KR20060118630A - Vaccines containing recombinant pilin against neisseria gonorrhoeae or neisseria meningitidis - Google Patents

Abstract

The pil E genes of each of Neisseria gonorrhoeae and Neisseria meningitidis are cloned and their corresponding recombinant pilin proteins are expressed. In addition, a chimeric pil E gene is constructed in which the region of the pil E gene of Neisseria meningitidis class I encoding the amino-terminal region of the pilin protein is replaced by the corresponding region of the pil E gene of Neisseria gonorrhoeae. The recombinant meningococcal chimeric class I pilin protein is expressed at higher levels than the pilin protein expressed by the full-length pil E gene of Neisseria meningitidis. Furthermore, a chimeric pil E gene is constructed in which the region of the pil E gene of Neisseria meningitidis class II encoding the carboxy-terminal region of the pilin protein is replaced by the corresponding region of the pil E gene of Neisseria gonorrhoeae. The recombinant pilin proteins are used in vaccines to protect against disease caused by Neisseria gonorrhoeae or Neisseria meningitidis.

Description

Vaccine Containing Recombinant PILIN AGAINST NEISSERIA GONORRHOEAE OR NEISSERIA MENINGITIDIS}

1 is cultured guinea pig antiserum against gonococcus rphyllin (strain Pgh3-1) (diluted 1:50 for 15 min), followed by incubation of auxiliary anti-guinea pig IgG conjugated to 12 nm colloidal gold (1 min for 30 min). Transmission electron micrographs of filiform cells of N. gonoria (strain I756 recA-) stained with: 5 dilution) followed by NanoVan. 1A shows anti-rphylline guinea pig immune serum (Note 6); 1B shows normal guinea pig serum (week 0); 1C shows the primary antibody free.

Figure 2 shows the effect of guinea pig antiserum on gonococcus rphyllin (of strain Pgh3-1) upon attachment of N. gonolia cells (strain I756 recA-) filthy to human cervical cells (ME180 cell line). 2A depicts inhibition of adhesion by guinea pig antiserum against rphylline (note 6); 2B depicts the inability of normal guinea pig antiserum to prevent attachment of gonococcal cells piling into cervical cells (Note 0). Representative masses of bacteria attached to cervical cells are circled in each panel. Each panel shows four different aspects of the same experimental conditions. Guinea pig antiserum is diluted 1: 10,000 per panel.

FIG. 3 shows the culture of guinea pig antiserum against meningococcal chimeric class I rphylline (strain H44 / 76) (diluted 1:60 for 30 min) followed by supplemental anti-guinea pig IgG conjugated to 12 nm colloidal gold. Transmission electron micrographs of filiform cells of N. meningitidis (strain H355) stained with NanoVan (1: 5 dilution for 30 min) are shown. 3A shows anti-rphylline guinea pig immune serum (Note 6); 3B shows normal guinea pig serum (week 0); 3C shows the primary antibody free. The cells are fixed before incubation with antisera.

The present invention is Nigeria ( Nisseria) gonorrhoeae ) or the use of recombinant pilin proteins in vaccines for protection against diseases caused by Niisseria meningitidis .

Nigerian gonoria (N. gonoria) and Nigeria meningitidis (N. meningitidis) are Gram-negative cocci. yen. Gonoria and N. meningitidis are generally very closely related, but the clinical signs of the diseases they cause are very different. N. gonoria causes gonorrhea, while N. meningitidis causes meningitis. These Nigerian species are parasitic on the mucosal surface of the body.

Type IV peels include a large number of Gram-negative bacteria, for example, Dichelobacter (formerly bacteroid ), Dichelobacter. nodous ), Eikenella corrodens , Kingella denitripicans denitrificans ), Moraxella bovis ), M. M. lacunata , M. M. nonliquefaciens , N. Gonoria, N. meningitidis, and Pseudomonas aeruginosa ) (Bibliography Entries 1,2) on non-flagella hairy structures on the surface. Vibrio cholera (Vibrio Toxin co-regulated pili of cholerae ) and bunching pili of Escherichia coli are considered to be somewhat distant relatives, showing a limited number of similarities with type IV pili (1,2). In the case of N. gonoria and N. meningitidis, the bacteria with fili are more attached to various endothelial cells of human origin than the cells without fili, which acts as a virulence factor by immobilizing the organism at the site of mucosal infection. It is considered to be.

Type IV filaments are 5-7 nM wide and 5 μM long or less. The filin protein subunits are linked in tandem to form long, thin polymers. In the case of pathogenic Nigeria, pili is clearly homopolymeric, consisting of a single structural subunit, ie, pilin protein. The filin protein has a molecular weight of 13,000 to 22,000 Daltons (1, 2, 3).

In essence, the filin protein aggregates in a bacterial outer membrane into a helical structure called pilus (plural), which has a molecular weight of approximately 10 ⁷ Daltons. When purified pili is dialyzed using pH 12 phosphate buffer, intact pili is irreversibly dissociated into a pilin protein mass called pilin oligomer (4). These fillin oligomer masses (molecular weight of approximately 600,000 Daltons) are smaller in size than intact fillers.

N. gonoria expresses a single fibrin protein that was originally isolated and sequenced by Schoolnik and his colleagues (5). Gonophyllin protein has three regions: (a) a highly conserved amino terminal region (residues 1-53); (b) a middle third region (residues 54-124) exhibiting a limited amount of sequence variation and (c) a carboxy third region (residues 125-160) of proteins containing a highly diverse disulfide loop.

During the course of the natural infection, the filli go through a high frequency phase and antigenic variation (3.6). The genetics of these variants have been unusually complex and extensively studied. Each strain of Nigerian gonoria has the ability to change the primary amino acid sequence of the pilin protein and thus the antigenicity of the pili. The molecular mechanism responsible for this variation involves non-homologous recombination between the expression site (pilE) and multiple (17-19), promoter-free, silent (pilS) genes (6). As the pilin sequence (or a portion thereof) moves from the pisS position to the expression site resulting in a new pilin variant, gonococci can express an extremely large number of pilin proteins.

Unlike N. gonoria, N. meningitidis expresses two different filin classes, called class I and class II. As in gonococcus, meningococcal class I pili undergoes an antigenic phase shift (3). Class I filins have been shown to be similar to gonococcal filins in terms of molecular weight (17-20 kd) and reactivity with monoclonal antibodies (SM1) that bind to highly conserved epitopes on gonococcal filins (3). Many class I filins were cloned and the amino acid sequence was shown to have a high degree of similarity with that of gonococcal filins (7). Class II filins, on the other hand, do not react with the SM1 antibody and have a lower molecular weight (13-16 kd). A number of strains expressing class II pilin have been shown to react with polyclonal antiserum against gonococcal pili (3). Aho et al. (7) recently determined the sequence of class II filins. The initial one-third of the Nigerian filins are essentially consistent. Class II pilin proteins differ from class I pilin and gonococcal pilin proteins in the hypervariable regions of proteins in which large amounts of deletion have occurred. Achtman et al. (8) have demonstrated that several serogroup A isolates from Africa bind to both antibodies using monoclonal antibodies specific for class I or class II filins. Strains expressing class II pili also demonstrated truncated, silent class I pilin genes (7). In addition, these data suggest the possibility that a single meningococcal cell expresses both pilin proteins simultaneously.

Because fil is believed to mediate initial contact with mucosal cells, there is considerable interest in using these structures as vaccine antigens to prevent diseases caused by filthy bacteria. Pilly vaccine against traveler's diarrhea and gonorrhea has been tested in humans (9). However, so far, this has only worked for homologous strains. Many Filiary vaccines are used for diseases that infect poultry, such as infectious keratoconjunctivitis (red eye disease) (1) in cows, subsidies (1,10) in sheep and diarrhea in piglets (9) or calves (9). Has been reported. In each of these examples, the Philly vaccine provides protection from the attack of strains that express homologous Philly but not heterologous Philly.

The first gonococcal vaccine contained whole organisms and provided little or no protection (11). Recent vaccine development for gonorrhea has focused on purified surface components, in particular pili (9,11) and porin proteins (P.I. or Por) (11). However, to date only Philly has been shown to protect humans from attack, which has been limited to protection against homologous strains (12). The denatured form of gonococcal phyll (4) has been shown to produce antibodies in mice or guinea pigs that bind heterologous phyllies in vitro. However, this is not regarded as an industrially viable approach, because it is difficult to grow filiform gonorrhea in liquid medium (lack of industrial production) (1). Based on the results of early human challenge studies, gonococcal filament vaccine was tested in a large, placebo-controlled double blind efficacy test (12). In this attempt, the vaccine failed to protect male volunteers from gonococcal infection. The most likely explanation for this is presumed to be heterogeneity of the pillars (12).

Indeed, antigenic variability of gonococcal and meningococcal filin proteins has been mentioned several times as a major obstacle in the development of phylogeny vaccines (3, 13, 14, 15, 16, 17). It has been suggested that the predominant immune epitope on the aggregated intact filaments is a disulfide loop showing the highest sequence variation (17,18). This is yen. The failure of the Korean field test with formalin-treated intact pili from gonoria strain Pgh3-2 can be explained (12). Additionally, this document contains a number of references that the anti-serum for purified fil, or filline fraction, is attached to denatured (western blot) or isolated Nigeria fil, but not to heterologous fil on the bacterial surface (16). , 17, 19, 20). Whether this is an antigenic variation or a hidden epitope in the aggregated pillars is not fully resolved. This is reinforced by a number of reports that describe that only monoclonal antibodies that exhibited functional activity in vitro were not attached to heterologous filaments (3). M. Intact pili of Vorbis (21) and D. nodosus showed that a protective immune response induced by the pili was possible (10). However, these vaccines can protect against the attack of bacteria expressing homologous filaments but not heterologous filaments (10, 21). The common opinion of the scientific community is that the phylloid vaccine (when possible) provides protection only against bacteria that express homologous phil.

Recombinant expression of aggregated pili has been described in the case of many organisms and depends on the proper transport and presence of assembly genes (22). In Nigeria, this approach is difficult because no genes encoding proteins involved in Philly aggregation and external transport are found in a single contiguous operon. Another alternative selected from European Patent 202,260 B1 is a bacterial host that already possesses the proteins necessary for the aggregation of several Type IV filaments; Eg in the expression of the type IV filin gene in Pseudomonas aeruginosa (23). However, as reported by Hoyne et al., The expression and aggregation of gonococcal fili in the outer membrane of Pseudomonas is unstable (24). When recombinant strains grow in liquid medium in the presence of selective antibiotics, wild type piliform pseudomonas is overgrown. The author is:

"The suitability of exogenous mePhe [N-methylphenylalanine] filament production in host strains will depend on the degree of diversity of host and donor filamentous agglutination systems. PAK / 2PfS [Pseudomonas aeruginosa strain K / 2PfS expressing gonococcal filament] ] May indicate that the limit of interspecies expression of mePhe Philly is approached in this example ”(24).

As a result, the expression of gonocoli fili in Pseudomonas is not seen as an industrially viable approach.

Elman, Egerton and their colleagues describe the development of vaccines for both subsidies using the full Phili of Dikelobacter nordosus. Field tests show that intact pili is protected from D. nodosus strains expressing homologous pili. This means that industrial vaccines need to contain 8 or 9 different pillars to achieve comprehensive protection. In an attempt at this viable approach, the D. nodosus filin gene was cloned and expressed in E. coli (25). Recombinant pilin proteins have been found to bind to the lining. When a vaccine consisting of sonicated E. coli cells expressing recombinant filins is tested in a dosing experiment, antibody titers similar to those seen for native filament in which recombinant E. coli cells have been purified produce 25 (25). However, the aggregation titers induced by recombinant E. coli cell vaccines are significantly lower than those seen for intact pili (690 vs 47,800) and lower than those associated with protection (5,000-10,000) (25,26).

After an active attack with D. nodosus, the recombinant pilin vaccine did not show significant protective activity unlike that seen with intact pili. Emery and his colleagues have demonstrated that intact Philly's metamorphosis eliminates her ability to induce protection in animals (27). In addition, phyllies dissociated using detergents (octyl-β-D-glucoside) or low pH (2.2) reduce the efficacy of proteins that induce protection against serious injury after attack (28). Nevertheless, antibody titers do not differ significantly between groups. The authors mentioned that there may be one or more epitopes with quaternary structures that are disturbed by treatment. These are additionally:

"Failure of E. coli expression products as vaccines may be due to physical blockade in the host cell membrane, but preliminary experiments have indicated that this is not a major cause of inability (unpublished data). E. coli expression products as vaccines Another explanation for the failure is that the monomeric prefillin units that are expressed cannot be combined in their native form for proper presentation of important epitopes due to the presence of the leader sequence ”(28).

Elleman and his colleagues demonstrated the importance of the presence of morphological epitopes on recombinant filins, and they need to increase the immune response by better presentation of antigens. They suggested two approaches: Purification of E. coli membrane protein, or protein expression in P. eruzinoza, to be able to aggregate into filamentous filaments on the cell surface. The latter approach is preferred because "this method greatly enhances immunity and simplifies the purification of proteins" (25).

In addition, Elleman and his colleagues also considered the use of pilin (Phili's subunit protein) as an inferior vaccine candidate against D. nodosus' mature pimbria (intact pili):

"Mature pimbria appears to elicit a stronger (and K-aggregated) immune response than an equivalent amount of pimbria subunit protein. A serological K-aggregation titer of about 5,000 generally results from infection of B. nordosus strains. It is regarded as the minimum response equivalent to adequate protective immunity. This response level (and up to the maximum) is readily achieved upon vaccination with mature pimbria rather than an isolated subunit protein that results in a weak level of serum K-aggregated antibody. " (23).

Additional data have recently been reported by Alves et al., Indicating that Pimbria (philus) subunit proteins are not viable vaccine candidates (29). Immunization of mice with polynucleotide vaccines encoding E. coli CFA / I pimbria attachment proteins (eg, filins) results in that the antibodies derived are distinguished from those induced by native, intact CFA / I pimbria. In addition, these antibodies to recombinant proteins did not show aggregate activity unlike antiserum against native proteins.

Despite all of the achievements described above, an effective Philotype gonococcal or meningococcal vaccine is still under development. Meningococcal vaccines are limited to those with serotype A, C, Y, W135 capsules.

Accordingly, it is necessary to identify the components included in the vaccine to protect against diseases caused by all serotypes of N. gonoria or N. meningitidis.

Summary of the Invention

Accordingly, it is an object of the present invention to identify suitable antigenic structures derived from N. gonoria and N. meningitidis, respectively, which can constitute a potent vaccine candidate against bacteria. These candidates must induce antibodies that recognize and bind to various isolates of individual pathogenic Nigerian organisms.

These and other objects of the present invention to be discussed below are achieved by cloning and expression of recombinant pilin proteins (rpilin) of N. gonoria and N. meningitidis, respectively.

The invention also relates to the construction of a plasmid expressing a recombinant meningococcal chimeric class I filin protein in which the amino-terminal region of the class I meningococcal pilin protein is replaced with the corresponding amino-terminal region of the gonococcal pilin protein. This plasmid expresses significantly higher amounts of meningococcal chimeric class I rpilin protein than class I meningococcal rpilin protein expressed from the full-length meningococcal pilE gene.

To obtain expression of the meningococcal chimeric class I rpilin protein, the chimeric DNA sequence is first inserted into a suitable plasmid vector. Suitable host cells are transformed or transfected with plasmids. In an embodiment of the invention, the host cell is an Escherichia coli strain. The host cell is cultured under conditions that allow for expression of the chimeric class I rpilin protein by the host cell.

The invention further relates to the construction of plasmids expressing the recombinant meningococcal chimeric class II filin protein in which the carboxy-terminal region of the class II meningococcal pilin protein is replaced with the corresponding carboxy-terminal region of the gonococcal pilin protein.

