JP2002527041A - Vaccine containing recombinant pilin against Neisseria gonorrhoeae or Neisseria meningitidis - Google Patents

Abstract

(57) each pil E gene Abstract: Neisseria gonorrhoeae (Neisseria gonorrhoeae) and meningococcal (Neiss ERIA meningitidis) was cloned and expressed their corresponding recombinant pilin proteins. Furthermore construct chimeric pil E gene, wherein the pilin protein amino - a region of pil E gene of terminal region encoding meningococcal (Neisseria meningitidis) class I, pil E of the corresponding Neisseria gonorrhoeae (Neisseria gonorrhea e) Replace with the region of the gene. Furthermore, to construct a chimeric p il E gene, wherein pilin protein carboxy - the region of the pil E gene of Neisseria meningitidis which encodes the end region (Neisseria meni ngitidis) class II, of the corresponding Neisseria gonorrhoeae (Neisseria gonorrhoeae) Replace with the pil E gene region. Recombinant pilin proteins used in vaccines to protect against diseases caused by gonococcal (Neisseriag onorrhoeae) or meningococcal (Neisseria meningitidi s).

Description

DETAILED DESCRIPTION OF THE INVENTION

TECHNICAL FIELD The present invention relates to Neisseria gonorrhoeae or Neisseria menin
gitidis ). The use of recombinant pilin proteins in vaccines to protect against diseases caused by gitidis ).

BACKGROUND Neisseria gonorrhoeae INVENTION (N. gonorrhoeae) and meningococcal (N. meningitidis) is a Gram-negative cocci. Neisseria gonorrhoeae (N. gonorrhoeae) and meningococcal (N. meningitidis) is are genetically very closely related, is very different clinical symptoms of the disease they cause. Neisseria gonorrhoeae (N. gonorrhoeae) causes gonorrhea, whereas Neisseria meningitidis (N.Meningitidi
s ) causes meningococcal meningitis. Such Neisseria bacteria inhibit the mucosal surface of the body.

[0003] Type IV pyridinium is, Jikerobakuta (Dichelobacter) (formally Bakutoroidesu (Bac
teroides)) Nodousu (nodous), Eikenera Korurodensu (Eikenella corro
dens), Kingera denitrificans (Kingella denitrificans), Morakusera bovis (Moraxella bovis), M. Rakunata (M.lacunata), M. Non-Riki tumefaciens (M.nonliquefaciens), Neisseria gonorrhoeae (N.gonorrhoeae), meninges Bacteria ( N.
meningitidis ) and numerous gram containing Pseudomonas aeruginosa-
Non-flagellated hair-like structure on the surface of negative bacteria (described in References 1 and 2). The toxin-derived pili derived from Vibrio cholera and the pili that form a bundle of the enteric pathogen Escherchia coli exhibit limited similarity to type IV pili, and It is considered more relevant (1, 2). For both N. gonorrhoeae (N. gonorrhoeae) and meningococcal (N. meningitidis), a variety of epithelial cells with pyridinium (pilliated) bacteria of human origin, no pyridinium (nonpiliated) higher binding than cells Actively attached, and thus it is believed that pili acts as a toxic factor by attaching microorganisms to the site of infection of the mucosa.

Type IV piri have a width of 5-7 nM and a length of 5 μM. The pilin protein subunits are connected in series to form long, thin polymers. In the case of the pathogen Neisseria, the pili are apparently homopolymeric in nature, a pilin protein consisting of a single structural subunit. This pilin protein is 13,000-
It has a molecular weight of 22,000 daltons (1, 2, 3).

[0005] In fact, pilin proteins Pirasu having a molecular weight of about ¹⁰⁷ daltons (pilus
) (Pyridinium: is a helical structure having a molecular weight of about ¹⁰⁷ daltons called plural) of Pirasu assembled in the outer membrane of the bacteria. When the purified pyr is dialyzed against a phosphate buffer at pH 12, the complete pyr is irreversibly dissociated into a condensate of pilin proteins called pilin oligomers (4). Such condensates of pyrin oligomers (approximately
(Molecular weight of 600,000 daltons) is much smaller in size than a perfect pilus.

[0006] Neisseria gonorrhoeae (N. gonorrhoeae) is first isolated and express a single pilin proteins are sequenced by Schoolnik and coworkers (5). The gonococcal pilin protein consists of three regions: (a) a highly conserved amino-terminal region (residues 1-53); (b) a central third (residue) that exhibits a limited amount of sequence variation. Bases 54 to 124)
And (c) 1/3 of the carboxy of the protein containing highly variable disulfide loops (residues 125-160).

[0007] During the natural process of infection, pili undergo a high frequency of phase and antigenic mutations (3,6).
). The genetic nature of this mutation is extremely complex and has been thoroughly studied. Each strain of Neisseria gonorrhoeae has the ability to alter the primary amino acid sequence of the pilin molecule, ie, to alter the antigenic nature of its pyri. The molecular mechanisms responsible for this mutation include the expressed locus ( pilE ) and numerous (17-19),
It involves non-reciprocal recombination between promoterless, silent ( pilS ) genes (6). The pilin sequences (or portions thereof) move from the pilS locus to the expressed locus, generating new pilin mutants, which in turn allow Neisseria gonorrhoeae to express a large number of pilin proteins.

[0008] In contrast to the Neisseria gonorrhoeae (N.gonorrhoeae), meningococcal (N.meningitidis) Class I
And express distinctly different classes of two pilins, designated class II.
As in N. gonorrhoeae, meningococcal class I piri undergo antigenic and phase mutations (
3). Class I pilins have been shown to be similar in molecular weight (17-20 kd) and reactivity with a monoclonal antibody (SM1) that binds to a highly conserved epitope on gonococcal pilins (3). A number of class I pilins have been cloned and the amino acid sequence has been shown to have a high degree of similarity to that of N. gonorrhoeae pilin (
7). In contrast, class II pilins do not react with the SM1 antibody and have a lower molecular weight (13-16 kd). Several strains expressing class II pilins have been shown to react with polyclonal antisera against gonococcal pili (3). Aho et al. (
7) Recently, the class II pilin was sequenced. The first one of Neisseria pilins
/ 3 are essentially the same. Class II pilin proteins differ from class I and gonococcal pilin proteins in the hypervariable region of the protein in which large deletions have occurred. Achtman et al. (8) demonstrated that some serogroup A isolated from Africa bound to both antibodies using monoclonal antibodies for class I or class II pilins. Strains expressing Class II pili also proved to have a shortened, silent Class I pilin gene (7). Taken together, such data suggest that a single meningococcal cell may express both pilin proteins simultaneously.

Since piri is believed to mediate initial contact with mucosal cells, the use of such structures as vaccine antigens to prevent diseases caused by pili-bearing bacteria is considerable. Interest has been raised. The piri vaccine was tested against traveler's diarrhea and gonorrhea in humans (9). But to date, they have been effective only against homologous strains. A number of vaccines based on pili have been used to detect infectious keratoconjunctivitis in cattle (infectious keratoconjunctivitis) (1), sheep foot rot (1, 10) and diarrhea in piglets (9) or calves (9). Diseases affecting healthy livestock were reported. In each such veterinary example, the pili vaccine provided protection against challenge by strains expressing homologous pili but not heterologous pili.

The first gonococcal vaccines contained whole organisms and protected little or no (11). Recent vaccine development against Neisseria gonorrhoeae has focused on purified surface components, particularly pyri (9,11) and porin protein (PI or Por) (11). However, to date, only Pili protected humans from challenge, but this defense was limited to protection against homologous strains (12). Degeneration state of gonococcal piri (4
) Has been shown to produce antibodies that bind heterologous pili in vitro in mice and guinea pigs. However, this was not considered a valuable method for mass sale because it was difficult to grow spicy gonococci in liquid media (necessary for industrial production) (1). Based on the success of the first human challenge experiment, the gonococcal piri vaccine was tested for efficacy in a large, placebo-controlled, double-blind study (12).
In this study, the vaccine failed to protect male volunteers infected with gonorrhea. This explanation has been postulated to be approximately the Helas heterogeneity (12).

Indeed, in both Neisseria gonorrhoeae and Neisseria meningitidis, antigenic variability of the pilin protein has been repeatedly stated to be a major obstacle in the development of pil-based vaccines (3
, 13, 14, 15, 16, 17). The dominant immune epitope on the assembled complete pilus is the disulfide loop, which represents the largest sequence variation (17, 18).
. This may be the cause of failure of field trials in Korea performed using formalin-treated whole pili from Pgh3-2 of N. gonorrhoeae (12). Furthermore, the technical literature states that a number of antisera to purified pyri or pyrin fragments bound to denatured (Western blot) or isolated Neisseria pyri but not to heterologous pyri on bacterial surfaces. Including literature (16, 17, 1
9, 20). Whether this was an antigenic mutation or the concealment of an epitope in assembled pilas was not completely elucidated. This was further confirmed by several reports demonstrating that only monoclonal antibodies displaying functional activity in vitro did not bind to heterologous pyri (3). M. bov
is ) and complete pili from D. nodosus have been shown to be capable of a protective immune response induced by pili (10). However, such vaccines could only protect against challenge with bacteria expressing homologous, but not heterologous, pili (10, 21). Consensus in the scientific community suggests that, where possible, pili-based vaccines will only protect against bacteria that express homologous pili.

[0012] Recombinant expression of assembled pili has been described for a number of microorganisms and depends on proper transport and the presence of assembled genes (22). In Neisseria, this approach is not feasible because genes encoding proteins involved in pili assembly and export are not found in a single, contiguous operon. An alternative method selected in EP 202,260 is to use a different type IV pili (eg Pseudomonas aeruginosa (
Pseudomonas aeruginosa )) was to express the type IV pilin gene in a bacterial host already possessing the proteins required for assembly (23). However, as reported by Hoyne et al., The expression and assembly of gonococcal piri in the outer membrane of Pseudomonas was unstable (24). When the recombinant strain was grown in liquid medium in the presence of selective antibiotics, wild-type, piliated Pseudomonas overgrown. The authors stated: "The suitability of exogenous mePhe [N-methylphenylalanine] pyrus production in a host strain will depend on the degree of divergent evolution of the host and the donor's pilus assembly system. PAK / 2PfS expressing P. gonorrhoeae observed [ Pseudomonas aeruginosa ]
The instability of mePhe piri may have been achieved in this example, as was the limitation of interspecific expression of mePhe piri. (24).

From these results, the expression of gonococcal piri in Pseudomonas is
It was not seen as an industrially valuable initiative.

[0014] Elleman, Egerton and co-workers have described Dicheloba Nodosas.
The development of a vaccine against sheep foot-and-mouth disease was described using whole pili from Cter nodosus ). In field tests, complete pili protected against D. nodosus, which expressed homologous pili . This meant that large-scale use vaccines would need to contain eight or nine different pili to achieve broad protection. For this approach to succeed, the pilin gene of D. nodosus was cloned and expressed in E. coli (25). The recombinant pilin protein was found to be at the inner membrane.
When a vaccine consisting of sonicated E. coli cells expressing recombinant pilin was tested in a challenge experiment, recombinant E. coli cells were found to be antibodies found in purified native pili. Antibody titers similar to the titers were generated (25). However, the agglutination titers induced by the recombinant E. coli cell vaccine were significantly lower than those seen for intact pili (690 vs. 47,800) and protection-related titers (5,000-5,000). 10,000) (25, 26).

After active challenge with D. nodosus , the recombinant pilin vaccine can show significant protective activity, in contrast to that seen for complete pili Did not. Emery and co-workers have shown that complete pili degeneration disrupts the ability of pili to induce protection in animals (27). In addition, pyri dissociated, either with detergents (octyl-β-D-glucoside) or at low pH (2.2), allow proteins to induce protection against the formation of severe damage following challenge. Potency was reduced (28). This was despite the fact that there were no significant differences in antibody titers between the groups. The authors stated that "there may be one or more epitopes associated with quaternary structures that are destroyed by processing." They further state: "The failure of the product expressed by E. coli as a vaccine is due to the fact that the product is physically sequestered within the host cell membrane, but in preliminary experiments, It was shown that this was not the main cause of non-efficacy (not published data) .Another explanation for the failure of the product expressed by E. coli as a vaccine was that The prepirin unit may not be able to associate with the natural configuration for proper presentation of key epitopes, presumably due to the presence of the leader sequence. "(28) Elleman and coworkers reported that a conformational epitope on the recombinant pyri was present. Confirmed the importance of the presence of, and that they would need to raise the immune response by better presentation of the antigen. They have proposed two methods: to purify proteins from E. coli membranes so that they can assemble into filamentous pili on the surface of cells, or to use P. aeruginosa ( P. aeruginosa ). The latter method was preferred because "the immunogenicity should be greatly improved and the purification of the protein should be simple" (25).

[0016] Furthermore, Elleman and coworkers also considered the use of pilin (a subunit protein of pili) as a vaccine candidate inferior to mature fimbriae (complete pili ) for D. nodosus : Intact fimbries appear to cause more potent and adequate (ie, K-aggregation) than equal doses of fimbrial-like subunit proteins. Generally B. nodosus
Is considered to be sufficient protective immunity against infection with the given strain and a minimum response of the same standard. The level of this response (and to a larger dimension) is easily achieved with vaccination with mature fimbriae, but isolated subunits that induce only a few poor levels of serum K-aggregated antibodies. Not achieved with proteins. (23).

Further data suggests that Alves et al. Recently reported that the subunit protein of fimbriae (Pillar) is not a valuable vaccine candidate (29). When mice are immunized with a polynucleotide vaccine encoding E. coli CFA / I fimbrial adhesion protein (eg, pilin), the antibodies induced are those derived from natural, intact CFA / I fimbriae. And obviously different. Furthermore, antibodies to the recombinant protein did not exhibit agglutinating activity, in contrast to antisera to the native protein.

