 PorA-siaD-Hsf 2595 1438 TbpA + MC58 PorA-siaD-Tr1Hsf 4383 2891 TbpA + MC58 PorA-siaD-Tr2Hsf 1568 742 TbpA + Negative Control 778 532

The results presented in the table above show that the first truncation (Tr1Hsf) elicits an immune response that can be combined with anti-TbpA antisera to produce higher serum bactericidal activity than when using full-sized Hsf. However, the degree of truncation is important, and truncation produced in Tr2 has an adverse effect compared to full-size Hsf. Enhanced bactericidal activity of Tr1Hsf was observed against both strains used.

Example 9: Serum bactericidal activity of antibodies against TbpA, Hsf and third meningococcal protein

N.meningitidis strain H66 / 76, in which PorA and capsular polysaccharides were negatively regulated as described above, was used as a background strain for positive regulation of TbpA and Hsf, LbpB, d15, PilQ or NspA using the method described above. Outer membrane vesicles were prepared from each strain as described above. Recombinant FhaB, FrpC, FrpA / C, and Nar were prepared using techniques well known in the art, as described in PCT / EP 99/02766, WO 92/01460 and WO 98/02547.

Outer membrane vesicle preparations and recombinant proteins were adsorbed onto Al (OH) ₃ and injected into mice on days 0, 21 and 28. On day 42, blood was taken from mice and serum was prepared. Serums against TbpA and Hsf of positively regulated BHM were mixed with sera of mice vaccinated with positively regulated LbpB, D15, PilQ or NspA OMVs or recombinant FhaB, FrpC, FrpA / C or Nar, and serum bactericidal analyzes were performed as described above.

results

The results are shown in the table below. In assays using the homologous strain H44 / 76, the addition of antibodies against the third meningococcal antigen, with the exception of FrpC, did not generate a higher serum bactericidal titer than the titer generated using antibodies against TbpA and Hsf only.

However, the addition of antibodies against the third antigen was useful in serum bactericidal analyzes using a heterologous strain. Antibodies against D15 (OMP85), Nar, Frp / C and LbpB were particularly effective in increasing serum bactericidal titer against strain CU 385.

Serum bactericidal titers A mixture of antisera H44 / 76 CU385 anti-TbpA-Hsf and non-immune serum 5378 2141 anti-TbpA-Hsf and anti-FhaB 5260 2563 anti-TbpA-Hsf and anti-Nar 4577 5150 anti-TbpA-Hsf and anti-FrpA / C 5034 4358 anti-TbpA-Hsf and anti-LbpB 5400 4834 anti-TbpA-Hsf and anti-D15 4823 4657 anti-TbpA-Hsf and anti-PilQ 4708 2242 anti-TbpA-Hsf and anti-NspA 4738 2518 anti-TbpA-Hsf and anti-FrpC 6082 2300

Example 10: Effect of FrpB KO in the vesicles of external membranes on their ability to induce a bactericidal immune response in homologous and heterologous strains

Two N.meningitidis strains H44 / 76 were used to prepare outer membrane vesicle preparations as described in WO 01/09350 using extraction with 0.1% DOC so that the VOC content was approximately 20%. Strain B1733 is siaD (-), PorA (-), has positive regulation of Tr1Hsf (Example 8), and lgtB is “knocked out”. Strain B1820 B1733 is siaD (-), PorA (-), has positive regulation Tr1Hsf, lgtB is “knocked out”, and FrpB is also “knocked out”. Both strains were cultured in a medium enriched with 60 μM Desferal so that iron-regulated proteins, such as LbpA / B and TbpA / B, were regulated positively.

Bubble preparations were adsorbed onto Al (OH) ₃ , and 5 μg was injected intramuscularly into groups of 30 mice on days 0 and 21. Blood samples were taken on day 28.

Serum bactericidal analyzes were performed on three L3 strains (wild type homologous strain H44 / 76 and two heterologous L3 strains: NZ124 and M97250687), as described in Example 6.

results Bubbles used for inoculation H44 / 76 M97250687 Nz124 GMT SC GMT SC GMT SC B1733 1518 30/30 151 11/30 70 4/29 B1820 781 19/30 1316 24/30 276 19/30

GMT indicates the geometric mean serum titer in SBA.

SC indicates the number of seroconverting mice (BSA titer greater than 1/100).

The results clearly show that FrpB KO (B1820) vesicles induce a better heterologous cross-bactericidal response than FrpB (+) vesicles (B1733). BSA titers were higher, and most mice had seroconversion in strains M97250687 and NZ124. The results in the homologous strain were not so good when FrpB was removed.

These data suggest that FrpB induces an immune response, but since this outer membrane protein is highly variable, antibodies against this protein can only induce the destruction of the homologous strain.