To obtain expression of the meningococcal chimeric class II rpilin protein, the chimeric DNA sequence is first inserted into a suitable plasmid vector. Suitable host cells are transformed or transfected with plasmids. In an embodiment of the invention, the host cell is an Escherichia coli strain. The host cell is cultured under conditions that allow for expression of the chimeric class II rpilin protein by the host cell.

In another aspect of the invention, isolated and purified rpilin protein (gonococcus, meningococcus or chimera) is used to prepare a vaccine composition that elicits a protective immune response in a mammalian host. The vaccine composition may further comprise an adjuvant, diluent or carrier. Examples of such adjuvants include aluminum hydroxide, aluminum phosphate, MPL ^™ , Stimulon ^™ QS-21, IL-12 and cholera toxin. The vaccine composition is administered to the mammalian host in an amount of an immunogen sufficient to protect the host from diseases caused by N. gonoria or N. meningitidis.

The present invention relates to a vaccine composition comprising the recombinant pilin protein of N. gonoria or N. meningitidis. Despite the teachings in the industry described above, it was decided to investigate the use of recombinant pilin proteins expressed in E. coli. Surprisingly, these recombinant pilin proteins showed the characteristics of vaccine candidates.

The first report describing the cloning of gonococcal pilE gene in E. coli was published in 1982 (30). Since then, the molecular properties of pilE have been performed in a number of experiments investigating genetic factors that control the expression of pilin proteins, the transport of pilin proteins, variations in the pilin sequences, and host adhesion of pili. However, no report describes the purification of recombinant pilin proteins and the immune response of recombinantly expressed pilin proteins.

Cloning and expression of the pilE gene encoding gonococcal recombinant pilin protein is described in Example 2 below. Expression was performed by transforming an E. coli strain named TOP10F 'using a plasmid containing the pilE gene. Following successful cloning and expression, the pilE gene was sequenced to confirm its presence with native sequences. To aid cloning, the NcoI site needed to modify 1 base was introduced. As a result, the second amino acid was changed from asparagine to aspartic acid in the 7 amino acid long signal peptide.

In Example 2 the plasmid containing the pilE gene (named pPX2000) contains an ampicillin resistance (Amp ^R ) marker. As described in Example 3, another plasmid was constructed to contain kanamycin resistance (Kan ^R ) markers instead of Amp ^R. The plasmid, designated pPX2002, transforms E. coli strain TOP10F 'and expresses gonococcus rphylline at a level similar to that obtained in pPX2000 containing Amp ^R markers.

As described in Example 4, a similar procedure is used to construct a plasmid designated pPX2003 containing the class I pilE gene of N. meningitidis. The NcoI site (CC ATG G) is introduced while spanning the initial of the gene encoding the signal peptide. This changes the second amino acid residue of the signal peptide from asparagine to aspartic acid (the first residue remains methionine). Amp ^R markers are also included. This construct expresses the class I rpilin of N. meningitidis after transforming E. coli strain TOP10F '. However, expression levels are significantly lower than for gonococcus rphylline obtained in pPX2000 or pPX2002. Without being bound by theory, this low expression level is likely due to the large number of reverse repeats present in recombinant class I pilE.

To increase the expression of meningococcal filins, chimeric plasmids are constructed as described in Example 5. The DNA of pPX2003, which encodes the initial 60 amino acids of meningococcal class I rpilin, is replaced by the same region of gonococcal DNA in pPX2002. The Amp ^R plasmid designated pPX2004 has the nucleotide sequence shown as SEQ ID NO: 1. Plasmid pPX2004 is used to transform E. coli strain K12 designated TOP10F '. After induction, the expression of chimeric rpilin comparable to the amount of meningococcal rpilin expressed from pPX2003 is significantly increased. The expression level of the chimeric construct is comparable to the amount of gonococcus rpilin expressed in pPX2002. Chimeric Class I rpilin is 167 amino acids in length (including signal) (SEQ ID NO: 2), according to the expected size.

A sample of E. coli strain K12 designated TOP10F 'containing the recombinant plasmid pPX2004 was deposited by the Applicant on January 27, 1998 to the American Type Culture Collection of Manassas University Boulevard 10801, Virginia, USA, and has an ATCC accession number. ATCC 98637 was granted.

As described in Example 6, a chimeric plasmid was constructed in which the 3 'end of the class II pilE gene of N. meningitidis was replaced with the corresponding region of N. gonoria. Specifically, the DNA of pPX8001 encoding the disulfide loop (the last 22 amino acids of meningococcal class II filin of N. meningitidis strain FAM18) has a similar (but larger) region + N. gonoria strain Pgh3-1 in pPX2000. Is replaced by an additional site of the carboxy-terminal region of a total of 44 amino acids of gonococcal (pilE) DNA. The Amp ^R plasmid designated pPX8017 has the nucleotide sequence shown in SEQ ID NO: 3, wherein nucleotides 1-378 are from N. meningitidis class II and nucleotides 379-510 are from N. gonoria. The plasmid pPX8017 is used to transform E. coli strain K12 designated TOP10F '. After induction, chimeric class II rphylline, which is 170 amino acids (SEQ ID NO: 4) (including a long signal of 7 amino acids) is expressed, wherein amino acids 1-126 are from N. meningitidis class II and amino acids 127-170 Is from N. Gonoria. These chimeric class II rpilins are of expected size. The NcoI site is introduced for cloning and the second amino acid in the signal sequence is changed from lysine to glutamic acid. Such changes are not expected to affect antigenicity or immunogenicity.

A sample of E. coli strain K12 designated TOP10F 'containing the recombinant plasmid pPX8017 was deposited by the Applicant on April 15, 1999 to the American Type Culture Collection of Manassas University Boulevard 10801, Virginia, USA, and has an ATCC accession number. ATCC 207199 was granted.

Various host cell-vector systems are suitable for use in expressing gonococcus, meningococcal and chimeric rphylline used in the vaccine of the present invention in addition to those described in Examples 2-6. Vector systems are compatible with the host cell used. Suitable host cells include bacteria transformed with plasmid DNA, cosamide DNA or bacteriophage DNA; Viruses such as vaccinia virus and adenovirus; Yeasts such as Pichia cells; Insect cells such as Sf9 or Sf21 cells; Or mammalian cell lines such as Chinese hamster egg cells; And other common organisms.

Various conventional transcription and translation factors can be used in the host cell-vector system. pilE DNA is inserted into the expression system and promoters and other regulatory factors are linked to specific sites in the vector so that when the plasmid vector is inserted into the host cell, the pilE DNA can be expressed by the host cell.

Plasmids are introduced into the host cell by transformation, transduction, transfection or infection depending on the host cell-vector system used. The host cell is cultured under conditions that allow for the expression of rpilin protein by the host cell.

The invention further comprises a DNA sequence encoding the meningococcal chimeric class I rpilin protein from which the amino-terminal region is from the gonococcal pilE gene and the central and carboxy-terminal regions are from the meningococcal pilE gene (SEQ ID NO: 1). Relates to an isolated and purified DNA sequence. Nucleotides 1-501 in SEQ ID NO: 1 encode the meningococcal chimeric class I rpilin protein prior to processing; Nucleotide 22-501 encodes meningococcal chimeric class I rpilin after processing as a mature protein. The invention additionally provides meningococcal chimeric class I rpilin protein having an amino acid sequence of amino acids 1-167 of SEQ ID NO: 2 or a mature protein after processing with an amino acid sequence of amino acids 8-167 of SEQ ID NO: 2 prior to processing. It is about. Approximately 10% of the total protein produced by the gonococcus rphilin or meningococcal chimeric class I rphylline constructs lacks signal sequences removed by processing.

The invention further provides a DNA sequence encoding a meningococcal chimeric class II rpilin protein from which the carboxy-terminal region is from the gonococcal pilE gene and the central and amino-terminal regions are from the meningococcal pilE gene (SEQ ID NO: 3). It relates to a separated and purified DNA sequence comprising. Nucleotide 1-501 in SEQ ID NO: 3 encodes the meningococcal chimeric class II rpilin protein prior to processing; Nucleotide 22-501 encodes meningococcal chimeric class II rpilin after processing with mature protein. The invention additionally provides meningococcal chimeric class II rpilin protein having an amino acid sequence of amino acids 1-170 of SEQ ID NO: 4 and a mature protein after processing with a mature protein prior to processing. It is about.

In addition to the chimeric DNA sequences contained in pPX2004 and pPX8017, which encode the meningococcal chimeric class I rpilin protein and the meningococcal chimeric class II rpilin protein, respectively, the present invention relates to sequences encoding the chimeric rpilin protein by duplication of genetic code. Biologically homologous DNA sequences, that is, these other DNA sequences are characterized by different nucleotide sequences than those described herein, but have the same amino acid sequence as encoded by the DNA sequence of SEQ ID NO: 1 or SEQ ID NO: 3 Encode your protein.

In particular, the present invention contemplates DNA sequences that fully overlap with the sequences of SEQ ID NO: 1 or SEQ ID NO: 3 to enable hybridization under standard high resolution Southern hybridization conditions as described by Sambrook et al. (31).

The invention also includes a DNA sequence (SEQ ID NO: 2 or SEQ ID NO: 4) that encodes an amino acid sequence that is biologically equivalent to one of these proteins, although different from that of meningococcal chimeric class I or class II rpilin protein. . Such amino acid sequences can be said to be biologically identical to those of the chimeric rpilin protein if these sequences differ only by slight deletions in the rpilin sequence, insertion into the rpilin sequence, or by substitution with the rpilin sequence, resulting in a third order of sequence The structure is essentially unchanged from that of the rpilin protein.

For example, the codons for the amino acid alanine, a hydrophobic amino acid, may be substituted by another weaker hydrophobic residue, such as glycine, or a codon encoding a larger hydrophobic residue, such as valine, leucine, or isoleucine. Likewise, replacement of one negatively charged residue with another negatively charged residue, such as aspartic acid to glutamine, or one positively charged residue with another positively charged residue, eg, lysine with arginine, and a numerical value. Changes based on similarity of residues in the therapeutic index may also be expected to produce biologically identical products. Nucleotides that result in alteration of the N-terminal or C-terminal region of a protein molecule are also not expected to alter the activity of the protein.

In addition, changes in known variable regions where the tertiary structure of the conserved region does not change essentially from that of the rpilin protein are biologically equivalent. Another definition of biologically equivalent sequence is one that can elicit a cross-reactive immune response. In particular, meningococcal chimeric class I and II recombinant pilins can be modified by lengthening or shortening the corresponding insertions from gonococcal pilin as long as the modified chimeric recombinant pilins can elicit a cross-reactive immune response.

Each of the proposed modifications is well known to those skilled in the art as determinants of the retention of the structural and biological activity of the encoded product. Thus, when the term "meningococcal chimeric class I rpilin protein" or "meningococcal chimeric class II rpilin protein" is used in the specification or claims, it is construed to include all modifications and variations that produce a biologically equivalent protein. do.

As described in Example 7, gonococcus rphylline protein is associated with and expresses the cell membrane of E. coli used. Various detergents include Empigen ^™ BB, Triton ^™ X-100, reduced Triton ^™ X-100, octyl-β-D-glucopyranoside (OG), Zwittergent ^™ 3-10 or 3-14. The filin protein can be selectively dissolved. After centrifugation, dialysis and columnar fractionation, purified rphylline is obtained.

As described in Example 8, chimeric class I rphylline is isolated and purified by disruption of E. coli cells, clarification by centrifugation, filtration, and fractionation on two columns.

As described in Example 9, meningococcal chimeric class II rphylline is isolated and purified by disruption of E. coli cells, clarification by centrifugation, dialysis and fractionation on two columns.

Purified gonococcus rphylline is analyzed for repeated N-terminal sequences as described in Example 10. Sequencing of 20-40 amino-terminal residues provides results consistent with missing amino acid sequences in the DNA sequence. The molecular weight of rphylline (with signal) was 18,006 Daltons according to mass spectral analysis when compared to the expected mass of 17,981 Daltons based on amino acid content. On the other hand, rphylline subjected to size exclusion column chromatography with detergent yields an apparent molecular weight of 68,899 daltons. This suggests that rphylline is aggregated. Dialysis of rphylline with PBS in an effort to remove the detergent yields a material with an apparent molecular weight of 452,349 Daltons as measured by gel filtration. This suggests that additional aggregation occurred.

As described in Example 11, immune serum is obtained by immunizing guinea pigs or mice with purified gonococcus rphylline. As described in Example 12, Western blot analysis shows that antisera against rphylline attached to intact cells lysates from gonococcal cells with Phil, but no binding is observed for cell lysates without Phil. On the other hand, antiserum against filin oligomers binds to both filiform cell lysates and fililess cell lysates.

As described in Example 13, when analyzed by ELISA, this pooled antiserum against gonococcus rphylline has a high endpoint titer for attachment to purified gonococcus rphylline protein. Example 13 details the efficacy of various adjuvants. Supplementing rphylline with MPL ^™ alone, MPL ^™ + aluminum phosphate, or Stimulon ^™ QS-21 yields an excellent humoral immune response in mice.

As described in Example 14, a full cell ELISA shows that antiserum against rphylline adheres to cells with fili, but not to cells without isogenic fili of certain gonococci.

As described in Example 15, mice are immunized intranasally with gonococcal rphylline with or without native cholera toxin. Significant immune responses were detected in antigen ELISA from pooled sera that occurred after mice were immunized with rphylline without adjuvant; This reaction is enhanced by the addition of native cholera toxin. Pooled sera have low ELISA titers to adhere to intact, filiform gonococcal cells; This binding is greatly enhanced when mice are immunized with native cholera toxin.

As described in Example 16, immunoelectron microscopy showed that the antibody against rphylline was attached along the length of the filament filament on the surface of the gonococcus. This shows that antibodies have attached to epitopes present on the surface of bacteria in vivo.

Example 17 shows higher titers than those obtained for rpilin antisera that adhere to heterologous piliform bacterial isolates compared to those obtained for antisera against recombinant pilin oligomers. rpilin is converted to rpilin oligomer by dialysis of rpilin against pH 12 phosphate buffer.

Philly mediates the early attachment of N. gonoria to human mucosal cells. Thus, if antigens can induce antibodies that inhibit the attachment of these bacteria to these cells, this provides evidence that these antigens are vaccine candidates.

As described in Example 18, guinea pig antibodies against rphylline significantly inhibit attachment of human germline expressing heterologous phylogenes to human cervical endothelial cells. The phylogeny of gonococcus correlates with the infectivity of these bacteria (2, 3, 32).

These data show that recombinant pili can induce antibodies that bind to various pili on intact gonococcal cells and that antiserum exhibits agonistic activity (inhibition of bacterial attachment) that protects immunized humans from colonization and infection of gonococcus (32,33). It has been previously reported that immunization with E. coli cells expressing recombinant filins from D. nodosus is immunogenic (23,25,28), but not protective against attack. Because of these results, these investigators turned interest in the use of recombinant subunit filins for the aggregated pillars. However, the data described herein suggest that after purification, the recombinantly expressed filin protein induces an immune response correlated with the protection of human from colonization of gonococci. Accordingly, these data support the view that rpilin is a potent vaccine candidate for N. gonoria.

As described in Example 19, the N-terminus of the meningococcal chimeric class I rpilin protein was sequenced. Sequence analysis of the 35 amino-terminal residues provided results consistent with the missing amino acid sequence in the DNA sequence. The molecular weight of the chimeric rphylline (with signal) was 17,659 daltons according to mass spectrum analysis, whereas the expected mass based on amino acid content was 17,676 daltons. On the other hand, size exclusion column chromatography of the meningococcal chimeric class I rphylline protein using a detergent yields an apparent molecular weight of 69,480 daltons. As in the case of gonococcus rphylline, this suggests that the meningococcal chimeric class I rphylline protein has aggregated.