[0018] Despite all the studies described above, effective Pilas-based gonococcal or meningococcal vaccines should be developed. Meningococcal vaccines are limited to those that possess the serotype A, C, Y, W135 capsular.

Therefore, there is a need to identify components to be included in a vaccine to protect against diseases caused by all serotypes of N. gonorrhoeae or N. meningitidis .

[0020] The purpose of summary the present invention as described above the invention is that each identify suitable antigenic structures derived from Neisseria gonorrhoeae (N. gonorrhoeae) or N. meningitidis (N. meningitidis), which are the A vaccine candidate capable of such bacteria can be constructed. Such candidates recognize various isolates of each pathogenic Neisseria microorganism,
And it must induce antibodies to bind.

These and other objects of the present invention, as described below, are directed to each of the gonococci ( N. gon
orrhoeae ) or the recombinant pilin protein (r-pilin) of N. meningitidis .

The present invention also relates to a class I meningococcus pilin protein having an amino-terminal region,
The present invention relates to the construction of a plasmid expressing a class I pilin protein of a recombinant meningococcal chimera which has been replaced by the corresponding amino-terminal region of the gonococcal pilin protein. This plasmid expresses significantly higher amounts of the meningococcal chimeric class I r-pilin protein than the class I meningococcal r-pilin protein expressed from the full-length meningococcal pilE gene.

To obtain the expression of the class I r pilin protein of the meningococcal chimera, the chimera D
The NA sequence is first inserted into a suitable plasmid vector. The appropriate host cells are then transformed or transfected with the plasmid. In an embodiment of the invention, the host cell is an Escherichia coli strain. The host cell is then cultured under conditions that allow the host cell to express the chimeric class Ir pilin protein.

Further, the present invention relates to a recombinant meningococcal chimera wherein the carboxy-terminal region of the class II meningococcal pilin protein is replaced by the corresponding carboxy-terminal region of the gonococcal pilin protein. And a plasmid expressing the class II pilin protein of the present invention.

To obtain expression of the class II r-pilin protein of the meningococcal chimera, the chimera D
The NA sequence is first inserted into a suitable plasmid vector. The appropriate host cells are then transformed or transfected with the plasmid. In an embodiment of the invention, the host cell is an Escherichia coli strain. The host cell is then cultured under conditions that allow the host cell to express the chimeric class II rpilin protein.

In another aspect of the invention, the isolated and purified r-pilin protein (G. gonorrhoeae,
Meningococcal or chimeric) to prepare a vaccine composition that induces 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 (
Trademark), Stimulon (TM) QS-21, IL-21 and cholera toxin. The vaccine composition is administered to a mammalian host in an amount that is immunogenic enough to protect the host against diseases caused by N. gonorrhoeae or N. meningitidis .

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a vaccine composition comprising a recombinant pilin protein of N. gonorrhoeae or N. meningitidis . Despite the teachings of the techniques discussed above, it was decided to investigate the use of such a recombinant pilin protein expressed in E. coli . Surprisingly, such recombinant pilin proteins have shown properties as vaccine candidates.

The first report describing the cloning of the gonococcal pilE gene in E. coli was made in 1982 (30). The molecular properties of pilE were then determined in a number of laboratories, examining the genetic factors controlling pilin protein expression, pilin protein transport, pilin sequence mutations, and host adhesion properties of pilin. However, there are no reports describing the recombinant pilin protein and the immune response of the recombinantly expressed pilin protein.

The cloning and expression of the pilE gene encoding the recombinant gonococcal pilin protein is described in Example 2 below. Expression was expressed in E. coli ( E.
coli ) strain was transformed with a plasmid containing the pilE gene. Following successful cloning and expression, sequencing with the pilE gene confirmed identity with the native sequence. To aid in cloning, an NcoI site was introduced, which required a single base modification. As a result, the second amino acid in the seven amino acid long signal peptide was changed from asparagine to aspartic acid.

In Example 2, the plasmid containing the pilE gene (designated pPX2000) contains an ampicillin resistance (Amp ^R ) marker. As described in Example 3, another plasmid was constructed to contain kanamycin resistance (Kan ^R ) instead of Amp ^R. This pP
After transformation with the E. coli TOP10F 'strain, the plasmid designated X2002, expressed r. Pyrin of Neisseria gonorrhoeae at the same level as obtained from pPX2000 containing the Amp ^R marker.

As described in Example 4, using similar procedures, N. meningitidi
A plasmid designated pPX2003 containing the class I pilE gene of s ) was constructed. Nco
An I site (CC ATG G) was introduced to extend the beginning of the gene encoding the signal peptide. This changed the second amino acid residue of the signal peptide from asparagine to aspartic acid (first residue left methionine). Amp ^R marker was also included.
This construct was transformed with the E. coli TOP10F 'strain, and
r-pilin was expressed. However, the expression level was significantly lower than that of gonococcal r-pilin obtained from pPX2000 or pPX2002. Without being bound by theory, this low expression level may be due to the number of inverted repeats present in recombinant class I pilE .

To increase the expression of N. meningitidis pilin, a chimeric plasmid was constructed as described in Example 5. The DNA in pPX2003, encoding the first 60 amino acids of meningococcal class Ir lipin, is replaced with an equivalent region from gonorrhoeae DNA in pPX2002. The resulting Amp ^R plasmid named pPX2004 has the sequence number 1
Has the nucleotide sequence described in Plasmid pPX2004 was used to transform E. coli K12 strain designated TOP10F '. After the introduction, the expression of chimeric r-pilin was significantly increased compared to the amount of r-pilin of N. meningitidis expressed from pPX2003. The expression level of the chimeric construct was comparable to the amount of gonorrhoeae r-pilin expressed from pPX2002. The chimeric class Ir pilin is 167 amino acids long (including the signal)
(SEQ ID NO: 2), which is consistent with the expected size.

A sample of the E. coli K12 strain designated TOP10F ′ harboring the recombinant plasmid pPX2004 was prepared by the applicant on January 27, 1998, USA, 20110-2209, Virginia, USA.
Manassas, Deposited at the American Type Culture Collection at Bolevart University 10801, and assigned ATCC deposit number ATCC98637.

As described in Example 6, a chimeric plasmid in which the 3 ′ end of the class II pilE gene of N. meningitidis was replaced with a corresponding region derived from N. gonorrhoeae was used. It was constructed. Specifically encodes a disulfide loop
DNA in pPX8001 ( N. meningitidis FAM18 strain Meningococcus class I
The last 22 amino acids of I-pilin) into a similar (but larger) region with additional pPX
Replace with a portion of the carboxy-terminal region of a total of 44 amino acids from the N. gonorrhoeae ( pilE ) DNA from N. gonorrhoeae strain Pgh3-1 in 2000. The resulting Amp ^R plasmid, designated pPX8017, has the nucleotide sequence set forth in SEQ ID NO: 3, wherein nucleotides 1-378 are derived from N. meningitidis class II and nucleotides 379-378. 510 is derived from N. gonorrhoeae . Plasmid pPX8017 was used to transform E. coli K12 strain designated TOP10F '. After introduction, a chimeric Class II r-pilin of 170 amino acids long (including a signal of 7 amino acids in length) (SEQ ID NO: 4) was expressed,
~ 126 is derived from N. meningitidis class II, and amino acids 127-1
70 is derived from N. gonorrhoeae . This chimeric IIr pilin was consistent with the expected size. An Nco I site was introduced for cloning considerations. This change that changes the second amino acid of the signal sequence from lysine to glutamic acid was not expected to have any effect on antigenicity or immunogenicity.

A sample of the E. coli K12 strain designated TOP10F ′ harboring the recombinant plasmid pPX8017 was filed by the applicant on April 15, 1999, USA, 20110-2209, Virginia, USA.
Manassas, deposited at the American Type Culture Collection at Bolevert University 10801, and assigned ATCC deposit number ATCC 207199.

Various host cell-vector systems, in addition to those detailed in Examples 2-6, for expressing gonococcal, meningococcal, and chimeric r-pilins for use in the vaccines of the invention Suitable for use. This vector system is compatible with the host cell used. Suitable host cells include bacteria transformed with plasmid DNA, cosmid DNA or bacteriophage DNA; viruses such as vaccinia virus and adenovirus; yeasts such as Pichia cells; insects such as Sf9 or Sf21 cells. Cells; or mammalian cell lines, such as Chinese hamster oocytes; as well as other common microorganisms.

A variety of customary transcription and translation elements can be used in a host cell-vector system. When the plasmid vector is inserted into a host cell, the pilE DNA as a host cell can express, insert the pilE DNA into an expression system, and linking the promoter and other control elements to a specific site in the vector.

The plasmid is introduced into the host cell by transformation, transduction, transfection or infection, depending on the host-vector system used. The host cell is then cultured under conditions that allow expression of the r-pilin protein by the host cell.

The present invention further encodes a class Ir pilin protein of a meningococcal chimera wherein the amino-terminal region is derived from the Neisseria gonorrhoeae pilE gene and the central and carboxy-regions are derived from the Neisseria meningitidis pilE gene. Isolated and purified DNA sequence comprising the DNA sequence (SEQ ID NO: 1). Nucleotides 1 to 501 of SEQ ID NO: 1
Encodes the meningococcal chimera class Ir pilin protein before processing; nucleotides 22-501 encode the meningococcal chimera class Ir pilin protein after processing to the mature protein. Further, the present invention relates to a meningococcal chimera having the amino acid sequence of amino acids 1 to 167 of SEQ ID NO: 2 before processing or having the amino acid sequence of amino acids 8 to 167 of SEQ ID NO: 2 after processing into a mature protein. Related to the class Ir pilin protein. Approximately 10% of the total protein produced by the Neisseria gonorrhoeae r-pilin or the meningococcal chimera class Ir pilin construct lacked the signal sequence, which was removed by processing.

The present invention further provides a class II r pilin protein of a meningococcal chimera in which the carboxy-terminal region is derived from the Neisseria gonorrhoeae pilE gene and the central and amino-terminal regions are derived from the Neisseria meningitidis pilE gene. For an isolated and purified DNA sequence comprising the encoding DNA sequence (SEQ ID NO: 3). Nucleotides 1 to 3 of SEQ ID NO: 3
510 encodes the pre-processing meningococcal chimera class II r pilin protein; nucleotides 22-510 encodes the meningococcal chimera class II r pilin protein after processing to the mature protein. Further, the present invention relates to a meningococcal chimera having the amino acid sequence of amino acids 1 to 170 of SEQ ID NO: 4 before processing or having the amino acid sequence of amino acids 8 to 170 of SEQ ID NO: 4 after processing to a mature protein. Related to the class II r pilin protein.

Meningococcal chimera class I pilin protein and meningococcal chimera class I
In addition to the chimeric DNA sequences contained in pPX2004 and pPX8017, which respectively encode the Ir pilin protein, the present invention further includes DNA sequences that are biologically equivalent to the sequence encoding the chimeric r pilin protein due to the redundancy of the genetic code. Consisting of
That is, such other DNA sequences differ from the nucleotide sequences described herein, but are characterized by a nucleotide sequence that encodes a protein having the amino acid sequence encoded by the DNA sequence of SEQ ID NO: 1 or SEQ ID NO: 3. is there.

In particular, the present invention is directed to hybridizing with the sequence of SEQ ID NO: 1 or SEQ ID NO: 3 under standard high stringency Southern hybridization conditions as described by Sambrook et al. DNA sequences that sufficiently overlap the sequence of SEQ ID NO: 1 or SEQ ID NO: 3 are contemplated (31).

The present invention also encodes an amino acid sequence that is different from the class I or class II r-pilin protein of the N. meningitidis chimera, and one of those described for such a protein (SEQ ID NO: 2 or SEQ ID NO: 4) It also comprises a DNA sequence that encodes a biologically equivalent amino acid sequence. Such amino acid sequences may be biologically modified if their sequences are only altered by small deletions, insertions or substitutions of the r-pilin sequence such that the quaternary arrangement of the sequence is essentially unchanged from the r-pilin protein. Specifically, it can be said that it is equivalent to such a chimeric r-pilin protein.

For example, the amino acid alanine, a codon for a hydrophobic amino acid, may be a codon encoding another less hydrophobic residue, such as glycine, or a more hydrophobic residue, such as valine, leucine or isoleucine. Can be replaced by Similarly, changing one negatively charged residue to another, for example, changing aspartic acid to glutamic acid, or changing one positively charged residue to another, for example, lysine to arginine. The resulting changes, as well as changes based on residue similarity in their hydropathic indices, are expected to produce biologically equivalent products. Nucleotide changes that result in alteration of the N-terminal or C-terminal portion of the protein molecule are not expected to alter the activity of the protein.

Furthermore, changes in known variable regions are known to be biologically equivalent if the quaternary configuration of the conserved region is not substantially altered from the r-pilin protein. Another definition of a biologically equivalent sequence is a sequence capable of producing a cross-reactive immune response. In particular, the class I and class II recombinant pilins of the meningococcal chimera are the corresponding inserts from the gonococcal pilin, as long as the modified chimeric recombinant pilin can still produce a cross-reactive immune response. May be modified by lengthening or shortening.

Each proposed modification is within the routine skill in the art, such as measuring the retention of structural or biological activity of the encoded product. Accordingly, the term "meningococcal chimera class Ir pilin protein" or "meningococcal chimera class II r pilin protein" as used in the description or in the claims, refers to all such biology It will be understood that it encompasses modifications and mutations that result in the production of highly equivalent proteins.

As described in Example 7, the gonococcal r-pilin protein is associated with the cell membrane of E. coli used to express it. EmpigenTM BB, Triton
X-100, reduced TritonTM X-100, octyl-β-D-glucopyranoside (OG)
, Zwittergent ™ 3-10 or 3-14, various surfactants have been used in E. coli (E. col.
The r-pilin protein derived from i ) can be selectively solubilized.

After centrifugation, dialysis and fractionation on the column, purified r-pilin is obtained.