Claims (56)

1. An immunogenic composition for use against a disease caused by infection with gram-negative bacteria, containing isolated transferrin-binding protein (Tbp) and an isolated Hsf-like protein or antigen fragments thereof that can elicit a protective response against infection caused by gram-negative bacterial strains from which they occur . 2. The immunogenic composition according to claim 1, wherein the transferrin-binding protein or fragment thereof and the Hsf-like protein or fragment thereof are derived from Neisseria. 3. The immunogenic composition of claim 1, wherein the transferrin-binding protein or fragment thereof is derived from N. meningitidis. 4. The immunogenic composition according to claim 1, in which the Hsf-like protein or its fragment is derived from N. meningitidis. 5. The immunogenic composition according to claim 1, in which the transferrin-binding protein or its fragment comes from N. meningitidis serogroup B. 6. The immunogenic composition according to claim 1, in which the Hsf-like protein or its fragment is derived from N. meningitidis serogroup B. 7. The immunogenic composition of claim 1, wherein the transferrin-binding protein or fragment thereof is derived from N. gonorrhoeae. 8. The immunogenic composition according to claim 1, in which the Hsf-like protein or its antigenic fragment comes from N. gonorrhoeae. 9. The immunogenic composition according to claim 1, in which the transferrin-binding protein is TbpA or its antigenic fragment. 10. The immunogenic composition according to claim 9, containing a high molecular weight form of TbpA or a low molecular weight form of TbpA or both a high molecular weight form of TbpA and a low molecular weight form of TbpA. 11. The immunogenic composition of claim 1, wherein the Hsf-like protein is Hsf or an antigenic fragment thereof. 12. The immunogenic composition of claim 1, comprising a Tbp fusion protein and an Hsf-like protein or antigenic fragments thereof. 13. The immunogenic composition according to item 12, containing a fusion protein containing TbpA and Hsf or antigenic fragments thereof that can cause a protective response against neisserial infection. 14. An isolated immunogenic composition containing an outer membrane vesicle preparation derived from gram-negative bacteria in which the expression of both the transferrin-binding protein and the Hsf-like protein is at least 1.5 times higher than the natural expression in unmodified gram-negative bacteria. 15. The immunogenic composition according to 14, in which the expression of the transferrin-binding protein is positively regulated by growth under conditions of iron depletion. 16. The immunogenic composition of claim 14, wherein at least a portion of the outer membrane vesicle preparation is derived from Neisseria. 17. The immunogenic composition of claim 14, wherein at least a portion of the outer membrane vesicle preparation is derived from Neisseria meningitidis. 18. The immunogenic composition of claim 14, wherein at least a portion of the outer membrane vesicle preparation is from Neisseria meningitidis serogroup B. 19. The immunogenic composition of claim 14, wherein at least a portion of the outer membrane vesicle preparation is derived from Neisseria gonorrhoeae. 20. The immunogenic composition of claim 14, wherein the host cell from which the outer membrane vesicle preparation is derived is designed such that the expression of one or more of LgtB and LgtE, preferably the first, is negatively regulated. 21. The immunogenic composition of claim 14, wherein the host cell from which the outer membrane vesicle preparation is derived is not capable of synthesizing capsular polysaccharides and is preferably designed to express one or more of siaD, ctrA, ctrB, ctrC, ctrD, synA (equivalent to synX and siaD), synB (equivalent to siaB and synC (equivalent to siaC)), most preferably siaD, is negatively regulated, and preferably one or more of siaD, ctrA, ctrB, ctrC, ctrD, synA (equivalent to synX and siaD) synB (equivalent to siaB and synC (equivalent to siaC)), most preferably siaD, yl deleted. 22. The immunogenic composition of claim 14, wherein the host cell from which the outer membrane vesicle preparation is derived is designed such that the expression of one or more of OPC, OA and PorA, preferably PorA, is negatively regulated, and that preferably one or more from OPC, OA and PorA, preferably PorA, was deleted. 23. The immunogenic composition of claim 14, wherein the host cell from which the outer membrane vesicle preparation is derived is designed so that expression of FrpB is negatively regulated, and that preferably FrpB is deleted. 24. The immunogenic composition of claim 14, wherein the host cell from which the outer membrane vesicle preparation is derived is designed such that expression of msbB and / or HtrB, preferably msbB, is negatively regulated, and that preferably msbB and / or HtrB, preferably msbB has been deleted. 25. The immunogenic composition of claim 14, wherein the outer membrane vesicle preparation contains LPS (lipopolysaccharide) that is conjugated to an outer membrane protein (BNM). 26. The immunogenic composition of claim 25, wherein the LPS is conjugated (preferably intravesically) with BNM in situ in an outer membrane vesicle preparation. 27. The immunogenic composition of claim 14, comprising an outer membrane vesicle preparation isolated from two or more strains of gram-negative bacteria. 