As described in Example 20, the pooled antiserum for meningococcal chimeric class I r pilin protein, as analyzed by ELISA, has a high endpoint titer for both meningococcal class I r pilin protein and piliform meningococcal cells. As described in Example 20, the adjuvant, in particular Stimulon ^™ QS-21, has a significant response for binding of antiserum to meningococcal chimeric class I rpilin protein with both meningococcal class I rpilin protein and piliform meningococcal cells. Cause

As described in Example 21, immunoelectron microscopy shows that antibodies against meningococcal chimeric class I rpilin protein bind along the length of meningococcal filament. This suggests that antibodies attach to epitopes present on the surface of bacteria in vivo.

In Example 4, antisera against gonococcus rphylline was recognized and shown to adhere to Philly meningococcal cells. In Example 22, antiserum generated against meningococcal chimeric class I rpilin protein is shown to adhere to Philly gonococcal cells.

As described in Example 23, passive immunization of childhood rats with guinea pig antiserum against meningococcal chimeric class I rpilin protein can help prevent meningococcal bacteremia in vivo. Colonization levels were significantly reduced in animals receiving these immune sera. In addition, immune responses generated using recombinantly expressed filins can protect against meningococcal bacteria that express heterologous filin protein in vivo.

As described in Example 24, meningococcal chimeric class I rpilin is immunized intranasally with or without cholera toxin, wherein the cholera toxin is in a mutant form in which glutamic acid at amino acid position 29 is replaced by histidine (CT-CRM). , E29H). Significant immune responses were detected in antigen ELISA from pooled sera that occurred after immunizing mice with rphylline in the absence of adjuvant; This response is enhanced by the addition of the mutant CT-CRM, E29H cholera toxin.

As described in Example 25, disruption of colonization of mouse nasopharynx by class I strain of N. meningitidis is demonstrated in mice immunized subcutaneously with meningococcal chimeric class I rpilin assisted with MPL ^™ .

As described in Example 26, Western blot analysis showed that antiserum obtained from guinea pigs immunized with meningococcal chimeric class II rpilin adhered to complete cell lysate from piliform meningococcal cells expressing either class I or class II pilin. Shows.

As described in Example 27, antiserum derived against partially purified meningococcal chimeric class II rphylline adheres to meningococcal cells from homologous bacterial strains.

In addition, these data support the view that rpyrins, in particular meningococcal chimeric class I and class II rphylline proteins, are potent vaccine candidates for N. meningitidis.

The gonococcus rphylline protein is useful for the production of vaccines that protect mammals from diseases caused by N. gonoria. Meningococcal rpilin protein, Meningococcal chimeric class I rpilin protein and Meningococcal chimeric class II rpilin protein are useful for the production of vaccines that protect mammals from diseases caused by N. meningitidis.

In addition, cross-protection against several Nigerian species can be achieved by immunizing with a vaccine containing gonococcus rphyllin protein or by diseases caused by N. gonoria to protect the mammal from diseases caused by N. meningitidis. Provided by immunization with a vaccine containing meningococcal rpilin protein, meningococcal chimeric class I rpilin protein or meningococcal chimeric class II rpilin protein to protect mammals from.

These vaccine compositions comprise separately purified rpilin protein, wherein the vaccine composition elicits a protective immune response in a mammalian host.

Vaccines containing rphylline proteins can be mixed with immunologically acceptable diluents or carriers in conventional manner for preparing injection solutions or suspensions. The levels of antibodies induced by the vaccine were: Stimulon ^™ QS-21 (Aquila Biophrmaceuticals, Inc., Framingham, Mass.), MPL ^™ (3-O-deacylated monophosphoryl lipid A; RIBI, Montana, USA According to ImmunoChem Research, Inc.), aluminum phosphate, aluminum hydroxide, IL-12 (Genetics Institute of Cambridge, Mass.) And cholera toxin (wild type or mutant form, for example US Provisional Patent Application No. 60 / 102,430 Glutamic acid at amino acid position 29 may be enhanced with an adjuvant such as another amino acid, preferably replaced with histidine.

The vaccine of the present invention is administered to humans by conventional injection methods such as subcutaneous, intraperitoneal or intramuscular injection, and orally, mucosal, intranasal or intravaginal, resulting in N. gonoria or N. meningitidis. Induce an active immune response for protection against. Dosage is determined by means known to those skilled in the art. Protection may be provided in a single dose of the vaccine, or multiple booster doses may be required.

In order to better understand the present invention, the following examples are described. The examples are illustrative only and are not intended to limit the scope of the invention.

Use standard molecular biology techniques according to the protocol described by Sambrook et al. (31).

Example  One

Bacteria and Cell Culture

Bacteria and Culture Conditions

Tampa Flora Tampa; Ottawa, Canada; Washington, D.C .; Washington Seattle; Rochester, NY; North Carolina Chapel Hill; And Illinois Evanston. Meningococcal isolates from Chapel Hill, North Carolina; And from Vilthoven, The Netherlands.

The bacteria are stored in lyophilized form until needed or frozen at -70 ° C. When grown in solid medium, the agar plates were incubated in an incubator at 37 ° C. containing moisture and 5% (volume / volume) CO ₂ . N. gonoria and N. meningitidis were grown in GC medium base (Difco Laboratories, Detroit, Mich.) Without hemoglobin, dextrose (400 mg / L), glutamine (10 mg / L), cocacarboxylase (20 Μg / L) and iron nitrate (500 μg / L). The liquid suspension culture of N. meningitidis was grown in a stirred incubator (70 RPM) at 37 ° C. in the same medium lacking agar. In experiments involving the culturing of meningococcal from mouse nasal tissue pulverization, bacteria were treated with the following antibiotic (Difco) mixture: colistin sulfate (75 μg / mL), nystatin (125 μg / mL), vancomycin (30 μg / mL) and trimethoprim lactate (50 μg / mL) in GC medium described above. Philly gonococci were confirmed by colony morphology and individual colonies passaged daily to maintain phenotype. The phylation status of meningococcal cells was evaluated by transmission electron microscopy of samples stained with NanoVan staining (Nanoprobes, Stony Brook, NY) for 30 seconds at pH 8. E. coli to 20 g / L Bacto tryptone (Difco), 5 g / L yeast extract (Difco), 0.6 g / L NaCl, 0.2 g / L KCl and 1% (weight / volume) agar (pH 7.5) It was grown on SOB agar composed or on SOB broth without agar added. In some experiments, Bacto tryptone was replaced with an equivalent amount of HySoy ^™ (Seffield Products, Norwich, NY).

Endothelial cell culture

The ME-180 cell line (ATCC, Beltsville, Maryland) is an epidermal tumor originally derived from cervical tumors. The cells were treated with 10% (volume / volume) fetal calf serum (Sigma, St. Louis, MO), penicillin G (1000 units / mL) in an atmosphere containing 5% (volume / volume) CO ₂ at 37 ° C. Gibco BRL), L-streptomycin (1 mg / mL) (Gibco BRL) and 2 mM L-glutamine supplemented in RPMI 1640 (Gibco BRL, Gatorsburg, Maryland). Cells were divided every 3 to 4 days.

Example  2

this. In Coli  Gonococcus pilE of Cloning  And expression

Frozen P. gorgonia strain Pgh3-1 samples were used as pilE DNA sources in PCR reactions. The pilE gene was amplified using the following primers recognizing the 3 'and 5' ends of the complete pilin protein (including the leader sequence): 5 'CCC CGC GCC ATG GAT ACC CTT CAA AAA GGC3' (PILEFWD) (SEQ ID NO: 5) and 5 ′ GGG CCT GGA TCC GTG GGA AAT CAC TTA CCG 3 ′ (PILEREV) (SEQ ID NO: 6). The PCR product contains a NcoI site and a BamHI site at the end of the pilE coding region. NcoI sites were introduced into the gene for cloning. This changes the second amino acid in the signal sequence from asparagine (AAT) to aspartic acid (GAT). Since amino acid 2 is part of the signal peptide cleaved during normal processing of the mature protein, this change is not expected to affect antigenicity or immunogenicity. PCR products were cloned into pCR ^™ II TA cloning vector (Invitrogen, Carlsbad, Calif.), Linked, and transformed with E. coli TOP10F ′ (Invitrogen). Colonies were selected on 100 μg / mL ampicillin containing plates or 50 μg / mL kanamycin containing plates. Plasmid DNA was isolated from the culture of these transformants overnight and analyzed by restriction digest using the enzymes EcoRI and NotI.

Four clones containing the correct size insert were provided for DNA sequencing to verify the presence of the pilE PCR DNA fraction. Clone # 17, designated pPX1999, was used as the pilE gene source. Plasmid DNA of pPX1999 and pTrcHisA (Invitrogen) were digested with NcoI and BamHI restriction enzymes, DNA fractions were gel separated, ligated and transformed with E. coli TOP10F ′. Ampicillin resistant colonies were selected, plasmid DNA of new transformants were isolated, and DNA restriction analysis was performed using BamHI and NcoI restriction enzymes. Two clones with the correct restriction pattern were provided for DNA sequencing. Both clones had the correct DNA sequence and named it pPX2000.

To test the expression of recombinant filins, cultures containing these clones were grown in stirred flasks or fermenters of SOB + 100 μg / mL ampicillin and 12 μg / mL tetracycline. When using a stirred flask, E. coli was grown in 1 L medium until A ₆₀₀ = 0.9-1.0 was obtained. Expression of recombinant filin was induced by addition of isopropyl-β-D-thiogalactopyranoside (IPTG) to a final concentration of 1 mM and continued growth for 1-4 hours, at which point centrifugation (4 ° C.) Cells were collected and stored at −20 ° C. for 20 minutes at 13,689 × g. In the case of fermenters, the plates were inoculated in flasks containing 500 mL medium grown overnight using culture medium overnight. This liquid culture was used to inoculate a Biostat B fermenter (Braun Biotech, Allentown, Pennsylvania) containing 8.9 L medium. Improved bacterial growth was obtained in the fermenter when HySoy ^™ -containing medium was supplemented with a final concentration of 1% (weight / volume) of dextrose. When the culture reached A ₆₀₀ = 1.0, IPTG was added at a final concentration of 1 mM, cells were grown for an additional 1-4 hours and harvested by centrifugation (13,689 xg for 20 minutes at 4 ° C). The medium was discarded and the cell pellet was stored at -20 ° C. The introduction of IPTG significantly increased the expression of rpilin protein, reaching a maximum level at late induction of 3-4 hours.

Induced culture samples were analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE). Recombinant filins were identified using Coomassie blue staining and their presence was confirmed by Western blot using monoclonal antibodies specific for Immunomyc fili (clone # A33020023, Biospacific, Emeryville, Calif.).

Example  3

Kanamycin  tolerance Marker  Recombinant Gonococcus pilE  Plasmid Construction

A plasmid was constructed in which the Amp ^R marker was replaced with the Kan ^R marker. The procedure of Example 2 was used except as noted below. TrcFXba Primer, 5 'GGC TCT PCR reactions were performed on pTrcHisA plasmid DNA using AGA CTG TCA GAC CAA GTT TAC TC 3 '(SEQ ID NO: 7), and TrcRXba primer, 5' GGC TCT AGA TTG AAG CAT TTA TCA GGG 3 '(SEQ ID NO: 8). Performed. Underlined sequences encode XbaI restriction sites. Approximately 3.5 kb PCR product contains the pTrcHisA DNA minus ampicillin coding region. Another PCR reaction was run on the pACYC177 plasmid DNA (New England Biolabs, Beverly, Mass.), KanFXba primer, 5 'GGC TCT. AGA TAA ACA GTA ATA CAA GGG G 3 '(SEQ ID NO: 9), and KanRXba Primer, 5' GGC TCT AGA TTA GAA AAA CTC ATC GAG C 3 ′ (SEQ ID NO: 10). In addition, the underlined sequences encode XbaI restriction sites. Approximately 860 bp PCR DNA product encodes the kanamycin resistance gene. Both PCR products were purified on agarose gels, DNA was digested with XbaI restriction enzymes, extracted and then linked. Part of the ligation reaction was transformed with E. coli TOP10F ′ and part of the transformation mix was placed on SOB plates containing 30 μg / mL kanamycin.

A total of 48 Kan ^R colonies were streaked twice on SOB plates containing ampicillin or kanamycin. As expected, all Kan ^R colonies were ampicillin sensitive. Because the cloning design is symmetrical, the two orientations of the kanamycin insert were separated. Kanamycin inserts of the same clockwise orientation as the original ampicillin gene were selected for future study and named pZ564. DNA regions containing the lacIq gene, trc promoter and multiple cloning sites in pZ564 were replaced with similar regions of the pPX2000 plasmid (containing pilE) in the following manner: Both pZ564 and pPX2000 were digested with SphI and XmnI restriction enzymes. Approximately 2.2 kb DNA fractions from pPX2000 and approximately 2.6 kb DNA fractions from pZ564 were gel purified, ligated and transformed with E. coli TOP10F ′. The exact plasmid generated was called pPX2002.

Similar induction time course and levels of recombinant pilin protein expression were observed when switching the selected antibiotics from ampicillin to kanamycin.

Example  4

this. In Coli Meningococcal  Class I pilE of Cloning  And expression

Due to the high homology of the DNA and amino acid sequences of gonococcus and meningococcal class I filins (7), the ability of gonococcus rphylline antisera to adhere to the phylogeny expressed in meningococcal cells was evaluated using a full cell ELISA. This antiserum shows titers 151,100 for attachment to N. meningitidis pili cells from strain H355.

This attachment is likely to indicate the filin protein, since Western blots of complete cell lysates from these strains show that only a single band moves with antiserum attached to the filin (data not shown). Many other meningococcal strains show low titers in whole cell ELISA, but the presence of pili was not confirmed by transmission electron microscopy. Based on these data, Class I filins from N. meningitidis were cloned and expressed recombinantly. Initially, the same procedure was followed as for pilE of N. gonoria. The procedure of Example 2 was used except as indicated below.

Class I pilE with the following primers: 5 'CCC CGC GCC ATG GAC ACC CTT CAA AAA GGT TTT ACC 3' (NMFPILE) (SEQ ID NO: 11), and 5 'GGG CCT GGA TCC GAG TGG CCG TGG AAA ATC ACT TAC CGC 3 (NMRPILE) (SEQ ID NO: 12) was used to amplify from genomic DNA of N. meningitidis strain H44 / 76. As expected, a PCR product of approximately 600 bp DNA was obtained. A portion of the PCR reaction product was cut with BamHI and NcoI restriction enzymes for insertion into pTrcHisA. The cleaved DNA was electrophoresed on agarose gel and the DNA fractions were gel purified. DNA fractions were linked and transformed with E. coli TOP10F '. Miniplasmid preliminary analysis of ampicillin resistant clones was performed using BamHI and NcoI restriction enzymes. Clones expressing the exact restriction cleavage pattern were called RZ1142 and the plasmid was called pPX2003.

After induction, the complete cell lysate was analyzed by SDS-PAGE and the presence of polypeptides stained with Coomassie blue with a molecular weight of approximately 15,000 Daltons was observed. Analysis of complete cell lysates in 4 of these clones by SDS-PAGE and Western blot demonstrated the presence of proteins of adequate mobility and reactivity with polyclonal antibodies against intact pili of N. gonoria strain LB2. Purified Class I rpilin is 167 amino acids in length. However, the expression level of such recombinant filins is significantly lower than that obtained using pPX2000 or pPX2002 grown under the same conditions. Analysis of the DNA sequence in recombinant class I pilE shows that there are a number of reverse repeats that explain the low level expression of these proteins in E. coli.

Example  5

this. In Coli  Gonococcus and Meningococcal  Class I chimera pilE of Construction  And expression

To increase the expression of meningococcal filin protein, DNA encoding the initial 60 amino acids in pPX2003 (meningococcal class I pilE construct described in Example 4) was replaced with pPX2002 (gonococcal pilE construct described in Example 3, 7 amino acids). (Including signal peptide) (SEQ ID NO: 4). The procedure of Example 2 was followed except as noted below.