As described in Example 8, chimeric class Ir pilins were obtained from E. coli .
Was isolated, purified by centrifugation, clarification by centrifugation, filtration and fractionation on two columns.

As described in Example 9, the class II r-pilin of the meningococcal chimera was E. coli (
E. coli ), isolated by clarification by centrifugation, filtration and fractionation on two columns and purified.

The purified gonococcal r-pilin was subjected to repeated N-terminal sequencing as described in Example 10. Sequencing of the 20-40 amino-terminal residues is DNA
The results were consistent with the amino acid sequence deduced from the sequence. The molecular weight of r-pilin (including the signal) was determined by mass spectrometry to be 18,006 daltons, which is in good agreement with the expected mass of 17,981 daltons based on amino acid content. In contrast, when r-pilin was subjected to size exclusion column chromatography, 68
An apparent molecular weight of, 899 daltons was obtained. This suggested that r-pilin was aggregated. In the dialysis of r-pyrine against PBS to remove detergent,
This resulted in a material having an apparent molecular weight of 452,349 daltons as determined by gel filtration. This indicated that it had undergone further aggregation.

As described in detail in Example 11, immune sera are obtained by immunizing guinea pigs or mice with purified r-pyrin of Neisseria gonorrhoeae. As described in Example 12, antiserum against r-pilin bound to whole cell lysates purified from gonococcal cells with piri, but no binding was observed in cell lysates without pili, as described in Example 12. Was. In contrast, antisera to pilin oligomers bound both cell lysates with and without pili.

As described in detail in Example 13, this pooled antiserum against gonococcal r-pilin, when analyzed by ELISA, has a high endpoint titer for binding to purified gonococcal r-pilin protein. It had. Example 13 also details the effects of various adjuvants. Good humoral immune responses were obtained in mice when r-pilin was supplemented with MPL ™ alone, or with MPL ™ plus aluminum phosphate, or with Stimulon ™ QS-21.

As described in Example 14, in a whole cell ELISA, the antiserum against rpilin binds to cells with pili but not to cells without the same genotype of pili in a particular gonorrhoeae strain. It has been shown.

As described in Example 15, mice were immunized intranasally with r-pyrin of Neisseria gonorrhoeae with or without native cholera toxin. There was a significant immune response detected by antigen ELISA from pooled sera generated after immunization of mice with r-pilin without adjuvant; this response was enhanced by adding native cholera toxin. Pooled sera has a low EL for binding to intact, spiky gonococcal cells
It had an ISA titer; this binding was also greatly enhanced when mice were immunized with native cholera toxin.

As shown in Example 16, immunoelectron microscopy showed that antibodies to r-pyrin bound along the length of the pyrifilament on the surface of Neisseria gonorrhoeae. This suggested that the antibody binds to an epitope present on the surface of the bacteria in vivo.

Example 17 demonstrates that the titers obtained for r-pilin antisera binding to isolates of bacteria with heterologous pili are similar to those obtained for antisera to recombinant pilin oligomers. It shows that it is relatively high. This r-pilin is r
The pilin is converted to the r-pilin oligomer by dialysis against a pH 12 phosphate buffer.

Pyri mediates the initial binding of Neisseria gonorrhoeae to human mucosal cells. Therefore, if an antigen could induce antibodies that inhibit the attachment of such bacteria to such cells, this would provide evidence that such antigens are vaccine candidates .

As discussed in Example 18, guinea pig antiserum to r-pilin significantly inhibited the binding of heterologous pili expressed by gonococci to human cervical epithelial cells.
Piling of gonococci is associated with the infectivity of this bacterium (2,3,3).
2).

Such data indicate that recombinant pilin is able to generate antibodies that bind to a variety of pili on intact gonococcal cells, and that the antiserum is capable of immunizing immunized humans with gonococcal colonization and It has been shown to exhibit functional activity to protect against infection (inhibition of bacterial adhesion) (32, 33). It was previously reported that immunization with E. coli cells expressing recombinant pilin from D. nodosus was immunogenic ( 23, 25, 28 ). ), It was not protective against provocation. These results have led these investigators to choose assembled pilas and reject the use of pilin, a subunit of the recombinant. However, the data described here suggest that, after purification, the recombinantly expressed pilin protein induces an immune response that must be related to protecting humans from gonococcal colonization. . That is, such data indicate that r-pilin is Neisseria
gonorrhoeae ).

As described in Example 19, the meningococcal chimera class Ir pilin protein was subjected to N-terminal sequencing. Sequencing of the 35 amino-terminal residues yielded results consistent with the amino acid sequence deduced from the DNA sequence. The molecular weight of the chimeric r-pyrine (including the signal) was determined by mass spectrometry to be 17,659 daltons, which fits well with the predicted mass of 17,676 daltons based on amino acid content. In contrast, when the meningococcal chimera class Ir pilin protein was subjected to size exclusion column chromatography using detergent,
An apparent molecular weight of 69,480 daltons was obtained. Like the gonococcal r-pilin, this suggested that the class I r-pilin protein of the meningococcal chimera was aggregated.

As described in detail in Example 20, this pooled antiserum against the meningococcal chimera class Ir pilin protein, as analyzed by ELISA, It had high endpoint titers for both meningococcal cells with pili. As also described in detail in Example 20, the adjuvant, in particular Stimulon ™ QS-21, is an antiserum against the meningococcal chimera class Ir pilin protein, A significant response was produced for binding to both the N. meningitidis cells.

As described in Example 21, immunoelectron microscopy showed that the class of meningococcal chimeras
Antibodies to the Ir pilin protein have been shown to bind along the length of N. meningitidis pili. This suggested that the antibody binds to an epitope present on the surface of the bacteria in vivo.

Example 4 showed that the antiserum against r. Pyrin of Neisseria gonorrhoeae recognizes and binds to pili-bearing meningococcal cells. Example 22 showed that antisera raised against the class Ir pilin protein of the N. meningitidis chimera binds to N. meningitidis cells with pili.

As described in Example 23, passive immunization of rat pups with a guinea pig antiserum to the class I pilin protein of the meningococcal chimera helps prevent meningococcal bacteremia in vivo. be able to. In animals receiving this immune serum,
There was a significant decrease in the level of colony formation. In addition, the immune response generated using the recombinantly expressed pilin can protect against meningococci expressing heterologous pilin proteins in vivo.

As described in Example 24, mice were immunized intranasally with a meningococcal chimera class Ir pilin with or without cholera toxin, where cholera toxin was glutamic acid at amino acid position 29. Indicates a mutant state in which histidine has been replaced (CT-CRM, E29H). There was a significant immune response detected by antigen ELISA from pooled sera generated after immunization of mice with r-pilin without adjuvant; this response was enhanced by adding mutant CT-CRM, E29H cholera toxin Was done.

As described in Example 25, the class of Neisseria meningitidis
Inhibition of murine nasopharyngeal colonization by strain I was demonstrated in mice immunized subcutaneously with MPL ™ -reinforced meningococcal chimera class Ir pilin.

As described in Example 26, antiserum from guinea pigs immunized with chimeric class II r-pilin of N. meningitidis expresses either class I or class II pilin in Western blot analysis It was shown to bind to whole cell lysates from meningococcal cells with pili.

As described in Example 27, antisera induced against the partially purified meningococcal chimera class II pilin bound to meningococcal cells from homologous bacterial strains.

Taken together, such data support the view that r-pilin, particularly the class I and II r-pilin proteins of N. meningitidis chimeras, are potential vaccine candidates against Neisseria meningitidis. are doing.

The gonococcal r-pilin protein is useful for the preparation of a vaccine that confers on mammals protection against diseases caused by Neisseria gonorrhoeae .
The meningococcal r-pilin protein, the meningococcal chimera class I r-pilin protein and the meningococcal chimera class II r-pilin protein are used in mammals to produce meningococcal (
N. meningitidis ) is useful in the preparation of a vaccine that confers protection against diseases caused by N. meningitidis .

In addition, cross-protection against various Neisseria species has been reported in mammals to induce meningococcal ( N.
By immunizing with a vaccine containing the r pilin protein gonococcal to confer protection against disease caused by meningitidis), or to confer protection of a disease caused by Neisseria gonorrhoeae (N. gonorrhoeae) in a mammal Immunized with a vaccine containing the r-pilin protein of N. meningitidis, the class I r-pilin protein of a N. meningitidis chimera or the class II r-pilin protein of a N. meningitidis chimera.

Such a vaccine composition comprises an isolated and purified r-pilin protein, wherein the vaccine composition elicits a protective immune response in a mammalian host.

The vaccine containing the r-pilin protein can be mixed in a conventional manner with an immunologically acceptable diluent or carrier to prepare an injectable liquid solution or suspension. The level of antibody induced by the vaccine was determined by Stimulon ™
QS-21 (Aquila Biopharmaceutic
als Inc.), Framingham, Mass., MPL ™ (3-o-deacylated monophosphoryl lipid A; RIBI ImmunoChem Re.
search, Inc.), Hamilton, Montana), aluminum phosphate, aluminum hydroxide, IL-12 (Genetics Institute)
Cambridge, Mass.) And cholera toxin (either in the wild-type or mutant state, for example, where glutamic acid is replaced by another amino acid, preferably histidine at amino acid position 29 according to US Provisional Application No. 60 / 102,430. Can be enhanced using certain adjuvants such as

The vaccines of the present invention may be administered by injection in a conventional manner, such as subcutaneous, intraperitoneal or intramuscular injection into humans, or orally, via mucosa, intranasally, or intravaginally. administered to induce an active immune response for protection against disease caused by Neisseria gonorrhoeae (N. gonorrhoeae) or N. meningitidis (N. meningitidis). The dose to be administered is determined by those skilled in the art by known means. Protection may be conferred by a single dose of the vaccine, or may require several booster doses.

The following examples are provided for a better understanding of the present invention. The examples are for illustrative purposes only and are not intended to limit the scope of the invention.

[0077]

【Example】

Standard molecular biology techniques are used according to the protocol described in Sambrook et al. (31).

Example 1 Bacteria and Cell Cultures Bacteria and Culture Conditions Isolates of Neisseria gonorrhoeae were obtained from Tampa, Florida; Ottawa, Canada; Washington, DC; Seattle, Washington; Rochester, New York; Chapel Hill, North Carolina; Obtained from Evanston, Illinois. Meningococcal isolates were obtained from Chapel Hill, North Carolina; and Biltven, The Netherlands.

Bacteria were stored lyophilized or frozen at -70 ° C until needed. When grown on solid medium, the agar plates were incubated at 37 ° C. in a moist atmosphere and a thermostat containing 5% (w / w) CO ₂ . Neisseria gonorrhoeae ( Ng
onorrhoeae ) and N. meningitidis do not contain hemoglobin, but contain dextrose (400 mg / L), glutamine (10 mg / L), cocarboxylate (2
Grow in GC medium (Difco Laboratories, Detroit, MI) supplemented with 0 μg / L) and iron nitrate (500 μg / L). Liquid suspension culture of N. meningitidis at 37 ° C in the same medium without agar
And grown in a shaking incubator (70 RPM). In experiments involving the culture of meningococci derived from homogenates of mouse nasal tissue, the bacteria were the following antibiotics (Deffuco):
Colistin sulfate (75 μg / mL), nystatin (125 μg / mL), vancomycin (30
μg / mL) and trimethoprim lactate (50 μg / mL). Pili-bearing gonococci were identified by colony morphology, and individual colonies were passaged daily to maintain phenotype. The condition of meningococcal cells with pili is
Samples stained with the NanoVan strain (Nanoprobes, Stony Block, NY) for 30 seconds at pH 8 were evaluated by transmission electron microscopy. E. coli contains 20 g / L Bacto tryptone (Defco), 5 g / L yeast extract (Defco), 0.6 g / L NaCl, 0.2 g / L KCl and 1% (vol / wt)%. Growing on SOB agar consisting of agar (pH 7.5) or SOB broth without agar.
For some experiments, Bacto Tripton was replaced with an equal amount of HySoy ™ (Sheffield Products, North Rich, NY). The epithelial cell culture ME-180 cell line (ATCC, Beltsville, MD) is an epidermoid carcinoma originally derived from cervical cancer. Cells were 10% (vol / vol) fetal calf serum (Sigma, St. Louis, Mo.), penicillin G (1000 units / mL) (Gibco BRL), L-streptomycin (1 mg / mL) (Gibco BRL). ) And 2 mM L-
RPMI1640 supplemented with glutamine (Gibco BRL, Gettysburg, MD)
The medium was grown at 37 ° C. in 5% (v / v) CO ₂ humidified. Cells are 3 ~
Divided every four days.

Example 2 Cloning and Expression of N. gonorrhoeae pilE in E. coli A frozen sample of the Pgh3-1 strain of N. gonorrhoeae harboring pil was used as a source of pilE DNA in the PCR reaction. used. This pilE gene was amplified using the following primers that recognize the 3 'and 5' ends of the complete pilin protein (including the leader sequence):

[0081]

[Table 1]

[0082] PCR products generated in the Nco I site at the beginning of the pilE coding region, and the terminal Ba
An mHI site was included. An Nco I site was introduced into the gene for cloning considerations. This changed 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 that cleaves during normal processing of the normal mature protein, this change was not expected to have any effect on antigenicity or immunogenicity. The PCR product was transferred to pCRTM IIT
A Cloned vector (Invitrogen, Carlsbad, CA), ligated and transformed in E. coli TOP10F '(Invitrogen). Colonies were selected on plates containing 100 μg / mL ampicillin or 50 μg / mL kanamycin. Plasmid DNA was isolated from an overnight culture of such transformants and analyzed by restriction digestion using EcoR I and Not I enzymes.