28. The immunogenic composition according to item 27, containing the preparation of the outer membrane vesicles, in which the transferrin-binding protein and the Hsf-like protein are positively regulated on different vesicles derived from different bacterial strains, or on the same vesicles originating from the same and the same bacterial strain. 29. The immunogenic composition of claim 14, comprising an outer membrane vesicle preparation in which enhanced expression of the transferrin-binding protein is derived from polynucleic acid introduced into gram-negative bacteria. 30. The immunogenic composition of claim 14, comprising an outer membrane vesicle preparation in which enhanced expression of an Hsf-like protein is derived from polynucleic acid introduced into gram-negative bacteria. 31. The immunogenic composition of claim 14, comprising an outer membrane vesicle preparation in which enhanced expression of a transferrin binding protein and an Hsf-like protein is derived from a polynucleic acid encoding both proteins that has been introduced into gram-negative bacteria. 32. The immunogenic composition of claim 14, wherein the bacterial strain has been genetically engineered by introducing the sequence of a stronger promoter in the opposite direction from the gene encoding the transferrin binding protein. 33. The immunogenic composition of claim 14, wherein the bacterial strain has been genetically engineered by introducing a sequence of a stronger promoter in the opposite direction from a gene encoding an Hsf-like protein. 34. The immunogenic composition of claim 14, wherein the bacterial strain has been genetically engineered by introducing the sequence of a stronger promoter in the opposite direction from the genes encoding the transferrin binding protein and the Hsf-like protein. 35. The immunogenic composition of claim 14, wherein the transferrin binding protein is TbpA, which is preferably high molecular weight TbpA, low molecular weight TbpA, or both high molecular weight TbpA and low molecular weight TbpA, most preferably N. meningitidis. 36. The immunogenic composition of claim 14, wherein the Hsf-like protein is Hsf from Neisseria meningitidis. 37. The immunogenic composition according to claim 1 or 14, containing an adjuvant. 38. The immunogenic composition according to clause 37, containing aluminum salts. 39. The immunogenic composition according to clause 37, containing 3D-MPL (3-de-0-acylated monophosphoryl lipid A). 40. The immunogenic composition according to clause 37, containing an adjuvant containing CpG. 41. A vaccine containing the immunogenic composition according to any one of claims 1 to 40 and a pharmaceutically acceptable excipient. 42. A method for preventing a disease caused by gram-negative bacteria, comprising administering a protective dose or an effective amount of a vaccine according to paragraph 41. 43. The method according to § 42, in which a neisserial infection is prevented. 44. The use of the immunogenic composition according to any one of claims 1 to 40 in the manufacture of a vaccine for the prevention of infection caused by gram-negative bacteria. 45. The use according to claim 44 in the manufacture of a vaccine for the prevention of neusserial infection. 46. A genetically engineered strain of gram-negative bacteria, from which vesicles of the outer membranes of the immunogenic composition according to any one of paragraphs.14-36 can be derived. 47. A method of manufacturing an immunogenic composition according to any one of claims 1 to 11, comprising the step of mixing together the isolated transferrin-binding protein and the isolated Hsf-like protein or antigenic fragments thereof. 48. A method of manufacturing an immunogenic composition according to any one of paragraphs.14-36, comprising the step of isolating the outer membrane vesicles from a culture of gram-negative bacteria. 49. The method of claim 48, wherein the step of isolating the outer membrane vesicles includes extracting 0-0.5%, 0.02-0.4%, 0.04-0.3%, 0.06-0.2%, 0.08-0.15% or preferably 0.1% detergent, preferably DOC (deoxycholate). 50. A method of manufacturing a vaccine according to paragraph 41, comprising the step of combining the immunogenic composition according to any one of claims 1 to 40 with a pharmaceutically acceptable excipient. 51. A method for producing immunoglobulin for use in the prevention or treatment of a neisserial infection, comprising the steps of immunizing a recipient with the vaccine of claim 41 and isolating the immunoglobulin from the recipient. 52. The immunoglobulin preparation obtained by the method according to item 51. 53. A pharmaceutical composition comprising an immunoglobulin preparation according to claim 52 and a pharmaceutically acceptable excipient. 54. A pharmaceutical composition comprising monoclonal antibodies against TbpA and Hsf from Neisseria meningitidis and a pharmaceutically acceptable excipient. 55. A method of treating or preventing infection with gram-negative bacteria, comprising the step of administering to the patient an effective amount of a pharmaceutical composition according to any one of claims 53 and 54. 56. The use of the pharmaceutical composition according to any one of paragraphs.53 and 54 in the manufacture of a medicament for the treatment or prevention of a disease caused by gram-negative bacteria.

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