The conserved 5 'terminal region of the meningococcal pilE gene was replaced with the same region of N. gonoria strain Pgh3-1 in the following manner. The BsmBI site was introduced into the meningococcal pilE gene as follows: DNA was primers: 5 'CCG GCG CGT CTC TCA CGG CGA ATG GCC CGG C 3' (CL-1ESPF) (SEQ ID NO: 13) and 5 'GGG CCT PCR amplification from pPX2003 using GGA TCC GAG TGG CCG TGG AAA ATC ACT TAC CGC 3 '(NMRPILE) (SEQ ID NO: 14) and Taq DNA polymerase. The plasmid generated by cloning the expected PCR DNA product directly with pCR2.1 (Invitrogen) and transforming with TOP10F ′ cells was termed pZ578. The BsmBI site was introduced into gonococcus pilE by the following method. Primers 5 'GCA TAA TTC GTG TCG CTC AAG GCG C 3' (SEQ ID NO: 15) and 5 'GCC GCG CGT CTC CCG TGA TTC AGG TAA TAC TCG G 3' (PILEESPR) (SEQ ID NO: 16) and pfu DNA polymerase was used to PCR amplify the 5 'end of the pilE gene of pPX2000. The resulting gonococcal PCR product and pZ578 were digested with BsmBI and linked. The linked DNA was transferred to 5 'GCA TAA TTC GTG TCG CTC AAG GCG C 3' (SEQ ID NO: 17) and 5 'GGG CCT GGA TCC GAG TGG CCG TGG AAA ATC ACT TAC CGC 3' (NMRPILE) (SEQ ID NO: 18) PCR amplification using primers.

The DNA PCR product was the expected size (approximately 850 bp) and was digested with NcoI and BamHI to obtain approximately 600 bp fraction. These fractions were gel isolated and cloned directly into NcoI- and BamHI-cut pPX2000 vectors by replacing gonococcal pilE gene. The resulting plasmid resistant to ampicillin was labeled with pPX2004 and used to transform TOP10F '. Analysis of these transformants demonstrates the presence of the desired chimeric pilE DNA. After induction with IPTG, there was a significant increase in the amount of meningococcal chimeric class I rpilin construct expressed relative to the amount of meningococcal rphylline expressed in pPX2003. Extraction with 1% octyl-β-D-glucopyranoside (OG) and the purification protocol described in Example 7 (TMAE Fractogel ^™ column at 10 mM Tris ^™ , 0.1% (weight / volume) Zwittergent PH 8.5 with ^TM 3-14) was used to obtain highly purified meningococcal chimeric class I rpilin protein (yield: approximately 5 mg / g cell wet weight). This material was> 90% pure when analyzed by SDS-PAGE and laser densitometry. SDS-PAGE showed the presence of large bands of approximately 15,000 Daltons in size. Meningococcal chimeric class I rpilin protein is 167 amino acids in length and comprises a signal sequence of seven amino acids according to sequencing of the amino-terminal 36 residues of the purified protein.

Example  6

this. In Coli  Gonococcus and Meningococcal  Class II chimera pilE of Construction  And expression

Initial cloning of meningococcal class II pilE was confirmed by Pilin. Isolation of chromosomal DNA of Meningitidis strain FAM18 cells and amplification of class II pilE DNA in PCR reactions. The following primers recognize class II pilE genes (including the leader sequence) of the 3 'and 5' ends of the complete filin protein: 5 'GCG GCC GCC ATG GAA GCA ATC CAA AAA GGT TTC ACC C 3' (PILE2FWD) (SEQ ID NO: : 19) and 5 'GCG GCC GGA TCC GGT CAT TGT CCT TAT TTG GTG CGG C 3' (PILE2REV) (SEQ ID NO: 23). In a method similar to Example 2, the resulting PCR product contains a NcoI site and a BamHI site at the beginning of the pilE coding region. NcoI sites were introduced into the gene for cloning. This changed the second amino acid of the signal sequence from lysine (AAA) to glutamic acid (GAA). As mentioned earlier, these changes are not expected to have any effect on antigenicity or immunogenicity. PCR products were cloned into the pCR2.1 cloning vector (Invitrogen), linked and then E. coli. E. coli was transformed with TOP10F '. Colonies were selected on 100 μg / mL ampicillin-containing plates or 50 μg / ml kanamycin plates. Plasmid DNA was isolated from the cultures of these transformants overnight and analyzed by restriction digest using the enzyme EcoRI.

Clone # 8, designated pPX8001, was used as the pilE gene source. Plasmid DNAs of pPX8001 and pTrcHisC (Invitrogen) were digested with NcoI and BamHI restriction enzymes respectively, and the resulting DNA fractions were gel separated, ligated and transformed with E. coli TOP10F ′. Ampicillin resistant colonies were selected, then plasmid DNA of the new transformant was isolated and DNA restriction analysis was performed using BamHI and NcoI restriction enzymes. Two clones with the correct restriction pattern were provided for DNA sequencing. Both clones have the correct DNA sequence named pPX8002.

To test the expression of recombinant class II filins, 10 mL cultures containing these clones, pPX8002, were subjected to A ₆₀₀ = 1.0 in a 50 mL tube of SOB containing 100 μg / mL ampicillin and 12 μg / mL tetracycline. Growing. Expression of the recombinant protein was induced by adding IPTG to a final concentration of 1 mM and the cultures were incubated continuously for 3 hours. When the complete cell lysates of the induced cells were separated by SDS-PAGE and stained with Coomassie Blue, no new (derived) bands were detected. This suggests that the FAM18 pilE gene product was expressed at levels below the level discernible using Coomassie blue. Cloning FAM18 pilE into the pET17b plasmid and transforming with E. coli BL21 (DE3) pLysS in the presence or absence of the pilE signal sequence, no significant expression of recombinant protein was detected. The same result was obtained when cloning the class II pilE genes of two different strains N. meningitidis (NmB, 2996) into the same pTrcHis plasmid and TOP10F 'expression system.

Specifically, strains NMB and 2996 have been shown to express class II filins based on PCR and sequencing data. The pilE gene was amplified from a number of N. meningitidis strains using Class I primer sets (NMFPILE and NMRPILE) and Class II primer sets (PILE2FWD and PILE2REV) as separate templates with chromosomal DNA or cells. PCR products were cloned into pTrcHisC and sequenced or directly sequenced. Sequence alignments were performed: sequences similar to those of the H44 / 76 strain were classified as Class I and sequences similar to those of the FAM18 strain were classified as Class II. No sequence was obtained while all strains were amplified. Preliminary classification was performed based on PCR data. Class I strains provide class I primers that are not class II primers and PCR products of the correct size, and class II strains provide class II primers that are not class I primers and PCR products of the correct size.

Based on experiments with meningococcal class I filins, the region encoding the initial 60 amino acids of the class II pilE (conserved amino-terminal region) was replaced with the corresponding region of N. gonoria strain Pgh3-1. Expression of the resulting chimeric pilE was examined in a number of E. coli expression strains using various promoters. The strains studied were PR13 (Rnase deficiency), BL21 (protease deficiency), KS474 (peripheral cytoplasmic protease deficiency), AD494 (Novagen, disulfide bond formation in the cytoplasm) and three strains of TOPP (Stratagene, useful for difficult to express proteins) Non-K12 strains). In all cases, no recombinant protein was detected in Coomassie blue-stained SDS-PAGE. The same results were obtained by examining other expression plasmids pET17B (including the T7 promoter).

Note that the native class II pilE gene sequence at pPX8002 ends at base 447. The DNA sequence found nucleotides 447-519 downstream of the native meningococcal class II pilE termination site, nucleotides 447-519, contains inverted repeats forming a stem and loop structure. Since stem and loop structures can be effective terminators of transcription, it is assumed that omission of this additional 3 'sequence (74 bases) in pPX8002 may affect the transcription of chimeric class II pilE messages in E. coli. Thus, all subsequent cloning restores this downstream 3 'terminal sequence.

To identify regions that inhibit expression, various sites of the class II pilE gene of the N. meningitidis strain were systematically replaced with corresponding regions of the pilE gene of the N. gonoria strain Pgh3-1. The replacement region starts at the 5 'or 3' end and grows in the direction of travel. Double replacements at the 5 'and 3' ends were constructed until only 84 nucleotides of internal region of native pilE class II remained. By replacing this region, rGC was recognized and, as expected, these clones expressed rpilin at Coomassie blue staining levels similar to pPX2000. The following regions of the FAM18 pilE gene (described as nucleotide numbers) were replaced with the corresponding regions of Pgh3-1 (shown in parentheses): Single region replacements were 1-108 (1-108), 1-181 (1-181). ), 1-294 (1-282), 439-499 (478-553), 379-519 (367-553), 295-519 (283-553), 295-378 (283-366); Dual-zone replacements are 1-294 (1-284) and 379-519 (367-553), 1-181 (1-181) and 439-499 (478-553), 1-181 (1-181) and 379-519 (367-553), 1-294 (1-282) and 439-499 (478-553).

When expressing these constructs in E. coli TOP10F 'using the pTrcHis expression system, both constructs produce detectable levels of recombinant protein with Coomassie blue: nucleotides (including disulfide loops and 3' extension). Initial protein containing a replacement for 379-519; And a second protein containing a replacement of nucleotides 1-181 (including the conserved 5 'region) and 379-519 (including the disulfide loop and 3' extension). These constructs (nucleotides 379-519) were selected for further investigation because replacement of only the 5 'region did not induce the expression of the recombinant protein and the initial construct retained most of the native meningococcal filin sequences. Although the amino acid sequences of meningococcal and gonococcal proteins differ significantly in this region, it has been reported that disulfide loops have significant antigenic variation (17, 18). Thus, the immune response to this region (eg disulfide loops) shows minimal cross-reactivity among meningococcal. Recently, because gonococcal inserts are nearly twice the size of the meningococcal disulfide loop (39 residues: 18 residues), the resulting chimeric protein migrates to an apparent molecular weight of approximately 19,000 Daltons on an SDS-PAGE gel.

The construction of this chimeric gene was performed by the following method. Primers below: 5 'GCG GCC GCC ATG GAA GCA ATC CAA AAA GGT TTC ACC C 3' (PILE2FWD) (SEQ ID NO: 19) and 5 'GCC GCG CGT CTC CGA ACC GGA GTT TTG TTT GCC 3' (REV-CYS) (SEQ ID NO: 20) was used to amplify pPX8002 (FAM18 class II pilE) to give a 5 'fraction. The gonococcal disulfide loop (ie, the 3 'end of the gonococcal gene) was transferred to primer 5' CCG GGC CGT CTC GGT TCG GTA AAA TGG TTC TGC 3 '(FWD-CYS) (SEQ ID NO: 21) and 5' GGG CCT GGA TCC GTG GGA AAT CAC TTA CCG 3 ′ (PILEREV) (SEQ ID NO: 22) was used to amplify on pPX2000. The resulting PCR products were each purified, digested separately with restriction enzyme BsmBI, and then linked to form a full length chimeric pilE and amplified using primers PILE2FWD and PILEREV. This PCR product was digested with restriction enzymes NcoI and BamHI and likewise linked with limited pTrcHisC and transformed into TOP10F 'campentant cells.

Transforms were cultured and analyzed using restriction enzymes NcoI and BamHI. Four clones with the correct size insert were analyzed using restriction enzyme StuI. Of these, three provide accurate restriction maps. Two of the three clones with the correct restriction pattern were sequenced. Clone # 5 had the correct DNA sequence and named it pPX8017. This clone contains the nucleotide sequence set forth in SEQ ID NO: 3, of which nucleotides 1-378 are from N. meningitidis and nucleotides 379-510 are from N. gonoria.

Expression was checked with 10 mL culture in a 50 mL tube. Cells were grown in SOB supplemented with 100 μg / ml ampicillin and 12 μg / ml tetracycline until A ₆₀₀ was approximately 1.0. Cultures were induced by the addition of IPTG to a final concentration of 1 mM. Continued growth for 3-4 hours, at which point cells were collected by centrifugation (13,689 xg for 20 minutes at 4 ° C) and stored at -20 ° C. In the fermentor, the plates were inoculated overnight using a culture medium or frozen stock in a flask containing 500 mL of medium grown overnight. This liquid culture was used to inoculate a Biostat B fermenter (Braun Biotech, Allentown, Pennsylvania, USA) containing 8.9 L medium. Supplementation of the final concentration of 1% (weight / volume) of dextrose in the HySoy ^™ -containing medium resulted in enhanced bacterial growth in the fermentor. When the culture reached A ₆₀₀ = 4.0-6.0, IPTG was added to a final concentration of 1 mM and cells were grown again for 2-4 hours and recovered by centrifugation (13,689 xg for 20 minutes at 4 ° C). The medium was discarded and the cell pellet was stored at -20 ° C. Induction with IPTG significantly increased the expression of the chimeric class II rpilin protein.

Samples of the induced cultures were analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE). Coomassie blue staining reveals recombinant meningococcal chimeric class II filins (approximately 19,000 Daltons of molecular weight) and their identification is based on the gonococcal peptide (Glu Ala Ile Leu Leu Ala) located in the conserved region of the amino terminus of class II filin proteins. Confirmed by Western blot using polyclonal antiserum against Glu Gly Gln Lys Ser Ala Val Thr Glu Tyr Tyr Leu Asn His Gly Lys (SEQ ID NO: 24). Meningococcal chimeric class II rphylline is 170 amino acids in length (including signal) (SEQ ID NO: 4), wherein amino acids 1-126 are from N. meningitidis and amino acids 127-170 are from N. gonoria It is.

7 to implementation

this. From coli  Recombinant gonococcus Filin's  Separation and Purification

The recombinant gonophylphylline obtained in Examples 2 and 3 was purified using the following procedure. This procedure was used to purify the meningococcal recombinant pilin obtained in Example 4 and initially used to purify the meningococcal chimeric class I rpilin protein obtained in Example 5. Subsequently, the isolation procedure of meningococcal chimeric class I rpilin protein was modified as described in Example 8 below.

Sub-cell fractionation of E. coli expressing rphylline shows that the protein binds to the cell membrane, mostly the inner membrane, based on the ability of 1% (volume / volume) Triton ^™ X-100 to dissolve this protein. In an attempt to remove contaminating E. coli protein in the presence of 0.05-0.1% (volume / volume) Triton ^™ X-100, it was found that rphylline did not bind consistently with the ion exchange column below pH 9.5. Accordingly, the ability of multiple detergents to selectively dissolve rphylline protein of E. coli membrane preparations was investigated.

Cell pellets (approximately 5 g wet weight) in 1 L culture were thawed by addition of 30 mL of 10 mM Hepes (pH 7.2) (Research Organics, Cleveland, Ohio), 1 mM EDTA and microfluidizer cell mill (Microfluidics International Corp., USA). Massachusetts Newton) is used to destroy the cells. Lysates were clarified by centrifugation (12,000 xg for 10 minutes) and the membrane was pelleted (288,652 xg for 1 hour). The membrane was resuspended in 33 mL of 10 mM Hepes, pH 7.4, 1 mM MgCl ₂ and extracted for one hour at room temperature with one of the following detergents: (a) Triton ^™ X-100 (TX100) (Calbiochem-Novabiochem International, San Diego, CA) ), (b) reduced Triton ^™ X-100 (Calbiochem), (c) octyl-β-D-glucopyranoside (OG) (Calbiochem), (d) Zwittergent ^™ 3-8 (Z3-8) (Calbiochem ), (e) Zwittergent ^TM 3-10 (Z3-10) (Calbiochem), (f) Zwittergent ^TM 3-12 (Z3-12) (Calbiochem), (g) Zwittergent ^TM 3-14 (Z3-14) ( Calbiochem), (h) Empigen BB ^™ (Calbiochem) or (i) Tween ^™ 80 (ICN, Cleveland, Ohio).