[0083] To confirm the presence of the pilE PCR DNA fragment, four clones containing the correct insert size were subjected to DNA sequence analysis. Clone # 17, designated pPX1999, was used as a source of the pilE gene. pPX1999 and pTrcHisA
The plasmid DNA from (Invitrogen) was digested with NcoI and BamHI restriction enzymes, gels of the DNA fragments were isolated, ligated, and E. coli TO
Transformed in P10F '. Ampicillin-resistant colonies were selected, new plasmid DNA of the transformants was isolated, and the D.D. was isolated using BamHI and NcoI restriction enzymes.
A restriction analysis of NA was performed. Two clones with the correct restriction pattern were subjected to DNA sequence analysis. Both clones had the correct DNA sequence and were named pPX2000.

To test for expression of recombinant pilin, the culture containing such clones was prepared by adding 100 μg / mL ampicillin and 12 μg / mL tetracycline to SOB in either a shake flask or a fermentor. Grew. When using shake flasks E. coli (E. coli) were grown to A ₆₀₀ = 0.9 to 1.0 is obtained in the medium 1 liter. Expression of the recombinant pilin was induced by adding isopropyl-β-D-thiogalactopyranoside (IPTG) at a final concentration of 1 mM and
The cells were grown for a period of time, at which time the cells were harvested by centrifugation (4 ° C, 13,689 xg for 20 minutes) and stored at -20 ° C. For the fermentor, the overnight culture was used to inoculate a flask containing 500 mL of medium, which was grown again overnight. This liquid culture is then converted to Biostat B containing 8.9 liters of medium.
It was used to inoculate a fermenter (Braun Biotech, Allentown, PA). Enhanced growth of bacteria in the fermentor was obtained when the medium containing HySoy ™ was supplemented with 1% (w / v) final concentration of dextrose. When the culture reached A ₆₀₀ = 1.0, IPTG was added to a final concentration of 1 mM and the cells were allowed to grow for an additional 1-4 hours before being harvested by centrifugation (4 ° C., 13,689 × g for 20 minutes). Discard medium and remove cell pellet to -20
Stored at ° C. When induced with IPTG, r-pilin protein expression is significantly increased,
The maximum level was reached 3 to 4 hours after the induction.

Samples from the induced culture were analyzed by polyacrylamide gel electrophoresis (SDS-PAGE) in the presence of sodium dodecyl sulfate. Recombinant pilin
Visualization using Coomassie blue staining and its identification was confirmed by Western blot using a monoclonal antibody specific for gonorrhoeae pili (clone # A33020023, Biospecific, Emilyville, Calif.). .

Example 3 Construction of Recombinant Pilon Plasmid of Gonorrhoeae Containing Kanamycin Resistance Marker 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. PCR reaction was performed on pTrcHisA plasmid DNA.

[0087]

[Table 2]

Was performed using The underlined sequence encodes an Xba I restriction site. About 3.5kb
PCR product contained pTrcHisA DNA without the ampicillin coding region. Another PCR reaction was performed using pACYC177 plasmid DNA (New England Biolabs (
New England Biolabs), Beverly, Mass.)

[0089]

[Table 3]

Was performed using Again, the underlined sequence encodes an Xba I restriction site.
The approximately 860 bp PCR DNA product contained the kanamycin resistance gene. Both PCs
The R product was purified from agarose gel, DNA was digested with Xba I restriction enzyme, extracted and ligated together. Aliquots of this ligation reaction were used for E. coli TOP1
Transformed with OF 'and aliquots of the transformation mix were plated on SOB plates containing 30 μg / mL kanamycin.

A total of 48 Kan ^R colonies were streaked on SOB plates containing either ampicillin or kanamycin in duplicate. As expected, all Kan ^R colonies were ampicillin resistant. Because the cloning design was symmetric, both directions of the kanamycin insert were isolated. The same clockwise kanamycin insert as the original ampicillin gene was selected for further experimentation and named pZ564. The DNA region containing the lacIq gene, trc promoter and multiple cloning site in pZ564 was replaced with a similar region from the pPX2000 plasmid (which also included pilE ) in the following manner: pZ564 and pPX2000 were replaced with Sph I and Xmn Digested with I restriction enzyme. The approximately 2.2 kb DNA fragment from pPX2000 and the approximately 2.6 kb DNA fragment from pZ564 were gel purified, ligated together, and E. coli ( E.
was transformed in .coli) TOP10F '. The resulting correct plasmid was named pPX2002.

A similar time course of induction and levels of recombinant pilin protein expression were seen when the selected antibiotic was changed from ampicillin to kanamycin.

Example 4 Cloning and Expression of N. meningitidis Class I pilE in E. coli Due to the highly homologous nature of the DNA and amino acid sequences of N. gonorrhoeae and N. meningitidis class I pilins. 7) The ability of gonococcal r-pilin antiserum to bind to pili expressed on N. meningitidis cells was evaluated using a whole-cell ELISA. This antiserum was tested for binding to N. meningitidis spiked cells from strain H355.
, Representing a titer of 100.

[0094] Western blots of whole cell lysates from this strain showed only a single band that co-migrated with pilin bound to antiserum (data not shown), so this binding appeared to be for pilin protein. Was done. Many other N. meningitidis strains are whole cell
Although the ELISA showed low titers, the presence of pili was not confirmed by transmission electron microscopy. Based on such data, class I pilins were cloned from N. meningitidis and determined to be expressed recombinantly. Initially, the same procedure was followed as with pilE from N. meningitidis . The procedure of Example 2 was used, except as noted below.

[0095] Class I pilE was amplified from the genomic DNA of N. meningitidis strain H44 / 76 using the following primers:

[0096]

[Table 4]

As expected, a PCR product of approximately 600 bp DNA was obtained. Aliquots of the PCR reaction product was digested with BamH I and Nco I restriction enzymes for insertion pTrcHisA. The digested DNA was electrophoresed on an agarose gel and the DNA fragment was gel purified. The DNA fragments are then ligated together and the E. coli TOP
Transformed in 10F '. Miniplasmid preps analysis of ampicillin resistant clones (Miniplasmid prep analysis) was performed using BamH I and Nco I restriction enzymes. A clone expressing the correct restriction digestion pattern was named RZ1142 and the plasmid was named pPX2003.

After induction, the presence of a polypeptide stained with Coomassie blue having a molecular weight of about 15,000 daltons was observed when whole cell lysates were analyzed by SDS-PAGE.
Analysis by SDS-PAGE and Western blot of whole cell lysates from such four clones, Neisseria gonorrhoeae (N. gonorrhoeae) Approximate mobility of polyclonal antisera against full pyridinium derived from LB2 strain and reaction The presence of a sex protein was indicated. The purified class Ir pilin was 167 amino acids long. However, the expression level of this recombinant pilin was similar to pPX2000 and p
Significantly lower than the levels obtained with any of PX2002. Recombinant class I p
Analysis of the ilE DNA sequence reveals a number of inverted repeats, which can be found in E. coli .
May explain the low expression levels of this protein in

Example 5 Construction and Expression of Neisseria gonorrhoeae and N. meningitidis class I chimeric pilE in E. coli To increase the expression of N. meningitidis pilin protein, use pPX2003 (Example 4).
DNA encoding the first 60 amino acids in the N. meningitidis class I pilE construct described in p. 3) was used as the pPX2002 (including the N. gonorrhoeae pilE construct described in Example 3, a 7 amino acid signal peptide) (SEQ ID NO. Replaced with 4). The procedure of Example 2 was followed, except as noted below.

[0100] The conserved 5 'terminal region of the pilE gene of Neisseria meningitidis, Neisseria gonorrhoeae (N. gonorrhoeae)
The same region from the Pgh3-1 strain was replaced in the following manner. A Bsm BI site was introduced into the pilE gene of N. meningitidis as follows: DNA was transferred to pP using the following primers:
PCR amplified from X2003;

[0101]

[Table 5]

And Taq DNA polymerase. The expected PCR DNA product was introduced directly into pCR2.1 (Invitrogen) and transformed into TOP10F 'cells, and the resulting plasmid was named pZ578. Next, the Bsm BI site was introduced into gonococcus pilE by the following method.

[0103]

[Table 6]

The 5 ′ end of pilE from pPX2000 was amplified using and Pfu DNA polymerase. The resulting gonococcal PCR product and pZ578 were then digested with BsmBI and ligated together. The ligated DNA is then

[0105]

[Table 7]

The primers were used for PCR amplification.

[0107] is the size the PCR product of DNA that was expected (approximately 850 bp), and Nco
Digestion with I and Bam HI resulted in an approximately 600 bp fragment. This fragment was gel isolated and cloned directly into pPX2000 vector cut with Nco I- and Bam HI-, replacing the pilE gene Neisseria gonorrhoeae. The resulting plasmid that was ampicillin resistant was labeled pPX2004 and used to transform TOP10F '. Analysis of this transformant indicated the presence of the desired chimeric pilE DNA. After induction with IPTG, the amount of meningococcal chimeric Class Ir pilin construct expressed was significantly elevated compared to the amount of meningococcal r pilin expressed from pPX2003. 1% octyl-
Extraction with β-D-glucopyranoside (OG) and the purification protocol described in Example 7 for recombinant gonococcal pilin (TMAE Fractogel ™ column, 0.1% (
Highly purified meningococcal chimeric Class Ir pilin protein was obtained using 10% Tris ™ (in pH 8.5) containing% w / v Zwittergent ™ 3-14 (approx. 5 mg / gram cell wet weight was obtained). This material was> 90% pure when analyzed by SDS-PAGE and laser densitometry. SDS-PAGE showed the presence of a major band with a size of about 15,000 daltons. The class Ir pilin protein of the N. meningitidis chimera is also 167 amino acids long and contains a signal sequence of 7 amino acids, as indicated by sequencing of the amino-terminal 36 residues of the purified protein.

Example 6 Construction and Expression of Neisseria gonorrhoeae and N. meningitidis class II chimeric pilE in E. coli The initial cloning of N. meningitidis class II pilE involved meningitis with pyri. Isolation of chromosomal DNA from fungal ( N. meningitidis ) strain FAM18 cells and amplification of class II pilE DNA in a PCR reaction were included. The class II pilE gene was amplified using the following primers that recognize the 3 'and 5' ends of the complete pilin protein (including the leader sequence):

[0109]

[Table 8]

In a manner analogous to Example 2, the resulting PCR product contains Nc at the beginning of the pilE coding region.
o Included an I site and a Bam HI site at the end. The Nco I site was introduced into the gene for cloning considerations. This changed the second amino acid in the signal sequence from lysine (AAA) to glutamic acid (GAA). As mentioned earlier, this change was not expected to have any effect on antigenicity or immunogenicity. The PCR product was cloned into the pCR2.1 cloning vector (Invitrogen), ligated and transformed in E. coli TOP10F '. Colonies were selected on plates containing 100 μg / mL ampicillin or 50 μg / mL kanamycin.
Plasmid DNA was isolated from an overnight culture of such transformants and analyzed by restriction digestion using the enzyme EcoRI .

Clone # 8, designated pPX8001, was used as a source of the pilE gene. pPX
8001 and plasmid DNA from pTrcHisC (Invitrogen), respectively Nc
o Digestion with I and BamHI restriction enzymes, gel isolation of the resulting DNA fragment,
Ligation and transformation in E. coli TOP10F '. After selecting for ampicillin resistant colonies were isolated and plasmid DNA of new transformants and was performed using BamH I and Nco I restriction enzyme restriction analysis of DNA. Two colonies with the correct restriction pattern were subjected to DNA sequence analysis. Both clones had the correct DNA sequence and were named pPX8002.

To test for expression of recombinant class II pilin, such clones (
pPX8002) containing 10 mL of culture, 100 μg / mL ampicillin and 12 μg
grown in SOB containing / mL tetracycline to A ₆₀₀ = 1.0 in a 50 mL tube. Expression of the recombinant protein was induced by adding IPTG at a final concentration of 1 mM, and the culture was continued for 3 hours. Whole cell lysates of the induced cells were separated by SDS-PAGE and no new (induced) bands were detected when stained with Coomassie Blue. This suggested that the FAM18 pilE gene product was expressed at lower levels than can be recognized by Coomassie blue. When FAM18 pilE was cloned into the pET17b plasmid and transformed in E. coli BL21 (DE33) pLysS with or without the pilE signal sequence, no significant expression of the recombinant protein was detected. Similar results were obtained when two other N. meningitidis strains (NmB, 2996) were cloned into the same pTrcHis plasmid and TOP10F 'expression system.

Specifically, it was determined that NMB and 2996 strains also expressed class II pilin based on PCR and sequencing data. The pilE gene was synthesized by using class I sets of primers (NMFPILE and NMRPILE) and class II sets of primers (PILE2FWD and PILE2REV) in separate reactions using either chromosomal DNA or cells as templates. From N. meningitidis strain. P
The CR product was cloned into pTrcHisC and sequenced or directly sequenced. Sequence alignments were performed: sequences similar to those from strain H44 / 76 were classified as class I, while sequences similar to sequences from strain FAM18 were classified as class II. Sequences were not obtained from all amplifications. Preliminary classification was also made based on the PCR data. Class I strains gave the correct size for PCR products using class I primers, but not for PCR products using class II primers, whereas class II strains used class II primers. PC using the class I primers that gave the correct size
Not given with R product.

Based on experiments with meningococcal class I pilin, the region encoding the first 60 amino acids (conserved amino-terminal region) of class II pilE was replaced with N. gonorrho
eae ) with the corresponding region of the Pgh3-1 strain. Expression of the generated chimeric pilE is
A number of E. coli expression strains were investigated using various promoters. The strains investigated were: PR13 (Rnase deficient), BL21 (protease deficient), KS474 (cellular protease deficient), AD494 (Novagen, which allows the formation of disulfide bonds in the cytoplasm) And three TOPP strains (Stratsgene)
(A non-K12 strain useful for making the protein difficult to express). In all cases, no recombinant protein was detected on Coomassie blue stained SDS-PAGE. Another expression plasmid pET17b, which contains the T7 promoter, was examined with the same results.