^TM Empigen BB (1% vol / vol) Zwittergent ^TM 3-10 (1% weight / volume), reduced Triton ^TM X-100 (1% vol / vol), octyl-glucoside (1% wt / vol) Zwittergent + ^{Each of TM} 3-10 (1% weight / volume) or 3-14 (0.1% weight / volume) selectively extracts recombinant filin protein minimally contaminated with E. coli protein. Zwittergent ^™ 3-12 dissolves both recombinant protein and a significant amount of E. coli protein even at 0.1% (weight / volume). Tween ^TM 80 failed to extract the recombinant protein in the test concentration (0.1-1% vol / vol).

Lysed protein was separated from insoluble membrane material by centrifugation (288,652 × g for 1 hour). The supernatant (containing rphylline) was dialyzed overnight at 4 ° C. with 10 mM Tris ^™ (pH 8.5) containing one of the following non-ionic detergents: (a) 0.1% (weight / volume) Zwittergent ^™ 3 -14, (b) 1% (weight / volume) Zwittergent ^™ 3-10 or (c) 1% (weight / volume) OG. The dialyzed material was fractionated in Fractogel ^™ EMD TMAE-650 (S) (EM Separations Technology, Rhode Island Wakefield) and the column was equilibrated in 10 mM Tris ^™ (pH 8.5) and individual detergents. Bound proteins were eluted with a linear gradient of 0 to 0.2 M NaCl in 10 mM Tris ^™ (pH 8.5) containing the appropriate detergent. Fractions containing rphylline were pooled and analyzed for purity and protein content. Occasionally, to increase the purity of rphylline, the pooled material was dialyzed with starting buffer and a second fractionation was performed on a TMAE column.

Selectively eluted rphylline on the column was highly purified (> 90% uniformity) as determined by laser densitometry analysis of Coomassie blue stained SDS-PAGE. Similar results were obtained when extraction and column chromatography were performed using 1% (weight / volume) Zwittergent ^™ 3-10, 1% (weight / volume) OG or 0.1% (weight / volume) Zwittergent ^™ 3-14. lost. The yield of rphylline, a significant proportion of total E. coli protein, is approximately 10 mg / L culture grown in a 1.5 L stirred flask with SOB medium. When recombinant E. coli (containing pPX2002) was grown in a fermenter using HySoy ^™ based medium, the yield of purified rpilin increased to approximately 30 mg / L of culture, which resulted in a 7 mg rpilin / g cell mass. Corresponds to. When 1% dextrose was included in the fermentor, the overall yield of rphylline increased to approximately 100 mg / L.

Purified rphylline was dialyzed with 140 mM NaCl (pH 7) (PBS) containing 10 mM sodium phosphate, 0.05% (weight / volume) Z3-14, sterile filtered and then stored at 4 ° C or at -20 ° C. Frozen.

Example  8

this. From coli Meningococcal chimera  Class I r Filin's  Separation and Purification

Large-scale cultures of E. coli cells containing pPX2004 were grown in the Biostat B fermenter described in Example 2. Bacterial cells (approximately 88 grams wet weight of E. coli pPX2004) were resuspended in 440 mL of 10 mM Hepes, 1 mM EDTA, pH 7.5 and destroyed using Microfluidizer Model 110Y (Microfluidics Corp., Newton, Massachusetts). . The destroyed cells were purified by centrifugation at 6,084 xg for 20 minutes at 10 ° C. The supernatant was pooled and the membrane fractions were separated by centrifugation at 205,471 xg for 1 hour at 10 ° C. The pellet was triturated in 220 mL 10 mM Hepes, 1 mM MgCl ₂ , 1% (weight / volume) octyl-β-D-glucopyranoside (pH 7.5) and resuspended and stirred at room temperature for 90 minutes. The suspension was centrifuged at 205,471 xg for 1 hour at 10 ° C. After centrifugation, the supernatant containing the dissolved chimeric class I rphylline was filtered through a 0.22μ Nalgene vacuum filter and stored at 4 ° C. The pH of the octylglucoside extract was adjusted to pH 8.5 with concentrated NaOH and subsequently equilibrated with 25 mM Tris ^TM , 0.1% (weight / volume) Zwittergent ^TM 3-14 (pH 8.5) to 200 mL TMAE Fractogel ^TM column (EM Separations Technology, Gibbstown, NJ. Unbound protein was washed through the column with 400 mL of additional equilibration buffer. Rphylline using a straight NaCl gradient (0-0.2 M NaCl) at an elution rate of 10.0 mL / min over 10 column volumes in 25 mM Tris ^™ , 0.1% (weight / volume) Zwittergent ^™ 3-14, pH 8.5 Eluted. Fractions containing chimeric class I rphylline were pooled and diluted 1: 1 with dH ₂ O and 40 μm equilibrated with 10 mM NaPO ₄ , 0.1% (weight / volume) Zwittergent ^™ 3-14 (pH 6) It was loaded onto 100 mL of a ceramic hydroxyapatite column (Bio-Rad, Hecules, Calif.). Unbound protein was washed through the column with an additional 200 mL of equilibration buffer. The chimeric class rphylline was eluted at a dissolution rate of 5.0 mL / min over 10 column volumes using a straight NaPO ₄ gradient (10-150 mM NaPO ₄ ) containing 0.1% (weight / volume) Zwittergent ^™ 3-14. . Fractions were selected by SDS-PAGE analysis and those containing chimeric class I rphylline were pooled. The purified material is at least 95% pure as measured by a laser densitometer of Coomassie blue-stained gel. The yield of purified chimeric class I rphylline is approximately 35 mg / g wet weight cells.

Example  9

this. From coli Meningococcal chimera  Class II r Filin's  Separation and Purification

All steps are performed at room temperature unless otherwise specified. Frozen E. coli cells were resuspended in 10 ml of 10 mM Hepes (pH 7.2), 1 mM EDTA / g cells and ground using a Microfluidizer cell mill to disrupt cells. Cell lysates were clarified by centrifugation at 13,689 × g for 30 minutes. The resulting supernatant was centrifuged at 388,024 xg for 30 minutes at 4 ° C. The supernatant was discarded and the pellet containing the membrane was frozen overnight at -20 ° C. The membrane pellet was resuspended in 9 mL / 10 mM Hepes tube, pH 7.2, 1 mM MgCl ₂ and extracted with 1% (weight / volume) Zwittergent ^™ 3-16 (Calbiochem) for 1 hour. The suspension was centrifuged at 388,024 xg for 30 minutes and the resulting pellet was extracted again with Zwittergent ^™ 3-16 as described previously. After centrifugation (388,024 × g 30 min), the pellet was resuspended in 9 mL 50 mM Tris ^™ (pH 8.0), 5 mM EDTA and stirred at 1% (w / v) N-laurylsarcosyl (g / v) with gentle stirring overnight at room temperature. Sigma). This dissolved meningococcal chimeric class II rphylline. Insoluble material was removed by centrifugation (388,024 xg 30 min) and discarded. Zwittergent ^TM 3-14 was added to the supernatant containing meningococcal chimeric class II rphylline to produce a final concentration of 1% (weight / volume) and the material to be 50 mM Tris ^TM (pH 8.0), 10 mM EDTA, 1% Zwittergent ^TM Dialysis overnight at 3-14. The dialyzed material Aliquots (1 mL, 1.3 mg ^{protein) 50 mM Tris TM (pH 8.0} ), 10 mM EDTA, a Mono-Q ^TM equilibrated in 1% (weight / vol) Zwittergent 3-14 ^TM (Pharmacia, New Jersey Fitzkata Way) column (5 × 10 mm) was passed at a flow rate of 0.5 mL / min. Unbound material containing chimeric class II rphylline was pooled and dialyzed overnight with 10 mM NaPO ₄ (pH 6.8), 1% (weight / volume) Zwittergent ^™ 3-14. This material was approximately 80% pure and was used for the irradiation described in Examples 26 and 27 below. Further purification of this material was obtained by passing it over a 1 mL hydroxyapatite column (Bio-Rad) equilibrated in 10 mM NaPO ₄ (pH 6.8), 1% (weight / volume) Zwittergent ^™ 3-14. Purified meningococcal chimeric class II protein was eluted using a linear gradient of 0-0.5 M NaPO ₄ containing 1% (weight / volume) Zwittergent ^™ 3-14. Fractions were selected for chimeric class II rphylline by SDS-PAGE using gels stained with Coomassie blue or silver. Both analyzes showed the presence of a single polypeptide band with a molecular weight of approximately 19,000 Daltons. This material shows a purity of at least 95% by laser densitometer analysis of polyacrylamide gels.

Example  10

Gonococcus r Filin  Analytical Method

 Protein content was determined using BSA as a standard, by non-sinuconic acid protein analysis (Pierce, Rockford, Ill.). Purity of the protein preparation was measured by polyacrylamide gel electrophoresis (SDS-PAGE) stained with Coomassie Brilliant Blue in the presence of SDS and analyzed by laser densitometer using Personal Densitometer SI (Molecular Devices). The presence of filin in the preparation was confirmed by western blotting using monoclonal antibodies against pili purified from N. gonoria strain P9 (Biospacific) described in Example 2. The N-terminus sequence of the filin protein was determined using the Applied Biosystems 477A Protein Sequencer. Both sequences are often detected when purified rphylline is provided for N-termination sequence analysis. The predominant sequence represents the complete filin protein comprising seven amino acid leader sequences. The minor sequence comprising 10-20% of the sample is rpilin protein, which has no leader sequence and the sequence starts with phenylalanine, the N-terminating residue of the mature gonococcal pilin protein. For both rphylline protein forms, sequencing of amino-terminal residues gives results consistent with sequences derived from DNA sequences.

The mass of the recombinant filins was measured by matrix assisted laser desorption ionization time on a Flight (MALDI-TOF) mass spectrometer using a Finnagan MAT Lasermat ^™ 2000 (San Jose, Calif.). The instrument was calibrated with horse myoglobin to within 0.01% of the expected mass of 16,951.5 Daltons. Recombinant pilin was mixed with an equal amount of cyano-4-hydroxycinnamic acid matrix (10 mg / mL in 70:30 acetonitrile: 0.1% (volume / volume) trifluoroacetic acid / water). An aliquot (1 μL) of this mixture was placed on the sample stage, air dried and analyzed by MALDI-TOF mass spectrometer. The mass of rpilin was measured by averaging data from 15 runs (each run sums 10 shots). The molecular weight of rphylline (with signal) was found to be 18,001 Daltons, comparable to the expected mass of 17,981 Daltons based on amino acid content. A small peak of mass 17,232 daltons (mean) was detected in each lot. The difference between the molecular weights of the two forms of the recombinant filin (769 Daltons) is considered to be the loss of the initial 6 amino acids (Met Asp Thr Leu Gln Lys) (SEQ ID NO: 2, amino acids 1-6) of the leader sequence with a mass of 774 Daltons.

Rphylline of very different apparent molecular weights was determined by size exclusion column chromatography using analytical Superose ^™ 12 columns (Pharmacia, Fitzkataway, NJ) equilibrated in BPS containing 0.05% (weight / volume) Zwittergent ^™ 3-14. Got it. Under these conditions, the protein eluted at a position corresponding to a molecular weight of 68,899. This means that the recombinant protein is aggregated. However, elution of proteins from size exclusion columns can be greatly affected by the shape of the proteins. The results of the rate settling centrifugation experiments demonstrate that the recombinant filins have a molecular weight in solution of approximately 45,000 Daltons. In an attempt to remove the detergent (Zwittergent ^™ 3-14), the recombinant protein was dialyzed with PBS alone. The dialyzed recombinant protein appears to be soluble and was not pelleted by high speed centrifugation (122,000 xg for 1 hour). There was no attempt to demonstrate complete separation of the detergent from the recombinant protein. Analysis of the material by gel filtration in PBS shows that the protein has an apparent molecular weight of 452,349 Daltons. This means that additional aggregation was done. The number of subunits in the mass was not measured.

Since the detergent may not be completely removed from the sample, the protein may still be in micelles, so the value of 452,349 should be considered an estimate. If rpilin is diluted approximately 15-30 fold when formulated in vaccine form, rpilin in the vaccine is believed to have an apparent molecular weight of approximately 450 kD.

Example  11

r From Philin  Preparation of Immune Serum

Immunogenicity studies were performed using guinea pigs (females, 200 g) immunized subcutaneously with 20 μg of purified gonococcal rphylline protein mixed with adjuvant. Adjuvants studied were (a) Stimulon ^™ QS-21 (25 μg / dose) in PBS (pH 6); (b) aluminum phosphate (Lederle Laboratories, New York Pearl River, 100 μg / dose) in PBS (pH 7); Or (c) PBS alone (pH 7). Initially, animals were immunized at weeks 0, 4 and 8 and serum was obtained at weeks 0, 4, 6 and 10. Analysis of immune response over time demonstrates that the third vaccination at 8 weeks did not elevate the immune response, so later studies with recombinant filins concluded with blood collection at 6 weeks.

To investigate the ability of adjuvants to modulate the immune response of gonococcus rphylline, mice (females, 5 or 10 animals / groups grown for 8 weeks) were subcutaneously immunized with 1-10 μg purified protein at 0, 4 and 6 weeks and 0 Serum was obtained at weeks 4, 6 and 8. RPMI 1640 (75 μL) was dropped into the vagina, sucked 3-4 times and vaginal washing was performed at 8 weeks. The washes from each group were pooled together and 50 μl of bovine serum was added to each pool.

For meningococcal chimeric class I rpilin protein, mice were immunized only at 0 and 4 weeks and serum was obtained at 0, 4 and 6 weeks. For all rpilin parenteral immunogenicity studies in mice, the following adjuvants were investigated: (a) Stimulon ^™ QS-21 (25 μg / dose) in PBS (pH 6); (b) aluminum phosphate (100 μg / dose) in PBS (pH 7); (c) MPL ^™ (50 μg / dose) in PBS (pH 7); (d) aluminum phosphate (100 μg / dose) and MPL ^™ (50 μg / dose) in PBS (pH 7); Or PBS alone (pH 7).

For meningococcal chimeric class II rpilin protein, guinea pigs were immunized with 20 μg protein supplemented with Stimulon ^™ QS-21 (25 μg / dose) in PBS (pH 6) at 0 and 4 weeks only, and 0, 4 and Serum was obtained at 6 weeks.

The ability of recombinant expressed filins (gonococcal or meningococcal chimeric class I) to induce a mucosal immune response was determined by (a) 1 or 10 μg of gonococcus rphylline in 2.5 μl of saline with or without 1 μg of native cholera toxin, or (b ) 5 μg of chimeric class I rphylline diluted in 10 μl of PBS (pH 7) with or without 1 μg of mutant CT-CRM, E29H cholera toxin was evaluated by intranasal immunization with mice. Immunization at 0, 2 and 3 weeks.

Example  12

Gonococcus r Filin  For immune response Weston Blot  analysis

Purified gonococcal rphylline was used to immunize guinea pigs according to the protocol described in Example 11. Antisera derived from guinea pigs immunized with gonococcus rphylline was first analyzed by Western blot (data not shown). These blots recognize bands corresponding to filin in the complete cell lysate of gonococcal cells with filiform antisera against gonococcus rphylline; Explain that no staining is seen in the filial cell lysate from the same gonococcus.

Comparative data were obtained from the antiserum of guinea pigs immunized with gonococcal filin oligomer. Intact pili as previously described was dissociated to give pilin oligomer (4). In brief, this involved dialysis of intact pili with 37 mM sodium phosphate (pH 12) for 48 hours at 4 ° C., followed by dialysis with 50 mM Tris ^™ , 145 mM NaCl (pH 8.0). Filin oligomers were clarified by centrifugation (100,000 × g for 1 hour). After centrifugation, the filin oligomer remained in the supernatant. Compared to the antiserum against gonococcus rphylline, the pilin oligomer antiserum also bound to a number of other bands in lysates of both pili and non-pili cells while binding to pilin in pilindaline cell lysates (data not shown). . These bands represent contaminants in the pilin oligomer preparation and do not seem to bind to pili.

Example  13

Recombinant gonococcus Filin  ELISA

Endpoint titers on purified protein or bacterial cells were determined by ELISA. In all ELISA procedures, the incubation is for 1 hour at room temperature unless otherwise noted. The end point titer is defined as the dilution inferred at the optical absorbance of 0.10 times greater than that of the blank wells (which do not contain the primary antibody). For guinea pig antisera analysis, purified recombinant filin was diluted in 0.1 M Tris ^™ (pH 8) to make a final concentration of 1 μg / mL. Aliquots (100 μl) were added to wells of microtiter plates (Immulon II, Nunc, Napusville, Ill.) And incubated overnight at 4 ° C. Plates were washed five times with PBS containing 0.05% (volume / volume) Tween ^™ -20 (PBS-T) using a Skanwash 300 plate washer (Skatron Instruments, Alexandria Virginia). Block wells using 200 μl of 1% (weight / volume) BSA in PBS-T, wash and add aliquots of antiserum (diluted in 0.1% (weight / volume) BSA in PBS-T) to wells did. Plates were washed and conjugated with 1: 2000 dilution diluted rabbit anti-guinea pig IgG (heavy and light chain) (Zymed Laboratories, South San Francisco, CA) in 0.1% (weight / volume) BSA in 50 mM Tris ^™ (pH 8). The bound primary antibody was detected using 100 μl of alkaline phosphatase. Plates were washed and developed using 100 μl / p-nitrophenol phosphate wells (Sigma) (2 mg / mL in 0.5 M diethanolamine, 0.25 mM MgCl ₂ , pH 9.8). After 30 minutes, 50 μl of 3 N NaOH was added to stop the reaction. Absorbance was read at 405 nm in a Thermomax ELISA plate reader (Molecular Devices, Sunnyvale, CA).

As can be seen by the antigen ELISA shown in Table 1, all animals immunized with rphylline responded very well.

Table 1

Purified recombinant gonophyl fibrin protein, endpoint titer for binding of pooled guinea pig antiserum to gonococcus rphylline ^*

Manufacture Immunogen Terminal titer Week 0 4 Weeks 6 Weeks One r Pgh3-1 filin (manufacture 1)  ≤100 51,345 494,805 2 r Pgh3-1 filin (manufacture 2) 54 30,237 594,298 3 r Pgh3-1 filin (manufacture 3) ≤100 24,830 546,682 Three different lots of rphylline are used as immunogens. Immunized (subcutaneously) guinea pigs at weeks 0 and 4 and collecting blood at weeks 0, 4 and 6. Analysis was performed on pooled serum.

Effects of Supplements on Immune Responses

The effect of the following adjuvant on the immune response to gonococcus rphylline was investigated in mice: (a) Stimulon ^™ QS-21 in PBS (pH 6); (b) aluminum phosphate in PBS pH 7; (c) MPL ^™ in PBS (pH 7); (d) aluminum phosphate and MPL ^™ in PBS pH 7; Or PBS pH 7 alone. For mouse antiserum analysis, the antigen ELISA protocol was modified as follows. Microtiter plates (Costar EIA / RIA, Corning Costar, Mass.) Were coated with 100 μl of 1 μg / mL rphylline in PBS overnight at 37 ° C. Plates were washed five times with PBS containing 0.1% (volume / volume) Tween ^™ -20 with a Skantron 300 plate washer. Wells were blocked with PBS containing 0.1% (weight / volume) gelatin and 0.02% (weight / volume) NaN ₃ . Primary antibodies were diluted in PBS containing 0.1% (weight / volume) gelatin, 0.05% (volume / volume) Tween ^™ -20 (PBS-TG) and 0.02% (weight / volume) NaN ₃ and 100 μl aliquots The amount was incubated in microtiter plates for 2 hours. After washing, bound using PBS-TG and biotinylated rabbit anti-mouse IgG (Fc region) diluted 1: 8000 in 0.02% (weight / volume) NaN ₃ (Brookwood Biomedical, Alabama Birmingham). Primary antibody was detected. Plates were washed and secondary antibody was prepared using streptavidin conjugated horseradish peroxidase diluted 1: 5000 (Zymed Laboratories) in PBS-TG and 0.02% (weight / volume) NaN ₃ (incubation for 30 minutes). Detected. The plates were washed and 0.5 mg / mL 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) in 0.1 M citrate (pH 4.2) containing 0.03% (volume / volume) hydrogen peroxide (ABTS) was used for 30 min and then monitored at 405 nm using an SLT 340 ATTC microplate reader (SLT Labinstruments, North Carolina Research Triangle Park). The data were plotted using log-log plots and endpoint titers measured as described above.

When MPL ^™ was supplemented with rphylline, a humoral immune response similar in size to that shown for Stimulon ^™ QS-21 was obtained in mice (Table 2a). Vaginal lavage assays from the same animal also show clear IgG titers in vaginal lavage of these animals.

Table 2a

Effect of Adjuvant on Humoral Immune Response to Gonococcus r-Philin ^* :

Antigen ELISA Full Cell ELISA Homologous strains Heterologous strain Supplements rphyllin Pgh3-1 LB2 # 11 T-1 I756 AlPO ₄ 361,260 249,410 93,444 141,622 42,535 89,615 MPL ^TM 1,262,735 902,891 499,223 586,642 165,301 321,340 AlPO ₄ + MPL ^TM 1,159,670 265,269 112,856 160,814 41,368 110,162 Stimulon ^TM QS-21 4,915,200 1,053,446 478,338 440,014 111,753 333,341

Table 2b

Effect of Adjuvant on Immune Response to Gonococcus rphylline ^* (vaginal lavage ^** ):

Supplements rphyllin titer Full Cell Titer ^*** IgG IgA LB2 P + LB2 P- AlPO ₄ 1,452 262 68  ≤50 MPL ^TM 6,409 26 1,128 0 AlPO ₄ + MPL ^TM 938 12 120 ≤50 Stimulon ^TM QS-21 2,716 175 311 37 Balb / c mice (5 / group) were immunized with 10 μg of supplemented rpilin as described previously at weeks 0, 4 and 6. Animals are bled at 0, 4, 6 and 8 weeks. Analysis was performed on pooled serum at week 8. End point titer at week 0 is ≤5000. ** Pooled vaginal lavage obtained at 8 weeks. *** Whole cell ELISA using dried cells. P +: filiform cells. P-: unphilized strain derived from piliform hair cells.

Example  14

Gonococcal whole cell ELISA

Expression of rpilin of a significant number of cross-reactive epitopes found on intact, aggregated pili was later demonstrated in full cell ELISA, and antiserum against purified rpilin showed high titers against a number of gonococcal expressing heterologous pili. . The ability of guinea pig antisera to bind to live gonococcal cells was done using the following protocol. Microtiter plates (Immunolon II) were incubated overnight at 100 ° C. using 100 μl of 0.01% (weight / volume) poly-L-lysine (Sigma) in PBS. Overnight agar cultures of bacteria were collected with PBS using a Dacron swab and the turbidity of the suspension was adjusted to A ₆₀₀ = 1.0. The poly-lysine solution was discarded from the microtiter plate and 100 μl aliquots of bacterial suspension were added to the wells. Plates were washed five times with PBS using a hand-held Nunc plate washer and blocked with 200 μl PBS (PBS-B) containing 1% (weight / volume) BSA. Plates were washed five times with PBS and 100 μl aliquots of antisera diluted in 0.1% (weight / volume) BSA in PBS were added to the wells. After 5 washes with PBS, bound antiserum was detected using 100 μl of the appropriate alkaline phosphatase conjugate previously loaded at a 1: 2000 dilution in 0.1% (weight / volume) BSA in PBS. The plate was washed, developed and the absorbance was read (developed for 30 minutes) as previously described.

When analyzed by full cell ELISA, guinea pig antiserum against rphylline binds to piliform isolates of various geographic locations, but not to corresponding piliform cells of the same gonococcus (Table 3).

TABLE 3

Endpoint titer for binding of guinea pig antiserum to rphylline into live gonococcal cells of various strains ^*

Strain source Pgh3-1 rphylline (Product 1) Pgh3-1 rphylline (Product 2) Pgh3-1 rphilin (manufacture 3) Pgh 3-1 P + Pittsburgh 2,940,971 2,461,272 2,373,641 Pgh 3-1 p- Pittsburgh <250 <250 <250 LB2 P + Unknown 505,439 481,815 825,517 LB2 P- Unknown 187 34 114 I-756 P + Ontario Canada 689,309 713,904 1,026,231 1948 P + Romania 290,367 259,487 325,640 1635P P + Panama 2,714,006 ≥546,750 ≥546,750 22714FB P + North Carolina Fort Bragg 397,763 346,895 498,882 T-1 P + Unknown 189,490 317,370 272,828 M-2 P + Unknown 124,906 117,734 151,966 3138K P + Korea 220,730 179,127 584,044 N-2 P + Virginia Norfolk 350,210 337,232 435,164 J474B P + Jamaica 238,640 205,247 380,970 New Clinical Isolates 52407 P + Florida Tampa 598,124 890,678 778,607 # 11 P + Rochester, New York 214,673 292,358 385,985 FA1090: 2-57-U17 P + North Carolina Chapel Hill 1,364,126 2,379,267 3,791,263 2-42-U14 P + North Carolina Chapel Hill 1,449,757 1,964,641 11,619,267 Three different lots of rphylline were used as immunogens. Guinea pigs are immunized (subcutaneously) as described previously and then blood is drawn. Analyzed pooled serum of 6 week old blood. The endpoint titer of serum at week 0 was ≦ 250. P +: filiform cells. P-: unphilized strain derived from piliform hair cells.

Complete cell ELISA analysis of mouse antiserum against rphylline (Table 2) was performed using microtiter plates, where bacteria were dried in wells by the following protocol. Overnight agar cultures of bacteria were recovered in PBS using a Dacron swab and the turbidity of the suspension was adjusted to A ₆₀₀ = 0.1. An aliquot (100 μl) of bacterial suspension was added to the wells and the plate was air dried at 37 ° C. After all liquids were evaporated, the plates were sealed and stored at 4 ° C. until use. The remaining assay was performed according to the protocol described above for antigen ELISA of mouse antiserum. Data from whole cell ELISA (Tables 2 and 3) suggest that rphilin induces antibodies that attach to epitopes conserved on the surface of Philly gonococci.

Example  15

Gonococcus r Filin  Induction of mucosal immune response to

Because gonococcal is a genital mucosal disease, it is important to investigate the mucosal immunization ability to induce a mucosal immune response. This was done in the following way. Mice were immunized intranasally with gonococcal rphylline (10 μl to 1 or 10 μg) in saline with or without 1 μg native cholera toxin at 0, 1 and 2 weeks. Five Swiss-Webster mouse groups were immunized intranasally with rphylline with or without cholera toxin at weeks 0, 1 and 2. Analysis was performed on pooled serum. The endpoint titer at week 0 is <50.

As can be seen in Table 4 below, when the animal was immunized with rphylline in the absence of adjuvant, a significant immune response was detected in antigen ELISA (titer at week 0 was <300). This reaction was enhanced by the addition of native cholera toxin.

Table 4

Purified recombinant Gonophilin protein, an endpoint titer for binding of pooled mouse antiserum to gonococcus rphylline

Antigen (dose) Adjuvant (dose) 22 days 36 days 50 days r Fillin (1) r Fillin (10) r Fillin (1) r Fillin (10) None None None Cholera Toxin (1) Cholera Toxin (1) Cholera Toxin (1) 1,684 29,046 4,673 107,011 <300 1,287 20,658 7,526 321,714 <300 2,291 71,067 3,273 280,079 <300

These sera were then examined for their ability to bind intact, golied gonococcal cells by ELISA performed on N. gonoria strain FA1090 cells. The cells were dried on the microtiter plates described above. As can be seen in Table 5, low titers were detected for rphylline alone (titer at week 0 was <300). Binding to filiform cells was greatly enhanced by the addition of cholera toxin.

Table 5

Endpoint titer for binding of pooled mouse antiserum to gonococcus rphylline, in complete, filiform gonococcal cells

Antigen (dose) Adjuvant (dose) 22 days 36 days 50 days r Fillin (1) r Fillin (10) r Fillin (1) r Fillin (10) None None None Cholera Toxin (1) Cholera Toxin (1) Cholera Toxin (1) 1,009 6,009 1,133 209,522 239 852 6,252 1,456 57,767 <500 729 5,564 1,048 38,127 <500

Example  16

Gonococcus immune Microscopy

Visualization of the binding of antiserum to the Philly bacteria was performed using the following protocol. The gold-coated grid recA ^-. With, Philippe yen was Kono Ria in late log-phase liquid culture medium 5 minutes spot with aliquots of the boot and remove excess fluid with a filter paper. The grid was blocked for 5 minutes with PBS-B and then for 10 minutes with 1% (weight / volume) fish gelatin (Fluka, Ronconcoma, NY) in PBS. The grid was incubated with polyclonal antiserum diluted in PBS-B for 1-60 minutes at room temperature. Grids were floated on PBS-B droplets to remove unbound antibody (4 × 30 sec). The bound primary antibody was detected by floating a grid over one release of 12 nm colloidal gold bound to secondary anti-guinea pig IgG (Jackson Research Labs, West Grove, Penn.) Diluted 1: 5 in PBS-B for 30 minutes. The grid was washed five times with PBS-B droplets as described previously. Samples were stabilized with 1% (volume / volume) glutaraldehyde in PBS (Electron Microscopy Sciences, Fort Wash., Pennsylvania), rinsed in dilute water for 5 x 1 min and then NanoVan staining (NanoProbes, Stony Brook, NY) (pH 8 Lightly dyed for 30 seconds. The grid was contacted with filter paper to remove all liquid and examined with a Zeiss 10C transmission electron microscope at 15-75,000 magnification using an acceleration voltage of 80 kv.

As can be seen in immunoelectron microscopy (FIG. 1A), antibodies to recombinant pilin were bound along the length of the heterologous filament on the surface of the gonococcus. This suggests that the antibody binds to epitopes present on the bacterial surface in vivo.

Example  17

Gonococcus r Filin Oligomer  Full Cell ELISA

To distinguish the biochemical and immunological properties of rphylline (or phyllo oligomer) of intact pili, purified rpilin was converted to rpilin oligomer by dialysis with pH 12 phosphate buffer. Antisera induced by this substance (rphyllin oligomer) was investigated for the ability to bind live, piliform gonococcus using whole cell ELISA. As can be seen in Table 6, the antiserum against rpilin oligomers has a significantly lower endpoint titer that binds to various piliform gonococcal cells as compared to the antiserum induced by untreated rpilin. This suggests that rpilin oligomers have lost a significant number of cross-reactive epitopes commonly present on rpilin protein.

Table 6

Effect of pH 12 ("oligomerization") on the endpoint titer on rpilin guinea pig antiserum binding to filiform N. gonoria cells ^*

Strain rphyllin rPhilin oligomer ** I-756 927,564 16,903 FA-19 107,100 1,982 FA1090 (2-57-U17) 721,786 6,737 LB2 905,711 4,205 # 11 225,602 6,999 #4 288,120 8,104 3138K 106,315 5,429 T-1 497,987 42,162 1848 166,864 8,616 J474B 576,640 25,002 * Guinea pigs (4 per group) were immunized (subcutaneously) with 20 μg supplemented rphylline antigen with 25 μl Stimulon ^™ QS-21 at weeks 0 and 4. Analyzed pooled serum at 6 weeks. ** Purified rphylline was dialyzed at 37 ° C. with 37 mM sodium phosphate (pH 12) for 48 hours and then dialyzed with PBS for 24 hours.