It should be noted that the native class II pilE gene sequence in pPX8002 ends at base 447. The DNA sequence found at nucleotides 447-519, downstream (3 ') from the natural meningococcal class II pilE termination site, contains inverted repeats that may form stem and loop structures. The stem and loop structure can be an effective terminator of transcription, and the omission of this will result in additional 3 ′ sequences in pPX8002 (
It was hypothesized that (74 bases) would affect the transcription of the chimeric class II pil E message in E. coli . Thus, all subsequent cloning preserved this downstream 3 'terminal sequence.

[0116] To identify the region that inhibits expression, p of the various parts of the Class II pilE gene from Neisseria meningitidis (N. meningitidis) strain, Neisseria gonorrhoeae (N.gonorrhoeae) Pgh3-1 strain
A systematic replacement with the corresponding region from the ilE gene was undertaken. The exchange region began at the 5 'or 3' end and became progressively larger. Double exchanges at the 5 'and 3' ends were also constructed until only 84 internal region nucleotides from the native pilE class II remained. This region was also exchanged, resulting in reconstitution of rGC, and, as expected, this clone expressed rpilin at the same Coomassie blue staining level as pPX2000. The following regions of the FAM18 pilE gene (listed in number of nucleotides) were replaced with the corresponding regions from Pgh3-1 (listed in parentheses): Single region exchanges 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), 295-3
78 (283-366); double domain exchange is 1-294 (1-282) & 379-519 (367-553)
, 1-181 (1-181) & 439-499 (478-553), 1-181 (1-181) & 379-519
(367-553), 1-294 (1-282) & 439-499 (478-533).

When such constructs were expressed in E. coli TOP10F 'using the pTrcHis expression system, the two constructs produced recombinant proteins at levels detectable by Coomassie Blue: One involves the exchange of nucleotides 379-519, which comprises a disulfide loop and a 3 'extension; and the second comprises 1-1-1.
81 (which comprises the conserved 5 'region) & 379-519 (which comprises the disulfide loop and the 3' extension). Since the exchange of the 5 'region alone did not lead to expression of the recombinant protein, and because the first construct retained most of the native meningococcal pilin sequence, this construct (nucleotide
379-519) were selected for further investigation. The amino acid sequences of N. meningitidis and N. gonorrhoeae proteins are significantly different in this region, so it is well documented that disulfide loops undergo significant antigenic mutations (17, 18). Therefore, any immune response to this region (eg, a disulfide loop) has minimal cross-linking between meningococcal strains.
Will show reactivity. Finally, since the insert of Neisseria gonorrhoeae is almost twice the size of the disulfide loop of N. meningitidis (39 residues versus 18 residues), the resulting chimeric protein is S
The DS-PAGE gel migrates to an apparent molecular weight of approximately 19,000 daltons.

The construction of this chimeric gene was performed in the following manner. The 5 'fragment is pPX8002
(FAM18 class II pilE ) was obtained by amplification using the following primers:

[0119]

[Table 9]

The gonococcal disulfide loop (ie, 3 ′ of the gonococcal gene) was amplified from pPX2000 using the following primers:

[0121]

[Table 10]

The resulting PCR products were each purified, separately digested with the restriction enzyme BsmBI , and then ligated to form the full-length chimeric pilE , which was combined with the primers PILE2FWD and PILERE.
Amplified using V. The PCR products were digested with a restriction enzyme Nco I and Bam HI, ligated into pTrcHisC vector treated similarly restricted, and transformed with TOP10F 'competent cells.

Transformants were cultured and analyzed using the restriction enzymes Nco I and Bam HI.
Four clones with the correct size insert were analyzed with the restriction enzyme StuI . Three of them provided the correct restriction maps. Two of the three clones with the correct restriction pattern were sequenced. Clone # 5 has the correct DNA sequence and was named pPX8017. This clone contains the nucleotide sequence described in SEQ ID NO: 3, wherein nucleotides 1 to 378 is derived from bacteria meningitis (N. meningitidis), and the nucleotide 379-510 is derived from Neisseria gonorrhoeae (N. gonorrhoeae).

[0124] Expression was examined by 10 mL culture in 50 mL tubes. Cells were grown to about A ₆₀₀ = 1.0 by supplementing SOB with 100 μg / mL ampicillin and 12 μg / mL tetracycline. Cultures were induced by adding IPTG at a final concentration of 1 mM. Growth is 3 ~
Cells were grown for 4 hours, at which time cells were harvested by centrifugation (4 ° C, 13,689 xg for 20 minutes) and stored at -20 ° C. For fermentors, flasks containing 500 mL of medium were inoculated using overnight culture from plates or frozen stocks, which were again grown overnight. This liquid culture was then used to inoculate a Biostat B fermenter (Braun Biotech, Allentown, PA) containing 8.9 liters of medium. Enhanced growth of bacteria in the fermentor was obtained when the medium containing HySoy ™ was supplemented with 1% (w / v) final concentration of dextrose. Culture is A ₆₀₀ = 4.0-6.0
When reached, IPTG was added at a final concentration of 1 mM, and the cells were allowed to grow for an additional 2-4 hours before being harvested by centrifugation (4 ° C., 13,689 × g for 20 minutes). Discard the medium,
The cell pellet was stored at -20 ° C. When induced by IPTG, chimeric class II
The expression of r-pilin protein was significantly increased.

Samples from the induced culture were analyzed by polyacrylamide gel electrophoresis (SDS-PAGE) in the presence of sodium dodecyl sulfate. The class II pilin of the recombinant meningococcal chimera was visualized using Coomassie blue staining (approximately 19
2,000 daltons apparent molecular weight), and its identification is a gonococcal peptide

[0126]

[Table 11]

Confirmed by Western blot using a polyclonal antiserum against (this peptide is located in the conserved region at the amino terminus of the class II pilin protein).
The class IIr pilin of the N. meningitidis chimera is 170 amino acids long (including the signal) (SEQ ID NO: 4), amino acids 1-126 from N. meningitidis , and amino acids 127-127. 170 is derived from Neisseria gonorrhoeae (N. gonorrhoeae).

Example 7 Isolation and Purification of Recombinant Neisseria gonorrhoeae pilin from E. coli The following procedure was used to purify the recombinant Neisseria gonorrhoeae pilins obtained in Examples 2 and 3 above. This procedure was also used to purify the meningococcal recombinant pilin obtained in Example 4 and the meningococcal chimeric class Ir pilin protein obtained in Example 5 above was first purified. Used to Next, the meningococcal chimera class
The isolation procedure for the Ir pilin protein was modified as described in Example 8 below.

For the cell fraction of E. coli expressing r-pilin, 1 (vol / vol)%
Based on the ability of Triton ™ X-100 to dissolve this protein, it was shown that this protein was bound to cell membranes, almost the inner membrane. 0.05-0.1% (vol / vol)% to remove E. coli proteins contaminating this experiment
It was found that rPyrine did not bind reliably to ion exchange columns below pH 9.5 when performed in the presence of Triton ™ X-100. Therefore, E. coli
The ability of a number of detergents to selectively dissolve r-pilin protein from different membrane preparations was investigated.

Cell pellet (about 5 g wet weight) from 1 liter of culture was added to 30 mL of 10 mM
Hepes (pH 7.2) (Research Organics, Cleveland, Ohio), lysed by adding 1 mM EDTA, and cells are microfluidized.
idizer Cell homogenizer (Microfluidics International, Inc.
Microfluidics International Corp.), Newton, Mass.). The lysate was clarified by centrifugation (12,000 g × g, 10 minutes) and the membrane was pelleted (288,652 g × g, 1 hour). Membrane is 33 mL of 10 mM Hepes (pH 7.4), 1 m
Resuspended in M MgCl ₂ and extracted for 1 hour at room temperature with one of the following surfactants: (a) Triton ™ X-100 (TX-100) (Calbiochem-Novabiochem International) -Novabiochem International), San Diego, Calif., (B) reduced Triton ™ X-100 (Calbiochem), (c) octyl-β-D-glucopyranoside (OG) (Calbiochem), (d) Zwittergent® ) 3-8
(Z3-8) (Calbiochem), (e) Zwittergent ™ 3-10 (Z3-10) (Calbiochem)
, (F) Zwittergent (trademark) 3-12 (Z3-12) (Calbiochem), (g) Zwittergent (trademark)
) 3-14 (Z3-14) (Calbiochem), (h) Empigen BB ™ (Calbiochem) or (
i) Tween ™ 80 (ICN Cleveland, Ohio).

Empigen BB ™ (1 vol / vol%), Zwittergent ™ 3-10 (1 wt / vol%), reduced Triton ™ X-100 (1 vol / vol%), Zwittergent ( (Trademark) 3-10
Octyl glucoside containing (1% w / v) or 3-14 (0.1% w / v)
1% w / v ) extracted the recombinant pilin protein with minimal contamination of each E. coli protein. Zwittergent ™ 3-12 dissolved both the recombinant protein and a significant number of E. coli proteins, even at 0.1% (w / v). Tween ™ 80 did not extract recombinant protein at any of the concentrations tested (0.1-1% w / w).

The solubilized proteins were separated from the insoluble membrane material by centrifugation (288,65
2g x g, 1 hour). The supernatant (containing r-pilin) was dialyzed overnight at 4 ° C. against 10 mM Tris ™ (pH 8.5) containing one of the following nonionic detergents: (a) 0.1
(Weight / volume%) Zwittergent (trademark) 3-14, (b) 1 (weight / volume%) Zwittergent
(Trademark) 3-10 or (c) 1 (weight / volume%) OG. The dialyzed material is 10 mM Tris (trademark)
) (pH 8.5) and Fractogel ™ EMD TME-650 (S) (EM
Separation Technology, Wakefield,
(Richmond) column. Bound protein was eluted with a linear gradient of NaCl from 0 to 0.2 M in 10 mM Tris ™ (pH 8.5) containing the appropriate detergent. Fractions containing r-pilin were pooled and analyzed for purity and protein content. The pooled material was dialyzed against starting buffer, optionally to increase the purity of the r-pyrine, and a second fractionation was performed on a TMAE column.

The r-pilin selectively eluted from the column was SDS-PA stained with Coomassie blue.
It was highly purified (> 90% homogeneous) as assessed by laser densitometry analysis of the GE gel. Similar results were obtained by extraction and column chromatography.
Obtained when performed with (weight / volume)% Zwittergent ™ 3-10, 1 (weight / volume)% OG or 0.1 (weight / volume)% Zwittergent ™ 3-14. The yield of r-pilin, which accounts for a significant proportion of total E. coli protein, was approximately 10 mg / L culture grown in 1.5 liter shake flasks containing SOB medium. When grown in medium Ri fermentor using based on recombinant E. coli (E. coli) (including PPX2002) the HySoy (R), the yield of the purified r pilin rises to approximately 30 mg / L Culture , Which corresponds to 7 mg of r-pyrine per gram of cell mass. When 1% dextrose was included in the fermentor, the total yield of r-pyrine increased to about 100 mg / L.

The purified r-pilin was dialyzed against 10 mM sodium phosphate, 140 mM NaCl (pH 7) (PBS) containing 0.05% (w / v) Z3-14, and sterile-filtered;
C. or frozen at -20.degree.

EXAMPLE 8 Isolation and Purification of Meningococcal Chimera Class Ir Pilin from E. coli A large-scale culture of E. coli cells containing pPX2004 is described in Example 2.
Grow in BioStat B fermenter as described in. Bacterial cells (about 8
Eight grams of wet weight of E. coli pPX2004) were added to 440 mL of 10 mM Hepes, 1 mM EDTA (
pH 7.5) and disrupted using a Microfluidizer model 110Y (Microfluidics International, Newton, Mass.). The disrupted cells were clarified by centrifugation at 6,084 g × g at 10 ° C. for 20 minutes. The supernatant was collected and the membrane fraction was isolated by centrifugation at 205,471 xg for 1 hour at 10 ° C. The pellet was resuspended by 10mM Hepes, 1mM MgCl _2, 1 homogenized in% (w / v) octyl -D- glucopyranoside (pH 7.5) of 220 mM, 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 solubilized chimeric class Ir pilin was filtered through a 0.22μ Nalgene vacuum filter and stored at 4 ° C. The pH of the octyl glucoside extract was adjusted to 8.5 using concentrated NaOH and subsequently equilibrated with 25 mM Tris ™, 0.1% (w / v) Zwittergent ™ 3-14 (pH 8.5). A 200 mL TMAE Fractogel ™ column (EM Separation Technology, Gibbstown, NJ) was applied to the column. Unbound protein was washed off the column with an additional 400 mL of equilibration buffer. r-pilin is 25 mM Tris ™, 0.1% (w / v)
A linear NaCl gradient (0-0.2 M NaCl) in Zwittergent ™ 3-14, pH 8.5, eluted at a flow rate of 10.0 mL / min over 10 column volumes. The fractions containing the class Ir pilin chimeric were pooled and 1 in dH ₂ O: 1 with water and 10 mM NaPO _4,
100 mL 40 μm equilibrated with 0.1% (w / v) Zwittergent ™ 3-14 (pH 6)
On a ceramic hydroxyapatite column (Bio-Rad, Herculus, CA). Unbound protein was washed out of the column with an additional 200 mL of equilibration buffer. The chimeric class Ir pilin is a linear NaPO ₄ gradient (10-150 mM NaPO ₄ ) containing 0.1% (w / v) Zwittergent ™ 3-14.
_{Using 4} ), elution was performed at a flow rate of 5.0 mL / min over 10 column volumes. Fraction SDS-PA
The fractions containing the chimeric class Ir pilin were examined by GE analysis and pooled. The purified material was at least 95% pure as determined by laser densitometry on Coomassie blue stained gels. Class of purified chimeras
The yield of Ir pilin was about 35 mg / g wet cells.

Example 9 Isolation and Purification of Meningococcal Chimera Class II r-pilin from E. coli Unless otherwise specified, all steps were performed at room temperature. Frozen E. coli cells were resuspended in 10 mL per gram of cells, 10 mM Hepes, pH 7.2, 1 mM EDTA, and homogenized using a Microfluidizer cell homogenizer to disrupt cells. Cell lysates were clarified by centrifugation at 13,689 g × 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 at -20 ° C overnight. 9 mL / tube of 10 mM Hepes (pH 7.2),
Resuspended in 1 mM MgCl ₂ and extracted with 1% (w / v) 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 above. After centrifugation (388,024 xg for 30 minutes), pellet the pellet in 9 mL of 50 mM Tris ™ (pH 8.0), 5 mM
Resuspend in EDTA and add 1% (w / v) N-lauryl sarkosyl (Sigma: Si
gma) and extracted overnight at room temperature with gentle stirring. This solubilized the class IIr pilin of the meningococcal chimera. Insoluble material is removed by centrifugation (38
8,024 xg for 30 minutes) and discarded. Zwittergent ™ 3-14 at a final concentration of 1% (w / v) was added to the supernatant containing the meningococcal chimera class IIr pilin and the material was added overnight to 50 mM Tris ™ (pH 8.0). ), 10mM EDTA, 1% (w / v) Zwitt
ergent ™ 3-14. Aliquots (1 mL, 1.3 mg protein) of the dialyzed material were aliquoted into 50 mM Tris ™ (pH 8.0), 10 mM EDTA, 1% (w / v) Zwitterg.
It was passed through a MonoQ column (Pharmacia, Piscataway, NJ) (5 × 10 mm) column (5 × 10 mm) equilibrated with ent ™ 3-14 at a flow rate of 0.5 mL / min. Unbound material containing the chimeric class II r pilin was pooled and 10 mM NaP
Dialysis overnight against O ₄ (pH 6.8), 1% (w / v) Zwittergent ™ 3-14. The material was approximately 80% pure and was used in the experiments described in Examples 26 and 27 below. Further purification of this material was achieved by passing it through a 1 mL hydroxyapatite column (Bio-Rad) equilibrated with 10 mM NaPO ₄ (pH 6.8), 1% (w / v) Zwittergent ™ 3-14. Obtained. Class I purified meningococcal chimeras
The I protein is a linear protein containing 1% (w / v) of Zwittergent ™ 3-14.
Elution was performed using a 0.5M NaPO ₄ gradient. Fractions were examined for chimeric class II r-pilin by SDS-PAGE stained with either Coomassie blue or silver. Both analyzes showed the presence of a single polypeptide band with a molecular weight of about 19,000 daltons. This material was> 95% pure by laser densitometry on polyacrylamide gel.

Example 10 Analysis of r-Pillin in Neisseria gonorrhoeae Protein content was determined by the bicinchoninic acid protein assay (Pierce, Rockford, IL) using BSA as a standard. The purity of the protein preparation was determined by polyacrylamide gel electrophoresis (SDS-PAGE) in the presence of Coomassie brilliant blue-stained SDS, and was measured using a Personal Densitometer SI (Molecular Device).
(ice). The identity of pilin in the preparation was confirmed by western blotting using the monoclonal antibody described in Example 2, which was raised against purified pili from the P9 strain of N. gonorrhoeae ( Biospecific). Applied N-terminal sequence of pilin protein
Determined using an Applied Biosystems 477A protein sequencer. When purified r-pyrin was subjected to N-terminal sequencing, two sequences were often detected. The major sequence represented the complete pilin protein including the seven amino acid leader sequence. The minor sequence, consisting of 10-20% of the sample, was the r-pyrin protein without the leader sequence and the sequence was phenylalanine, starting at the N-terminal residue of the mature gonococcal pilin protein. For both r-pilin protein forms, sequencing of the amino-terminal residue yielded results consistent with the sequence expected from the DNA sequence.

The mass of the recombinant pilin was determined by matrix-assisted laser desorption time-of-flight (MALSI-TOF) mass spectrometry using a Finnagan MAT Lasermat ™ 2000 (San Jolla, CA). The instrument was calibrated with equine myoglobin to 0.01% of its expected 16,951.5 dalton molecular weight. Recombinant pilin was mixed with an equal volume of a cyano-4-hydroxycinnamic acid matrix (10 mg / mL, 70:30 acetonitrile: 0.1 (vol / vol)% trifluoroacetic acid / water). An aliquot (1 μL) of this mixture was deposited on the stage of the sample, air-dried and subjected to MALSI-TOF mass spectrometry analysis. 1
The five data (each analysis represents the sum of 10 shots) were averaged to determine the mass of r-pilin. The molecular weight of r-pilin (with signal) was determined to be 18,001 daltons, which fits well with the predicted mass of 17,981 daltons based on amino acid content. 17,2
A minor peak with a mass of 32 daltons (average) was detected in each lot. The difference in molecular weight of the two forms of recombinant pilin (769 daltons) is due to the loss of the first six amino acids of the leader sequence with a mass of 774 daltons (Met Asp Thr Leu Gln Lys)
(SEQ ID NO: 2, amino acids 1-6).

A very different apparent molecular weight of r-pyrine is 0.05% (w / v) of Zwitterg
Obtained by size exclusion column chromatography using an analytical Superose ™ 12 column (Pharmacia, Piscataway, NJ) equilibrated in PBS containing ent ™ 3-14. Under these conditions, the protein is 68,899
Eluted at a position corresponding to the molecular weight of This suggested that the recombinant protein was aggregated. However, elution of the protein from the size exclusion column can greatly affect the shape of the protein. Velocity sedimentation ce
Results from the experiments showed that the recombinant pilin had a molecular weight of approximately 45,000 daltons in solution. Detergent (Zwittergent ™ 3-14) was removed and protein was dialyzed exhaustively against PBS only. The dialyzed recombinant protein was soluble and did not pellet by high speed centrifugation (122,000 × g, 1 hour). No confirmation of complete removal of detergent from the recombinant protein was made. Analysis of this material by gel filtration in PBS showed that the protein was 4%.
It was shown to have an apparent molecular weight of 52,349 daltons. This suggested that the protein was further aggregated. The number of subunits in the aggregate was not measured.

The value of 452,349 should be considered presumed because the protein may still be micelles, as it is not known whether the detergent has been completely removed from the sample. When formulated as a vaccine, if r-pilin is diluted about 15-30 fold, the r-pilin in the vaccine will have an apparent molecular weight of about 450 kD.

Example 11 Preparation of Immune Serum from r-Pillin Immunogenicity experiments were performed by immunizing subcutaneously (sc) guinea pigs (female, 200 g) with 20 μg of purified gonococcal r-pilin protein mixed with adjuvant. ). The adjuvants tested were: (a) Stimulon ™ S in PBS (pH 6)
Q-21 (25 μg / dose); (b) Aluminum phosphate in PBS (pH 7) (Lederle Laboratories, Pearl River, NY, 100 μg / dose)
(Dose); or (c) PBS (pH 7) alone was used. Animals were initially immunized at weeks 0, 4, and 8 and sera were obtained at weeks 0, 4, 6, and 10. Analysis of the time course of the immune response showed that giving the third vaccination at week 8 did not enhance the immune response, and therefore experiments following the use of recombinant pilin at week 6 The blood collection was completed.

To investigate the ability of the adjuvant to modulate the gonococcal r-pilin immune response, mice (female, 8 weeks old, 5 or 10 animals per group) were injected with 1-10 μl
g of purified protein was immunized subcutaneously at weeks 0, 4, and 6, and serum was obtained at weeks 0, 4, 6, and 8. Vaginal irrigation was performed by instillation of RPMI 1640 (75 μL) into the vagina at week 8 and aspiration 3-4 times. The wash fluids from each group were pooled together, and 50 μL of fetal calf serum was added to each pool.

For the meningococcal chimera class Ir pilin protein, mice were immunized only at weeks 0 and 4, and sera were obtained at weeks 0, 4, and 6. The following adjuvants were tested for parenteral immunogenicity experiments of r-pyrine in mice: (a) Stimulon ™ SQ-21 (25 μg / dose) in PBS (pH 6); (b)
Aluminum phosphate (100 μg / dose) in PBS (pH 7); or (c) in PBS (pH 7)
MPL ™ (50 μg / dose); (d) Aluminum phosphate (100 μg / dose) in PBS (pH 7) and MPL ™ (50 μg / dose); or PBS (pH 7) only.

For the meningococcal chimera class II r-pilin, guinea pigs were tested for Sti in PBS (pH 6).
Immunization of 20 μg protein supplemented with mulon ™ SQ-21 (25 μg / dose)
Received only at weeks 0 and 4, and sera were obtained at weeks 0, 4, and 6.

The ability of recombinantly expressed pilins (gonococcal or meningococcal chimeras class I) to induce a mucosal immune response has been demonstrated in mice by intranasally (a) containing 1 μg of natural cholera toxin 1 or 10 μg of gonococcal r-pilin with or without (2.5 μL of saline)
Or (b) by immunization with 5 μg of chimeric class Ir pilin diluted in 10 μL of PBS (pH 7) with or without 1 μg of mutant CT-CRM, E29H cholera toxin. Immunizations were given at 0, 2 and 3.

Example 12 Western Blot Analysis of the Immune Response to Neisseria gonorrhoeae r-pilin Guinea pigs were immunized using the purified Neisseria gonorrhoeae r-pilin according to the protocol described in Example 11. Antisera from guinea pigs immunized with r-pyrin of Neisseria gonorrhoeae were first analyzed by Western blot (data not shown).
These blots showed that the antiserum against gonococcal r-pilin recognized a band corresponding to pili in whole cell lysates from cells of P. gonorrhoeae with piri; There were no stained bands seen in cell lysates without.

Comparative data was obtained from antisera from guinea pigs immunized with gonococcal pilin oligomers. Pyrine oligomers were obtained by complete pyri dissociation as described previously (4). Briefly, this involves dialyzing whole pili at 37C for 4 hours against 37 mM sodium phosphate (pH 12) for 48 hours, followed by dialysis against 50 mM Tris ™, 145 mM NaCl (pH 8.0). Was included. The pilin oligomer is then centrifuged (100,000 xg, 1 hour)
For clarification. After centrifugation, pilin oligomers were present in the supernatant. Compared to the antiserum against r. Pyrin of Neisseria gonorrhoeae, the pilin oligomeric antiserum binds to pyr in cell lysates with piri, while lysing from both cells with or without pili. Also bound to a number of other bands in the product (data not shown). These bands represent contamination in the pilin oligomer preparation and are not expected to be related to pili.

Example 13 Recombinant Neisseria gonorrhoeae pilin ELISA Endpoint titers against purified proteins or bacterial cells were determined by ELISA. Incubation was 1 hour at room temperature unless otherwise specified in all ELISA procedures. The end-point titer was determined by optical absorption in blank wells (1
(Excluding the secondary antibody) was determined by extrapolation dilution greater than 0.10. For analysis of anti-serum of guinea pigs, purified recombinant pilin was added to 1 μg / mL with 0.1 M Tris ™ (pH 8).
To a final concentration of. Aliquots (100 μL) are transferred to microtiter plates (Immulo
nII, Nunc, Napalville, Illinois) plus 4 wells
Incubation at 0 ° C. overnight. Plates are 0.05% (vol / vol)% Tw
Washed 5 times with PBS containing een ™ -20 (PBS-T) using a Skanwash 300 plate washer (Skatron Instruments, Alexandria, VA). Wells were blocked with 200 μL of 1% (w / v) BSA in PBS-T, washed, and aliquots of antiserum (0.1% in PBS-T).
(Weight / volume) diluted in% BSA) was added to the wells. The plate was then washed and the bound primary antibody was washed in rabbit anti-mogmot IgG (heavy & light chains) (Zymed Laboratories, South San Francisco, CA) (50 mM Tris ™ (pH 8)). Detection was performed using 100 μL of alkaline phosphatase conjugated to 0.1 (w / v)% BSA (1: 2000 dilution). Plates were washed and color development was performed using 100 μL per well of p-nitrophenol phosphate (Sigma) (2 mg / mL, 0.5 M diethanolamine, 0.25 mM MgCl ₂ , pH 9.8). After 30 minutes, the reaction was stopped by adding 50 μL of 3N NaOH. Absorption is T
hermomax ELISA plate reader (Molecular Device
ces), Sunnyvale, CA).

All animals immunized with the r-pilin protein responded very well, as shown by the antigen ELISA shown in Table 1.

[0150]

[Table 12]

• Three different lots of r-pyrine were used as immunogens. The guinea pigs were immunized at 0 and 4 weeks (sc) and bled at 0, 4 and 6 weeks. Analysis was performed on pooled sera. Effect of Adjuvants on Immune Response The following adjuvants were tested in mice on the immune response to gonococcal r-pilin: (a) Stimulon ™ SQ-21 in PBS (pH 6); (b) PBS (pH7)
(C) MPL ™ in PBS (pH 7); (d) Aluminum phosphate and MPL ™ in PBS (pH 7); or (e) PBS (pH 7) only. For analysis of mouse antisera, the antigen ELISA protocol was modified as follows. Microtiter plates (Costar, EIA / RIA, Corning Coster (Corning Cos
tar), Cambridge, Mass.) with 100 μL of 1 μg / mL r-pyrine (P
(In BS) at 37 ° C. overnight. Plates are 0.1% (vol / vol) Tween® (trademark)
Washed 5 times with PBS containing) -20 using a Skantron 300 plate washer. Wells contain 0.1% (w / v) gelatin and 0.02% (w / v) N
and blocked with PBS containing aN _3. The primary antibody was 0.1% (w / v) gelatin, 0.05% (v / v) Tween ™ -20 (PBS-TG) and 0.02 (w / v).
Diluted in PBS containing 5% NaN ₃ and aliquots of 100 μL were incubated in microtiter plates for 2 hours. After washing, the bound primary antibody was biotinylated rabbit anti-mouse IgG (Fc region) (Brookwood Biomedical, Birmingham, Ala.) (PBS-TG and 0.02% (w / v)) (1: 8000 dilution with NaN ₃ ). Plates were washed and secondary antibody, then with PBS-TG and 0.02 (w / v)% NaN ₃ 1: 5
Detection was performed using 000-fold diluted streptavidin-conjugated horseradish peroxidase (Zymed Laboratories) (30 minute incubation). The plate is washed and 0.5 mg / mL of 2,2'-azino-bis (3-ethylbenzothiazoline in 0.1 M citric acid (pH 4.2) containing 0.03% (vol / vol) hydrogen peroxide. -6-sulfonic acid) (ABTS) for 30 minutes, and SLT340 A
TTC microplate reader (SLT Lab Instruments)
Research Triangle Park, NC) at 405 nm. Data were plotted using a log-log plot, and endpoint titers were determined as described previously.