Example  18

Inhibition of adhesion to human cervical cells

Since pili mediates the early binding of N. gonoria to human mucosal cells, the ability of rpilin antibodies to inhibit the adhesion of these bacteria to endothelial cells was investigated. ME-180 cells derived from cervical cancer were selected. In addition, in these experiments, they were grown in liquid suspension culture in order to minimize the agglomeration of filiform bacteria. This requires using recA ^- derivatives of gonococcal Pgh3-1 or I756 to maintain phyl expression. These recA ^- strains do not show significant mass during 4-5 hours growth in liquid culture to make the results easier to interpret.

In these experiments, ME-180 cells were seeded in 8 well chamber slides (Nunc) to allow cells to be 80-90% adherent on the day of the experiment. Overnight cultures of recA ^- gonococcal cells were swapped with PBS (warmed at 37 ° C) and used to inoculate flasks of liquid GC medium supplemented with 0.4% (weight / volume) NaHCO ₃ to a final A ₆₀₀ = 0.2. The cell culture is stirred at 37 ° C. at 120 RPM for approximately 4 hours, at which point the culture reaches approximately A ₆₀₀ = 800. The bacterial cell suspension was diluted 1: 8 in RPMI 1640. The wells of the secondary 8 well chamber were incubated with RPMI 1640 and fetal calf serum 300 μL for at least 1 hour. Discard RPMI 1640 block and add 40 μl of antiserum or RPMI 1640 (without calf serum), then add 260 μl of diluted bacterial suspension (= 8 × 10 ⁷ CFU) and 37 ° C./5% (volume / volume) CO Incubated at ₂ for 1 hour. Chamber well slides with ME180 cells were washed once with antibiotic-free RPMI 1640. Then, a precultured mixture of bacteria and antiserum was added onto the ME180 cells and the slides were incubated at 37 ° C. for 30 minutes. Medium containing unbound bacterial cells was removed from the cervical cell monolayer and the wells washed gently three times with RPMI 1640. After the last wash, the chamber wells were removed from the slides, cells were fixed in methanol for 30-60 seconds and stained with Wright-Giemsa staining (VWR Scientific, West Chester, Pennsylvania). After bleaching in water, coverslips were mounted on each well.

The slides were examined by light microscopy using an oil immersion and a person outside the test serum photographed a representative sight. The photo was analyzed by someone who did not know about the presence of the sample. In addition, since Philly gonococcus binds to endothelial cell monolayers as agglomerates, the effect of antisera on binding of gonococcal cells was quantified by counting bacterial agglomerates instead of individual bacteria. The number of piliform bacterial masses observed in 10 random ranges across each well was measured. The percent difference between wells containing immune and normal serum was measured. In addition, this analysis was performed independently by investigators who did not know about the sample to be analyzed.

Initial results show that pili cells and pili cells have different binding patterns for adhesion monolayers of ME180 cells. Two pili gonococci typically bound in the form of bacterial masses to selected endothelial cells within the adhesion monolayer. On the other hand, the corresponding isophilic Philobacteria did not bind to endothelial cell monolayer (Pgh3-1) or showed low levels of distributed binding (I756) to endothelial cell monolayer. Thus, the aggregation of bacteria on endothelial monolayer correlates with the presence of pili.

Next, the ability of guinea pig antisera against Pgh3-1 rphylline to inhibit the piliated cells of strain I-756 binds to ME-180 was tested. Representative photographic analysis (compare FIG. 2A (week 6) and FIG. 2B (week 0)) shows that the antibody against rphylline inhibits the binding of Philly gonococcal to cervical endothelial cells. In contrast, rpilin antiserum did not affect the binding of philly free cells of the same strain as compared to normal guinea pig serum (data not shown). Although the number of bacteria bound under these conditions could not be determined, adhesion force was quantified by counting the bound bacterial masses in the presence of normal or immune serum. Using this method, it was concluded that approximately 60% reduction in antiserum against rphylline occurred in the mass of bacteria bound to endothelial cells compared to normal guinea pig serum.

Although these assays do not provide easily quantifiable data, the estimates obtained by counting bacterial masses were within the range of measurements of the effect of antiserum. This is due to binding mediated by other cell surface components (eg Opa proteins) that cannot be overcome using antiserum against rphylline alone.

Example  19

Meningococcal chimera  Class I r Filin  Analytical Method

The analytical method described in Example 10 for the chimeric meningococcal class I rpilin was used. As measured by a MALDI-TOF mass spectrometer, the subunit molecular weight of meningococcal chimeric class I rpilin protein is 17,659 Daltons, which is very similar to the expected mass of 17,676 Daltons based on amino acid content. The protein was analyzed by size exclusion chromatography using a Superose ^™ 12 column equilibrated in PBS containing 0.05% (weight / volume) Zwittergent ^™ 3-14, and the chimeric class I rphylline had an apparent molecular weight of 69,480 Daltons. . This is essentially consistent with the apparent molecular weight (68,899 Daltons) of gonococcus rphylline analyzed under the same conditions.

When the N-terminus sequence of the purified Meningococcal Class I rpilin protein was determined by Edman degradation, the results (from three different samples) were consistent with the expected protein sequence. At least 35 residues were measured for all sequences.

Example  20

Meningococcal chimera  Class I r Filin  ELISA

Endpoint titers on purified protein or bacterial cells were measured by ELISA using the methods described in Examples 13 and 14. ELISA was performed using serum pooled from individual blood collections. Whole cell ELISA was performed on heat killed (56 ° C. 60 min) meningococcal cells or dried directly on monotiter plates. The cell suspension was diluted with an absorbance of 0.1 at 620 nm and 100 μl aliquots were placed in the wells of a microtiter plate. Each plate was dried at 37 ° C. or room temperature, sealed and stored at 4 ° C. until use. The protocol for whole cell ELIDA was modified as follows: (1) Primary and secondary antiserum were diluted in PBS containing 0.1% (volume / volume) Tween ^™ -20 and 0.1% (weight / volume) BSA and ; (2) Plates were washed five times with PBS containing 0.05% (volume / volume) Tween ^™ -20 using a Skanwash 300 plate washer.

As can be seen by the antigen ELISA shown in Table 7, all guinea pigs immunized with chimeric class I rpilin responded very well.

TABLE 7

Endpoint titer for binding of meningococcal chimeric class I rpilin antiserum to purified meningococcal chimeric class I rpilin ^*

Blood collection: Supplements: Stimulon ^TM QS-21 AlPO ₄ none Week 0 23 12 32 4 Weeks 26,607 12,067 4,829 6 Weeks 1,519,956 372,539 302,911 Guinea pigs (4 per group) (a) 25 μl Stimulon ^™ QS-21 in PBS (pH 6); (b) 100 μl of AlPO _{4 in} saline; Or (c) immunized 0 and 4 weeks (subcutaneously) with 20 μg of chimeric class I rpilin supplemented with PBS alone (pH 7) alone. Animals are bled at weeks 0, 4 and 6. Pooled blood is used for all assays.

Significant responses to piliform cells were confirmed by ELISA as shown in Table 8.

Table 8

Endpoint titer for binding of meningococcal chimeric class I rpilin to piliform cells of N.meningitidis (strain H355) ^*

Blood collection: Supplements: Stimulon ^TM QS-21 AlPO ₄ none Week 0 28 19 53 4 Weeks 1,293 1,052 381 6 Weeks 61,497 25,477 16,467 * Kill cells with heat.

Effects of Supplements on Immune Responses

The effect of the adjuvant on the immune response to meningococcal chimeric class I rphylline was investigated in mice using the method described in Example 13. As shown in Table 9, most important responses to the binding of antisera with meningococcal chimeric class I rpilin were achieved by the addition of Stimulon ^™ QS-21.

Table 9

Endpoint titer for binding of meningococcal chimeric class I rpilin antiserum to purified meningococcal chimeric class I rpilin ^*

Blood collection: Supplements: Stimulon ^TM QS-21 AlPO ₄ MPL ^TM AlPO ₄ / MPL ^TM none Week 0 <50 44 <50 31 <50 4 Weeks 44,011 29,925 40,146 110,093 <250 6 Weeks 707,084 103,437 284,455 137,686 115,022 * (10) 25 μl Stimulon ^™ QS-21 in PBS (pH 6); (b) 100 μl of AlPO _{4 in} saline; (c) 50 μg MPL ^™ in PBS (pH 7); (d) 100 μg and 50 μg MPL ^™ in AlPO ₄ in saline; Or (e) immunized at 0 and 4 weeks (subcutaneously) with 10 μg of meningococcal chimeric class I rpilin supplemented with PBS alone (pH 7) alone. Animals are bled at weeks 0, 4 and 6. Pooled blood is used for all assays.

As shown in Table 10, the most important response to the binding of antiserum to phyllocytes of N. meningitidis was achieved by the addition of Stimulon ^™ QS-21.

Table 10

Endogenous titers for binding of meningococcal chimeric class I rpilin antiserum to piliform cells of N.meningitidis (strain H355) ^*

Blood collection: Supplements: Stimulon ^TM QS-21 AlPO ₄ MPL ^TM AlPO ₄ / MPL ^TM none Week 0 172 146 153 160 <50 4 Weeks 6,088 2,310 4,205 5,647 311 6 Weeks 171,718 25,135 52,053 17,039 16,617 * Kill cells with heat.

Further analysis demonstrated that the antiserum against chimeric class I rphylline binds to meningococcal cells expressing class I filin or, in some cases, class II filin. The results shown in Table 11 demonstrate this partial cross-reactivity.

Table 11

End point titer for binding of meningococcal chimeric class I rpilin to piliform cells of N.meningitidis ^*

Strain Expressed Fillin Class 0 days Supplements Stimulon ^TM QS-21 AlPO ₄ MPL ^TM H355 M982 CDC1521 I I II 409 217 988 127,383> 36,540 2,602 41,190> 36,540 1,345 102,987> 36,540 1,768 FAM18 II 3,518 > 36,540 26,513 > 36,540 * Dry cells directly (at room temperature) on microtiter plates without killing the cells with heat.

Example  21

Meningococcal chimera  Class I r Filin  Immune electron Microscopy

Visualization of the binding of chimeric meningococcal class I rpilin antiserum to the filiform cells of N. meningitidis strain H355 was performed using transmission-electron microscopy as follows. Colonies of overnight cultures of N. meningitidis were carefully selected using a sterile loop and modified in a Franz medium [1.3 g / L glutamic acid, 20 mg / L cysteine, 10 g / L Na ₂ HPO ₄ .7H ₂ O, 90 mg / L KCl, 6 g / L NaCl, 2 g / L yeast dialysate and dextrose (4 g / L), glutamic acid (100 mg / L), cocarboxylase (200 μg / L) and iron nitrate ( Supplemented with 5 mg / L)] in a microfuge tube containing 0.5-1.0 mL. Gold plated grids were spotted with an aliquot of the cell suspension for 5 minutes and excess fluid was removed using filter paper. Bacterial cells were fixed at 4% (volume / volume) paraformaldehyde, 0.1% (volume / volume) glutaraldehyde in PBS for 30 minutes at room temperature. The grid was incubated in sequence using (a) PBS-B for 5 minutes, (b) PBS containing 1% (weight / volume) fish gelatin in PBS for 10 minutes, and (c) 0.2 M glycine for 5 minutes. Blocked grids were probed with antisera against the meningococcal chimeric class I rpilin protein described in Example 16. As shown in FIG. 3A, antibodies to meningococcal chimeric class I rpilin protein bound along the length of the pili. In contrast, normal serum (week 0) did not appear to bind to Phili (FIG. 3B).

Example  22

Meningococcal chimera  Class I r Filin  Antiserum and Gonococcus Philly  Run of cells cross Reactivity

Based on the sequence similarity between meningococcal class I fibrino and gonococcal pilin, antisera against gonococcus rphylline recognizes and binds to the meningococcal cells with Philly, and is shown in Example 4 above. In this example, it has been demonstrated that antiserum against meningococcal chimeric class I rpilin binds to gonococcal cells. Data from mouse and guinea pig experiments are summarized in Tables 12 and 13, respectively.

Table 12

Endpoint Titers for Binding of Mouse Antiserum to Meningococcal Chimera Class I rpilin with Philly D. Gonoria Cells

                  Antigen / adjuvant GC strain Nm r Class I Filin GC rphylline + MPL ^TM Stimulon ^TM QS-21 AlPO ₄ MPL ^TM AlPO ₄ / MPL ^TM none I756 4,527,943 79,927 114,958 56,627 57,426 356,936 FA1090 531,627 40,406 97,224 31,267 38,122 219,600

Table 13

Endpoint titer for binding of guinea pig antiserum to meningococcal chimera class I rpilin with filiform N. gonolia cells

                     Antigen / adjuvant GC strain Nm r Class I Filin GC pilrin r + Stimulon ^TM QS-21 Stimulon ^TM QS-21 AlPO ₄ none I756 301,969 122,714 78,111 46,424 FA1090 322,311 262,422 170,842 108,094

GC-N. gonoria; Nm-N. meningitidis

Example  23

Meningococcal chimera  Class I r Filin  By antiserum Meningococcal From bacteremia  Manual protection

A validated animal model evaluating the ability of a vaccine to protect against meningococcal bacteremia is the childhood rat model originally described by Saukkonen and Leinonen (34). The ability of guinea pig antisera against meningococcal chimeric class I rpilin (supplied with Stimulon ^™ QS-21) to protect against bacteremia caused by meningococcal expressing filins was tested. On day 0, Sprague-Dawley childhood rats (grown for 4-5 days) were treated with 0.1 mL of guinea pig antiserum (6 weeks) against chimeric class I rpilin diluted 1: 5, 1:10 or 1:20 in PBS ( Intraperitoneal) passive immunization. The control group was injected with 0.1 mL of normal guinea pig serum (week 0) diluted 1: 5 in PBS. After 24 hours, animals were intraperitoneally administered approximately 5 × 10 ⁵ colony forming units (cfu) to 0.1 mL of Phil. En. Meningitidis (strain H355). Three hours after dosing, animals were killed and cardiac blood samples diluted and placed on GC agar plates. Plates were incubated at 37 ° C. for 5% CO ₂ for 18-24 hours. Bacterial colonies were counted and levels of bacteremia were measured. One analysis of the variable t test was used to compare the control group to receive normal serum (week 0) and the group to receive immune serum (6 weeks). As shown in Table 14 below, animals passively immunized with guinea pig antiserum specific for meningococcal chimeric class I rpilin showed a log-normal reduction in the level of bacteremia compared to animals immunized with normal guinea pig serum. This difference is statistically significant with a p value of <0.05.

Table 14

Filippi with yen. Menin GT display (strain H355) is of guinea pig anti-serum for meningococcal class of chimera to prevent bacteria hypertriglyceridemia in rats administered childhood I r pilrin ability ^*

Blood collection Dilution Average cfu ± std Week 0 1: 5 4,87 ± 0.18 6 Weeks 1: 5 3.55 ± 0.48 ** 6 Weeks 1:10 3.63 ± 0.36 ** 6 Weeks 1:20 3.98 ± 0.75 ** Antiserum against meningococcal chimeric class I rpilin protein is obtained from the guinea pigs listed in Table 7. ** p <0.05. cfu ± std = colony forming unit ± standard deviation

Example  24

Meningococcal chimera  Class I r Filin  Induction of mucosal immune response to

 Immunization of mice with meningococcal chimeric class I rpilin (10 μl to 5 μg) in saline with or without 1 μg of cross-reactive mutant form of cholera toxin (CT-CRM, E29H) at 0, 1 and 2 weeks I was. As shown in Table 15 below, immunization of animals with rphylline in the absence of adjuvants detected a significant immune response in antigen ELISA. This reaction was enhanced by the addition of cholera toxin.