[0152] When r-pilin was supplemented with MPL ™, a humoral immune response was obtained in mice, similar to the scale seen with Stimulon ™ QS-21 (Table 2A). Analysis of vaginal irrigation of the same animals revealed demonstrable IgG titers in vaginal irrigation fluids of these animals (Table 2B).

[0153]

[Table 13]

[0154]

[Table 14]

• Balb / c mice (5 per group) were immunized at 0, 4 and 6 weeks with 10 μg of r-pilin reinforced as described above. Animals were bled at 0, 4, 6, and 8 weeks. Analysis was performed on pooled sera at 8 weeks. Week 0 endpoint titer < 5,000
Met. ★★ Pooled vaginal washes were obtained at 8 weeks. ★★★ Whole cell ELISA using dried cells. P +: cells having pili. P-: a mutant having no pili, which was generated from a parent cell having pili.

Example 14 N. gonorrhoeae whole cell ELISA r Significant number of cross-reactive epitopes found on complete assembled pili
Expression by pilin was subsequently confirmed by whole-cell ELISA, where the purified antiserum against r-pyrin exhibited high titers against a number of gonococcal strains expressing heterologous pyri. The ability of the guinea pig antiserum to bind live gonococcal cells was performed using the following procedure. Microtiter plates (Immulon II) were incubated overnight at 4 ° C. with 100 μL of 0.01% (w / v) poly-L-lysine (Sigma) in PBS. The overnight bacterial agar culture was collected with a Dacron swab in PBS and the turbidity of the suspension was adjusted to A ₆₀₀ = 1.0. Poly-
Discard the lysine solution from the microtiter plate and add 10
An aliquot of 0 μL was added to the well. Plates were washed 5 times using a hand held Nunc plate washer with PBS and blocked with 200 μL PBS containing 1% (w / v) BSA (PBS-B). The plates were then washed 5 times using PBS and 100 μL aliquots of the antiserum diluted in 0.1% (w / v) BSA in PBS were added to the wells. After washing 5 times with PBS, the bound antiserum was
A suitable alkaline phosphatase conjugate (0.1% (w / v) BSA in PBS)
(1: 2000 dilution). Plates were washed, developed, and read for absorbance as above (30 minutes after development).

When analyzed by whole-cell ELISA, guinea pig antisera to r-pilin bound to pili-bearing isolates from various regions but bound to cells without the corresponding pili of the same gonococcal strain. No (Table 3).

[0158]

[Table 15]

[0159]

[Table 16]

* Three different lots of r-pyrine were used as immunogens. Guinea pigs were immunized (sc) and bled as previously described. Pooled sera from week 6 blood draws were assayed. Week 0 endpoint titer < 250. P +: cells having pili. P-: Pili-free mutant derived from the parent cell having Pili.

[0161] Whole cell ELISA analysis of mouse antiserum to r-pilin (Table 2) was performed using microtiter plates with bacteria dried in the wells by the following procedure.
The overnight culture of bacteria is collected with a Dacron swab in PBS,
The turbidity of the suspension was adjusted to A ₆₀₀ = 0.1. Aliquot of bacterial suspension (
100 μL) was added to the wells and the plates were air dried at 37 ° C. After all liquids had evaporated, the plates were sealed and stored at 4 ° C. until use. The rest of the procedure was performed according to the procedure already described for the antigen ELISA for mouse antisera. Data from whole cell ELISA (Tables 2 and 3) suggest that r-pilin induces antibodies that bind to conserved epitopes on the surface of gonococci with pili.

Example 15 Induction of Mucosal Immune Response to Neisseria gonorrhoeae rpilin Since gonorrhea is a disease of the genital mucosa, it was interesting to investigate the ability of mucosal immunization to induce mucosal immune responses. This was done in the following manner. Neisseria gonorrhoeae r-pilin (1 or 10 μg in 10 μL in saline) was immunized intranasally in mice at 0, 1 and 2 weeks with and without 1 μg of natural cholera toxin. 5
Groups of Swiss-Webster mice received 0, with or without cholera toxin.
Immunizations were given intranasally at 1 and 2 weeks. Analysis was performed on pooled sera. Week 0 endpoint titers were <50.

As shown in Table 4 below, when animals were immunized with r-pyrine in the absence of adjuvant, a significant immune response was detected by antigen ELISA (0 week titer <300). This reaction was enhanced by adding natural cholera toxin.

[0164]

[Table 17]

Next, the ability of these sera to bind to the complete, pilin-bearing gonococcal cells of these sera was tested on cells derived from the N. gonorrhoeae strain P1090.
Investigated by A. Cells were dried on microtiter plates as described above. As shown in Table 5, low titers were detected with r-pyrine alone (0 week titer <
300). Binding to cells with pili was greatly enhanced by the addition of cholera toxin.

[0166]

[Table 18]

Example 16 Immunoelectron Microscopy of Neisseria gonorrhoeae Visualization of antiserum binding to pili-bearing bacteria was performed using the following procedure. Gold coated grids, late log phase of recA ^- pyridinium reduction gonococcus (N.Gonorrh
An aliquot from the liquid culture of E. oeae ) was spotted for 5 minutes and excess fluid was removed with a piece of filter paper. Grids were blocked with PBS-B for 5 minutes, followed by 1% (w / v) fish gelatin (Fluka, Ronkonkoma, NY) in PBS. The grid was then incubated for 1-60 minutes at room temperature with polyclonal antiserum diluted in PBS-B. Unbound antibody was removed by floating the grid over a drop of PBS-B (4 × 30 seconds). The bound primary antibody was a drop of 12 mm colloid conjugated to donkey anti-guinea pig IgG (Jackson Research Labs, West Grove, PA) diluted 1: 5 in PBS-B. Detected by floating grid over gold. The grid was then washed five times on the PBS-B droplets as described above. Next sample is PBS
1% (vol / vol) glutaraldehyde (electron microscopy)
Stabilize in Electron Microscopy Science, Fort Washington, Pa. For 3 minutes, then rinse in distilled water for 5 × 1 minute, and Nano
Light staining was performed for 30 seconds with Van staining (NanoProbes, Stony Block, NY) (pH 8). All liquids were removed by contacting the grit with a piece of filter paper and using an accelerating voltage of 80 kv at 15-75,000X.
eiss 10C transmission electron microscopy.

As shown by immunoelectron microscopy (FIG. 1A), antibodies to recombinant pilin bound along the length of the heterologous pyrifilament on the surface of the gonorrhea. This suggested that the antibodies bind to such epitopes that would be present on the surface of bacteria in vivo.

Example 17 Whole Cell ELISA for Neisseria gonorrhoeae r-pilin Oligomers To identify the biochemical and immunological properties of r-pilins derived from such intact pyri (or pilin oligomers), purified r-pilins were purified. It was converted to r-pilin oligomer by dialysis against a phosphate buffer of pH 12. The antiserum elicited by this material (r-pyrine oligomer) was examined using a whole cell ELISA for its ability to bind live goniosis with pilus. As shown in Table 6, antisera to r-pilin oligomers had significantly lower endpoint titers compared to antisera induced by untreated r-pilin with respect to binding to variegated gonococcal cells. This suggests that the r-pilin oligomer has lost a significant number of cross-reactive epitopes normally present on the r-pilin protein.

[0170]

[Table 19]

• Guinea pigs (4 per group) were immunized (sc) at 0 and 4 weeks with 20 μg of r-pilin antigen supplemented with 25 μg of Stimulon ™ QS-21. Six week pooled sera were analyzed. ★★ The purified r-pilin was dialyzed against 37 mM sodium phosphate (pH 12) at 4 ° C. for 48 hours, followed by 24 hours against PBS.

Example 18 Inhibition of Human Cervical Adhesion Pyri mediates the initial binding of N. gonorrhoeae to human mucosal cells, so that the r-pilin antibody binds to such bacterial epithelial cells. The ability to inhibit adhesion was investigated. ME-180 cells, which were derived from cervical cancer, were selected. Cells were also grown in liquid suspension culture to further minimize the aggregation of cells with pili during such experiments. This includes the relocation of P. gonorrhoeae Pgh3-1 or I756 strains to maintain expression of pili.
It was necessary to use the cA ^- derivative. Such recA ^- strains are 4 in liquid culture.
It shows no significant aggregation during 5 hours of growth, which makes interpretation of the results easier.

In such experiments, ME-180 cells were placed in 8-well chamber slides (Nunc).
Was seeded so that the cells were 80-90% confluent on the day of the experiment. recA^-Neisseria gonorrhoeae
The cells are swabbed in PBS (warmed to 37 ° C.) and 0.4% (w / v) NaHCO_Three
Into a flask of liquid GC medium supplemented with₆₀₀= 0.2
Used. Incubate at 37 ° C at 120 RPM for about 4 hours while shaking the cell culture.

[0174]

[Outside 1]

The liquid was diluted 1: 8 in RPMI 1640. The wells of the second 8-well chamber were incubated for at least 1 hour with 300 μL of RPMI 1640 and fetal calf serum. Discard the RPMI1640 block and add 40 μL of antiserum or RPMI1640 (fetal blood

[0176]

[Outside 2]

FU) was added and incubated for 1 hour at 37 ° C./5 (vol / vol)% CO ₂ . Chamber well slides containing ME180 cells are RPMI without antibody
Washed once with I1640. The mixture of pre-incubated bacteria and antiserum was then added to ME180 cells and slides were incubated at 37 ° C. for 30 minutes. The medium containing unbound bacterial cells was removed from the cervical cell monolayer and the wells were gently washed three times with RPMI1640. After the last wash, the chamber wells were removed from the slides, cells fixed with methanol for 30-60 seconds and stained with Wright-Giema stain (VWR Scientific, West Chester, PA). After decolorization with water, coverslips were placed on each well.

The slides were examined by light microscopy using oil immersion, and representative photographs were taken by individuals without judgment about the test area. The obtained photographs were also analyzed by a person without judgment on the identification of the sample. In addition, the effect of antiserum on gonococcal cell binding was quantified by counting bacterial agglutination instead of individual bacteria, since gonococci with pilus bound to the aggregated epithelial monolayer. The number of flocks of bacterial bacteria with pili observed in 10 random scans across each well was determined.
The percent difference between wells binding immune and normal serum was determined. Again, this analysis was performed individually by individuals without judgment on the sample being analyzed.

Initial results indicate that cells with or without pili are confluent with ME18
0 cells had a different binding pattern to the monolayer. Both strains of N. gonorrhoeae with pili typically bound selected epithelial cells within a confluent monolayer as bacterial aggregates. In contrast, bacteria without the corresponding genotype of pili have different binding to the epithelial monolayer (I756), either unbound (Pgp3-1) or exhibiting low levels. Thus, bacterial aggregation on epithelial monolayers correlates with the presence of pili.

Next, the ability of guinea pig antisera to Pgh3-1 r-pilin to inhibit the binding of pili-bearing cell line I-756 to ME-180 cells was tested. Analysis of a representative photograph (FIG. 2B)
FIG. 2A (comparison with week 6) vs. (week 0) showed that antibodies to r-pilin significantly inhibited the binding of pyriform gonococci to cervical epithelial cells. In contrast, r
The pilin antiserum was ineffective at binding non-pili-free cells from the same strain when compared to normal guinea pig serum (data not shown). Although the number of bacteria bound under these conditions could not be determined, adhesion was quantified by counting the aggregation of bound bacteria in the presence of either normal or immune serum.
Using this method, antisera to r-pilin reduced 凝集 60% of bacterial bound to epithelial cells compared to normal guinea pig serum.

Although this assay did not yield quantitative data that was easily read, the predictions obtained by counting bacterial agglutination probably resulted in an underestimation of the efficacy of the antisera. This is because the binding is mediated by other cell surface components (eg, Opa protein) that would not be overcome using antisera to rpyrin alone.

Example 19 Assay for Class Ir Pilin of Meningococcal Chimeras The assay described in Example 10 was used for chimeric Neisseria gonorrhoeae class Ir pilin. M
As determined by ALDI-TOF mass spectroscopy, the subunit molecular weight of the class Ir pilin protein of N. meningitidis chimera is 17,659 daltons, which is based on the amino acid content.
Very similar to the expected mass of 17,676 daltons. This protein, when analyzed by size exclusion chromatography using a Superose ™ 12 column equilibrated with PBS containing 0.05% (w / v) Zwittergent ™ 3-14, showed a class of chimeras.
Ir pilin has an apparent molecular weight of 69,480 daltons. This is essentially identical to the apparent molecular weight (68,899 daltons) of the gonorrhoeae r-pyrin analyzed under the same conditions.

The N-terminal sequence of the purified meningococcal class Ir pilin protein was determined by Edman degradation and the results (from three different samples) were consistent with the expected protein sequence. At least 35 residues were determined for all sequences.