Table 15

Endpoint titer for binding of pooled mouse antiserum to meningococcal chimeric class I rpilin with purified recombinant meningococcal chimeric class I rpilin protein ^*

r Fillin, no supplement rphilin + CT-CRM Serum IgG IgA Bronchial Wash ** IgG IgA Nasal Wash ** IgG IgA Vaginal Wash ** IgG IgA  6,168 490 <10 <10 <10 12 174 15  1,181,871 3,940 580 19 98 236 70 687 Analyzed pooled serum at 4 weeks. Endpoint titers for pooled serum at week 0 for IgG and IgA were <50. ** Washing is performed as follows: Bronchial: Lungs are washed 5 times with 1 mL RPMI 1640, 50 μl fetal bovine serum (FBS) is added to the sample, purified by centrifugation (2,000 × g × 5 min) Store at 20 ° C. Nasal: Nasal wash was washed once with 0.5 mL RPMI 1640 and 20 μl of FBS was added to the sample and stored at -20 ° C. Vagina: Wash the vagina five times with 0.075 mL of RPMI 1640, add 10 μl of FBS to the sample and store at -20 ° C.

Example  25

Meningococcal chimera  Class I r Filin  Antisera Meningococcal From colonization  Proactive protection

The early stage of meningococcal disease in humans is colonization of the nasopharynx by bacteria. In this process, Philly seems to play an important role in mediating early adhesion to endothelial cells. Many investigators have described the process of colonizing nasopharynx in neonatal animals, but no one has investigated this as a model to test the efficacy of meningococcal vaccines (35). To evaluate the present invention described herein, a nasal colonization model of N. meningitidis was developed using an adult two-crossed mouse. Swiss-Webster mice were immunized with MPL ^™ subcutaneously assisted meningococcal chimeric class I rpilin at weeks 0, 4 and 8. At 10 weeks, animals were intraperitoneally injected with 2 mg iron dextran (Sigma) and intranasally administered with 10 μl of approximately 1 × 10 ⁷ cfu meso-logophylled meningococcal containing 40 μg iron dextran. . One day after dosing, an additional intraperitoneal injection of 2 mg iron dextran was added to half of the number of animals. The number of living bacteria in the nose was measured on days 1 and 2 after administration of plated nasal tissue grounds on GC agar plates containing selective antibiotics. The results are shown in Table 16.

Table 16

Number of live bacteria recovered from nasal crushes of mice administered N. meningitidis strain H355 (cfu) ^*

Antigen (dose μg) * cfu / nose 1 day** 2 days** H355 Complete Cells (25) Class I rPhilin (10) Saline 1,165 6,866 17,943 67 63 3,406 * All vaccines are formulated at 100 μg MPL ^™ / dose. Each group consists of 5 mice. ** days after intranasal administration

Example  26

Meningococcal  Class II chimera  r Filin  For immune response Weston Blot  analysis

 Purified meningococcal class II chimeric rpilin was used to immunize guinea pigs according to the protocol described in Example 11. Antisera derived from guinea pigs immunized with meningococcal class II chimeric rphyllin was first analyzed by Western blot (data not shown). These blots indicate that antiserum against meningococcal class II chimeric rphylline recognizes a band corresponding to pilin in complete cell lysates of piliform meningococcal cells expressing either class II filin (FAM18) or class I filin (H355). Shows. In contrast, antiserum against blood from E. coli containing only the pTrcHis vector did not react with filin bands in these western blots.

Example  27

Philly  Run Meningococcal disease  Into the cell Meningococcal chimera  Class II r Filin  Antiserum binding

Antisera derived from partially purified meningococcal chimeric class II rphylline was shown to bind to meningococcal cells of FAM18, a homologous strain with a titer> 36,450 (0 week titer is 473).

references

The present invention provides a vaccine composition comprising the recombinant pilin protein of N. gonoria or N. meningitidis.

<110> American Cyanamid Company <120> Vaccines Containing Recombinant Pilin Against Neisseria          Gonorrhoeae or Neisseria Meningitidis <130> 33377-00 / PCT <150> PCT / US 60 / 083,405 <151> 1998-04-29 <160> 24 <170> KopatentIn 1.71 <210> 1 <211> 504 <212> DNA <213> Unknown <220> <223> Aritificial Sequence <400> 1 atggataccc ttcaaaaagg ctttaccctt atcgagctga tgattgtgat cgccatcgtc 60 ggcattttgg cggcagtcgc ccttcccgcc taccaagact acaccgcccg cgcgcaagtt 120 tccgaagcca tccttttggc cgaaggtcaa aaatcagccg ttaccgagta ttacctgaat 180 cacggcgaat ggcccggcaa caacacttct gccggcgtgg catcttcttc aacaatcaaa 240 ggcaaatatg ttaaggaagt tacagtcgca aacggcgtca ttaccgccac aatgctttca 300 agcggcgtaa acaaagaaat ccaaggcaaa aaactctccc tgtgggccaa gcgtcaagac 360 ggttcggtaa aatggttctg cggacagccg gttacgcgca ccgacgccaa agccgacacc 420 gtcgccgccg ccgccaagac cgccgacaac atcaacacca agcacctgcc gtcaacctgc 480 cgcgacgcaa gtgatgccag ctaa 504 <210> 2 <211> 167 <212> PRT <213> Unknown <220> <223> Aritificial Sequence <400> 2 Met Asp Thr Leu Gln Lys Gly Phe Thr Leu Ile Glu Leu Met Ile Val   1 5 10 15 Ile Ala Ile Val Gly Ile Leu Ala Ala Val Ala Leu Pro Ala Tyr Gln              20 25 30 Asp Tyr Thr Ala Arg Ala Gln Val Ser Glu Ala Ile Leu Leu Ala Glu          35 40 45 Gly Gln Lys Ser Ala Val Thr Glu Tyr Tyr Leu Asn His Gly Glu Trp      50 55 60 Pro Gly Asn Asn Thr Ser Ala Gly Val Ala Ser Ser Ser Thr Ile Lys  65 70 75 80 Gly Lys Tyr Val Lys Glu Val Thr Val Ala Asn Gly Val Ile Thr Ala                  85 90 95 Thr Met Leu Ser Ser Gly Val Asn Lys Glu Ile Gln Gly Lys Lys Leu             100 105 110 Ser Leu Trp Ala Lys Arg Gln Asp Gly Ser Val Lys Trp Phe Cys Gly         115 120 125 Gln Pro Val Thr Arg Thr Asp Ala Lys Ala Asp Thr Val Ala Ala Ala     130 135 140 Ala Lys Thr Ala Asp Asn Ile Asn Thr Lys His Leu Pro Ser Thr Cys 145 150 155 160 Arg Asp Ala Ser Asp Ala Ser                 165 <210> 3 <211> 510 <212> DNA <213> Unknown <220> <223> Aritificial Sequence <400> 3 atggaagcaa tccaaaaagg tttcaccctg atcgagctga tgatcgtcat cgccatcgtc 60 ggtatcttgg cagccgtcgc cctgcccgca taccaagact acaccgcgcg cgcccaaatg 120 tccgaagccc tgactttggc agaaggtcaa aaatccgcag tgatcgagta ttattccgac 180 aacggcacat tcccgaacag caatacttcc gcaggtattg ctgcctctaa cgagattaaa 240 ggtaagtatg tggcatcggt taaggttgaa ggtaatgcct ctgttgcttc tattaccgct 300 accatgaact ctagtaatgt gaataaggac atcaaaggta aaaccttggt actcgtcggc 360 aaacaaaact ccggttcggt aaaatggttc tgcggacagc cggttacgcg cgacaacgcc 420 gacaacgacg acgtcaaaga cgccggcaac aacggcatcg aaaccaagca cctgccgtca 480 acctgccgcg atacgtcatc tgatgccaaa 510 <210> 4 <211> 170 <212> PRT <213> Unknown <220> <223> Aritificial Sequence <400> 4 Met Glu Ala Ile Gln Lys Gly Phe Thr Leu Ile Glu Leu Met Ile Val   1 5 10 15 Ile Ala Ile Val Gly Ile Leu Ala Ala Val Ala Leu Pro Ala Tyr Gln              20 25 30 Asp Tyr Thr Ala Arg Ala Gln Met Ser Glu Ala Leu Thr Leu Ala Glu          35 40 45 Gly Gln Lys Ser Ala Val Ile Glu Tyr Tyr Ser Asp Asn Gly Thr Phe      50 55 60 Pro Asn Ser Asn Thr Ser Ala Gly Ile Ala Ala Ser Asn Glu Ile Lys  65 70 75 80 Gly Lys Tyr Val Ala Ser Val Lys Val Glu Gly Asn Ala Ser Val Ala                  85 90 95 Ser Ile Thr Ala Thr Met Asn Ser Ser Asn Val Asn Lys Asp Ile Lys             100 105 110 Gly Lys Thr Leu Val Leu Val Gly Lys Gln Asn Ser Gly Ser Val Lys         115 120 125 Trp Phe Cys Gly Gln Pro Val Thr Arg Asp Asn Ala Asp Asn Asp Asp     130 135 140 Val Lys Asp Ala Gly Asn Asn Gly Ile Glu Thr Lys His Leu Pro Ser 145 150 155 160 Thr Cys Arg Asp Thr Ser Ser Asp Ala Lys                 165 170 <210> 5 <211> 30 <212> DNA <213> Neisseria gonorrhoeae <400> 5 ccccgcgcca tggataccct tcaaaaaggc 30 <210> 6 <211> 30 <212> DNA <213> Neisseria gonorrhoeae <400> 6 gggcctggat ccgtgggaaa tcacttaccg 30 <210> 7 <211> 29 <212> DNA <213> Neisseria gonorrhoeae <400> 7 ggctctagac tgtcagacca agtttactc 29 <210> 8 <211> 27 <212> DNA <213> Neisseria gonorrhoeae <400> 8 ggctctagat tgaagcattt atcaggg 27 <210> 9 <211> 28 <212> DNA <213> Neisseria gonorrhoeae <400> 9 ggctctagat aaacagtaat acaagggg 28 <210> 10 <211> 28 <212> DNA <213> Neisseria gonorrhoeae <400> 10 ggctctagat tagaaaaact catcgagc 28 <210> 11 <211> 36 <212> DNA <213> Neisseria Meningitidis Class I <400> 11 ccccgcgcca tggacaccct tcaaaaaggt tttacc 36 <210> 12 <211> 39 <212> DNA <213> Neisseria Meningitidis Class I <400> 12 gggcctggat ccgagtggcc gtggaaaatc acttaccgc 39 <210> 13 <211> 31 <212> DNA <213> Neisseria Meningitidis Class I <400> 13 ccggcgcgtc tctcacggcg aatggcccgg c 31 <210> 14 <211> 39 <212> DNA <213> Neisseria Meningitidis Class I <400> 14 gggcctggat ccgagtggcc gtggaaaatc acttaccgc 39 <210> 15 <211> 25 <212> DNA <213> Neisseria gonorrhoeae <400> 15 gcataattcg tgtcgctcaa ggcgc 25 <210> 16 <211> 34 <212> DNA <213> Neisseria gonorrhoeae <400> 16 gccgcgcgtc tcccgtgatt caggtaatac tcgg 34 <210> 17 <211> 25 <212> DNA <213> Neisseria Meningitidis Class I <400> 17 gcataattcg tgtcgctcaa ggcgc 25 <210> 18 <211> 39 <212> DNA <213> Neisseria Meningitidis Class I <400> 18 gggcctggat ccgagtggcc gtggaaaatc acttaccgc 39 <210> 19 <211> 37 <212> DNA <213> Neisseria Meningitidis Class II <400> 19 gcggccgcca tggaagcaat ccaaaaaggt ttcaccc 37 <210> 20 <211> 33 <212> DNA <213> Neisseria Meningitidis Class II <400> 20 gccgcgcgtc tccgaaccgg agttttgttt gcc 33 <210> 21 <211> 33 <212> DNA <213> Neisseria gonorrhoeae <400> 21 ccgggccgtc tcggttcggt aaaatggttc tgc 33 <210> 22 <211> 30 <212> DNA <213> Neisseria gonorrhoeae <400> 22 gggcctggat ccgtgggaaa tcacttaccg 30 <210> 23 <211> 37 <212> DNA <213> Neisseria Meningitidis Class II <400> 23 gcggccggat ccggtcattg tccttatttg gtgcggc 37 <210> 24 <211> 22 <212> PRT <213> Neisseria gonorrhoeae <400> 24 Glu Ala Ile Leu Leu Ala Glu Gly Gln Lys Ser Ala Val Thr Glu Tyr   1 5 10 15 Tyr Leu Asn His Gly Lys              20  

Claims (13)

Nigerian gonoria having an amino acid sequence of amino acids 1-170 of SEQ ID NO: 4 or a mature protein prior to processing, or having an amino acid sequence of amino acids 8-170 of SEQ ID NO: 4 after processing, or a biologically equivalent amino acid sequence And a chimeric recombinant pilin protein of class II Nigeria Meningitidis, Triggering a protective immune response in a human host, Vaccine composition for the treatment or prevention of Nigeria infection. Have, or are biologically equivalent to, the amino acid sequence of amino acids 1-170 of SEQ ID NO: 4 or a mature protein before processing under standard high resolution Southern hybridization conditions An isolated and purified DNA comprising a DNA sequence that hybridizes with a DNA sequence encoding a chimeric recombinant pilin protein of Nigerian gonoria and Class II nigerian meningitidis having an amino acid sequence. The isolated and purified DNA of claim 2, wherein the DNA sequence hybridizes to a DNA sequence having nucleotide sequences 1-510 or 22-510 of nucleotides SEQ ID NO: 3 under standard high resolution Southern hybridization conditions. Nigerian gonoria having an amino acid sequence of amino acids 1-170 of SEQ ID NO: 4 or a mature protein prior to processing, or having an amino acid sequence of amino acids 8-170 of SEQ ID NO: 4 after processing, or a biologically equivalent amino acid sequence And an isolated and purified DNA comprising a DNA sequence encoding a chimeric recombinant phyllin protein of class II nigeria meningitidis. Have, or are biologically equivalent to, the amino acid sequence of amino acids 1-170 of SEQ ID NO: 4 or a mature protein before processing under standard high resolution Southern hybridization conditions A plasmid containing isolated and purified DNA comprising a DNA sequence that hybridizes with a DNA sequence encoding a chimeric recombinant pilin protein of a Nigerian gonolia and a class II Nigerian meningitidis having an amino acid sequence. 6. The plasmid of claim 5, wherein the plasmid contains a DNA sequence that hybridizes to a DNA sequence having a nucleotide sequence of nucleotides 1-510 or 22-510 of SEQ ID NO: 3 under standard high resolution Southern hybridization conditions. A plasmid containing isolated and purified DNA encoding the chimeric recombinant pilin protein of the Nigerian gonolia and class II Nigerian meningitidis comprising the DNA of claim 4. 8. The plasmid of claim 7, wherein the plasmid is named pPX8017 (ATCC 207199). A host cell transformed with the plasmid of claim 5. 10. The host cell of claim 9, wherein the host cell is an Escherichia coli strain. The host cell of claim 10, wherein the plasmid is named pPX8017 (ATCC 207199). A chimeric recombinant pilin protein of Nigeria goniria and class II Nigerian meningitidis comprising transforming or transfecting the host cell with the plasmid of claim 5 and culturing the host cell under conditions in which the host cell expresses the chimeric recombinant pilin protein. Manufacturing method. Nigerian gonoria having an amino acid sequence of amino acids 1-170 of SEQ ID NO: 4 or a mature protein prior to processing, or having an amino acid sequence of amino acids 8-170 of SEQ ID NO: 4 after processing, or a biologically equivalent amino acid sequence And separately purified chimeric recombinant filin protein of class II Nigeria meningitidis.

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