Example 20 Class I r-pilin of meningococcal chimeras Endpoint titers against purified proteins or bacterial cells were determined by ELISA using the methods described in Examples 13 and 14. This ELISA was performed using pooled sera from each blood. Whole cell ELISA was performed with heat (60 ° C at 56 ° C).
Min) or dried directly on a microtiter plate for meningococcal cells. The cell suspension was diluted to an absorbance of 0.1 at 620 nm and a 100 μL aliquot was placed in a well of a microtiter plate. Each plate was dried at 37 ° C. or room temperature, sealed, and stored at 4 ° C. until use. Whole cell ELISA
The procedure was modified as follows: (1) Primary and secondary sera were in PBS containing 0.1% (v / v) Tween ™ -20 and 0.1% (w / v) BSA. Diluted; and (2) Plates were washed 5 times with a Skanwash 300 plate washer using PBS containing 0.05% (v / v) Tween ™ -20.

All guinea pigs immunized with the chimeric class Ir pilin reacted very well as evidenced by antigen ELISA as shown in Table 7.

[0186]

[Table 20]

★ Guinea pigs (4 per group) consisted of (a) 25 μg of Stimulon (PBS, pH 6)
Immunization (sc) at 0 and 4 weeks with (2) 100 μg of AlPO _{4 in} salt solution; or (c) 20 μg of chimeric class Ir pilin supplemented with PBS (pH 7) only. )
did. Animals were bled at 0, 4 and 6 weeks. Pool serum was used for all analyses.

Significant responses were also seen in ELISA against cells with pili, as shown in Table 8.

[0189]

[Table 21]

★ Cells were killed by heat. Effect of Adjuvant on Immune Response The effect of adjuvant on the immune response to N. meningitidis chimera against class I r-pilin was studied in mice using the method described in Example 13. As shown in Table 9, the most significant response for binding of the antiserum to the meningococcal chimera class I r-pilin was achieved with the addition of Stimulon ™ QS-21.

[0191]

[Table 22]

* Mice (10 per group) were: (a) 25 μg of Stimulon ™ (trademark) in PBS (pH 6)
) QS-21; (2) 100 μg of AlPO _{4 in} salt solution; (c) 50 μg of MPL in PBS (pH 7)
(D) 100 μg of AlPO ₄ and 50 μg of MPL ™ in salt solution; or (e) PBS (
Immunization (sc) was performed at 0 and 4 weeks with 10 μg of meningococcal chimera class Ir pilin supplemented with pH 7 only. Animals were bled at 0, 4 and 6 weeks. Pool serum was used for all analyses.

As shown in Table 10, the most significant response for binding of antiserum to pili-bearing cells from N. meningitidis was also achieved with the addition of Stimulon ™ QS-21. Was done.

[0194]

[Table 23]

★ Cells were killed by heat.

In a further analysis, this antiserum to the chimeric class Ir pilin was raised to class I
It has been shown to bind to pilins or, optionally, to class II pilins. The results, shown in Table 11, demonstrate this partial cross-reactivity.

[0197]

[Table 24]

★ The cells were dried directly (room temperature) on microtiter plates without heat killing.

Example 21 Immunoelectron microscopy of class I pilin of N. meningitidis chimerism N. meningitidis H355 of antiserum of class I r pilin of chimerism N. meningitidis
Visualization of binding to cells with pili from the strain was performed using a transmission electron microscope as follows. Colonies from the overnight culture of N. meningitidis were carefully removed using a sterile loop, and 0.5-1.0 mL of modified Franz
Medium [1.3 g / L glutamate, 20 mg / _{L-cysteine, 10g / L Na 2 HPO 4} · 7H 2 O, 90mg / 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 (5 mg / L)
) Was added to microcentrifuge tubes containing An aliquot of the cell suspension was spotted on the gold-coated grid for 5 minutes, an aliquot of the cell suspension was spotted for 5 minutes, and excess fluid was removed with a piece of filter paper. The bacterial cells were then fixed with 4% (v / v) paraformaldehyde, 0.1% (v / v) glutaraldehyde in PBS for 30 minutes at room temperature. Continuous grid (a) PB
Incubation with SB for 5 minutes, (b) 10% (w / v) fish gelatin in PBS for 10 minutes, and (c) 10 minutes with PBS containing 0.2 M glycine. The blocked grid was probed with antiserum to class I pilin protein of N. meningitidis chimeras as described in Example 16. As shown in FIG. 3A)
Antibodies against the class I pilin protein of the meningococcal chimera bound along the length of the pili. In contrast, normal serum (week 0) showed no binding to pili (FIG. 3B).

Example 22 Cross-reactivity of Meningococcal chimera class Ir pilin antiserum with gonococcal piri-bearing cells Based on the sequence similarity of meningococcal class Ir pilin and gonococcal pilin, In Example 4, the antiserum against gonococcal r-pilin recognizes meningococcal cells with pili,
And it was shown to combine. In this example, the meningococcal chimera class Ir
Antiserum raised against pilin is shown to bind to gonococcal cells with pili.
The data from the mouse and guinea pig experiments are summarized in Tables 12 and 13, respectively.

[0201]

[Table 25]

[0202]

[Table 26]

[0203] GC- gonococcal (N. gonorrhoeae); Nm meningococcal (N. meningitidis) Example 23 Neisseria meningitidis (N. meningitidis) meningitis class Ir pilin antisera chimeric Kinkinchisho An animal model accepted to evaluate the vaccine's ability to protect against passive protection against meningococcal bacteremia was originally described by Saukkonen and Leinonen (34)
It is a rat model of a child. Guinea pig antiserum against Meningococcal chimera class Ir pilin (supplemented with Stimulon ™ QS-21) was tested for its ability to protect against bacteremia caused by pilin-expressing meningococcal strains . On the 0th, Spragu
Rats (4-5 days old) of e-Dawley pups were 0.1 mL of guinea pig antiserum against chimeric class Ir pilin (6 weeks) (diluted 1: 5, 1:10 or 1:20 in PBS). Was used for passive immunization (ip). The control group received an injection of 0.1 mL of normal guinea pig serum (week 0) diluted 1: 5 in PBS. Twenty-four hours later, the animals were challenged (ip) with about 5 × 10 ⁵ colony forming units (cfu) in 0.1 ml of N. meningitidis (H355 strain) with pili. Three hours after challenge, animals were sacrificed and aliquots of heart blood were diluted and plated on GC agar plates. Then plate at 37 ° C, 5%
Incubated with CO ₂ for 18-24 hours. Count bacterial colonies,
And the level of bacteremia was determined. As one method of analysis, variance tt test
est) was used to compare a group receiving immune serum (6 weeks) with a control group receiving normal serum (0 weeks). As shown in Table 14 below, animals passively immunized with a guinea pig antiserum specific for the class Ir pilin of the meningococcal chimera were compared to animals immunized with normal guinea pig serum. Showed more than a log reduction in the level of bacteremia. This difference is substantially significant, with a p less than 0.05
Value.

[0204]

[Table 27]

★ Antisera to the meningococcal chimera class I pilin protein were obtained from guinea pigs as described in Table 7. ★★ p <0.05 cfu + std = colony forming unit ± standard deviation Example 24 Induction of mucosal immune response to meningococcal chimera class Ir pilin Mice were injected with meningococcal chimera class Ir pilin in saline 5μg) (
Cholera toxin was immunized intranasally at 0, 1 and 2 weeks with 1 μg of the cross-reactive mutant form (with or without the cross-reactive mutant form (CT-CRM, E29H)). As shown in Table 15 below, when animals were immunized with r-pyrine with or without adjuvant, a significant immune response was detected in the antigen ELISA. This response was enhanced by adding cholera toxin.

[0206]

[Table 28]

*: Pooled sera from week 4 were analyzed. The end point titer of pooled sera from week 0 was Ig
<50 for G and IgA. ★★: Washing was performed as follows: Bronchi: Lungs were washed 5 times with 1 mL RPMI 1640, then 50 μL fetal bovine serum (FBS) was added to the sample, which was centrifuged (2,000 × g × 5 min). ) And stored at -20 ° C.

Nasal: Nasal passages were washed once with 0.5 mL RPMI 1640 and stored at −20 ° C. after adding 20 μL of FBS to the samples.

Vagina: The vagina was washed 5 times with 0.075 mL RPMI 1640 and stored at −20 ° C. after adding 10 μL of FBS to the sample.

Example 25 Active Protection Against Meningococcal Colonization by Meningococcal Chimera Class Ir Pilin Antiserum The first step in meningococcal disease in humans is bacterial colonization in the nasopharynx. It is. In this process, the pill is thought to play an important role in mediating initial adhesion to epithelial cells. A number of investigators have described procedures for nasopharyngeal colonization in neonatal animals, but none have investigated this as a model to test the efficacy of meningococcal vaccines (35) . To evaluate the present invention as described herein, a nasal colonization model for N. meningitidis was developed using adult outbred mice. Swi
The ss-Webster mice were immunized at weeks 0, 4, and 8 with MPL ™ -reinforced meningococcal chimera class Ir pilin. At week 10, animals were injected intraperitoneally with 2 mg of dextran iron (Sigma) and with a volume of 10 μL meningococcus (including 40 μg dextran iron) with a metaphase of about 1 × 10 ⁷ cfu. An attack was induced in the nose. One day after challenge, half of the animals received a further intraperitoneal injection of 2 mg dextran iron.
The number of surviving bacteria in the nose was determined by plating nasal tissue homogenates on GC agar plates containing the selected antibiotics one and two days after challenge. FIG. 16 shows the results.

[0211]

[Table 29]

* All vaccines were formulated with 100 μg MPL ™ per dose. Each group consisted of 5 mice. ★★ Number of days since the nose attack was triggered.

Example 26 Western blot analysis of immune response to meningococcal class II chimeric r-pilin
Use meningococcal Class II chimeric r Pilin was analyzed purified, guinea pigs were immunized according to the procedure described in Example 11. Antisera from guinea pigs immunized with meningococcal class II chimeric r-pilin were first analyzed by Western blot (data not shown). These blots show whole cell lysis of meningococcal cells with pili expressing either class II pilin (FAM18) or class I pilin (H355) against antiserum against meningococcal class II chimeric r-pilin. This indicates that a band corresponding to pilin in the product was recognized. In contrast, E. coli containing only the pTrcHis vector ( Ec
oli ) did not react to any pilin bands in such Western blots.

Example 27 Meningococcal cells with antiserum pili against class II r-pilin of meningococcal chimera
The sera raised against the class II r-pilin of the purified meningococcal chimera was 36,4
It was shown to bind to meningococcal cells from allogeneic strain FAM18 at titers greater than 50 (0 week titer is 473).

[0215]

[Table 30]

[0216]

[Table 31]

[0219]

[Table 32]

[0218]

[Sequence list]

[Brief description of the drawings]

FIG. 1: Guinea pig antiserum against gonococcal r-pilin (derived from strain Pgh3-1) (1:50 dilution for 15 minutes), followed by donkey anti-guinea pig IgG (1), conjugated to 12 nm colloidal gold.
: 5 incubation for 30 minutes) and at a dilution, and stained Neisseria gonorrhoeae (N.gonorrhoeae) (I756 recA in NanoVan ^- represents a transmission electron micrograph of cells with pyridinium strains). FIG. 1A represents anti-r-pilin guinea pig immune serum (6 weeks); FIG. 1B represents normal guinea pig serum (week 0); FIG. 2C represents no primary antibody.

[Figure 2] guinea pig antiserum against gonococcal r pilin (derived from Pgh3-1 strain) is, Neisseria gonorrhoeae with pyridinium (N. gonorrhoeae) cell ^- to (I756 recA strain) human cervical cells (ME180 cell line) in The effect on the adhesion of FIG. 2A shows guinea pig serum against r-pilin (
FIG. 2B shows that normal guinea pig antiserum is incapable of preventing the attachment of piliated gonococcal cells to cervical cells (week 0). Aggregates measuring the size of representative bacteria bound to cervical cells are circled in each panel. Each panel shows four different views of the same experimental conditions. Guinea pig antiserum was diluted 1: 10,000 in each panel.

FIG. 3. Class Ir pilin of N. meningitidis chimera ( derived from H44 / 76 strain)
Guinea pig antiserum (1:60 dilution for 35 min), followed by donkey anti-guinea pig IgG conjugated to 12 nm colloidal gold (1: 5 dilution for 30 min) and stained with NanoVan 1 shows a transmission electron micrograph of cells having pili derived from N. meningitidis (H355 strain). FIG. 3A represents anti-r-pilin guinea pig immune serum (6 weeks); FIG. 3B represents normal guinea pig serum (0 weeks);
C represents no primary antibody. Cells were fixed before incubation with antiserum.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12N 1/21 C12P 21/02 C C12P 21/02 C12N 15/00 ZNAA (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, C , DE, DK, EE, ES, FI, GB, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR , TT, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventor Oy, Peggy 14506 Mendon Main Street Fushiyazu, New York, USA 494 F-term (reference) 4B024 AA01 AA13 BA31 CA03 CA07 CA20 DA06 EA04 GA11 GA19 GA27 HA03 4B064 AG31 CA02 CA19 CC01 CC24 CE02 CE03 CE06 CE07 CE08 CE09 CE11 DA01 DA15 4B065 AA01X AA01Y AA26X AB01 AC14 BA02 BA25 BD15 BD16 BD17 CA24 CA45 CA46 4C085 AA03 AA02 FF23 FF02 CC GG04 GG0 6 GG08 4H045 AA11 AA20 AA30 BA10 BA41 CA11 DA86 EA31 EA52 FA74 GA01 GA10 GA15 GA22 GA23 GA24

Claims (42)

[Claims] 1. A vaccine composition comprising an isolated and purified Neisseria recombinantly expressed pilin protein wherein the vaccine composition induces a protective immune response in a human host. 2. The vaccine composition according to claim 1, wherein the pilin protein is derived from Neisseria gonorrhoeae species. 3. The vaccine composition according to claim 1, wherein the pilin protein is derived from Neisseria meningitidis species. 4. The vaccine composition according to claim 3, wherein the pilin protein is a class I pilin protein of Neisseria meningitidis . 5. The vaccine composition according to claim 3, wherein the pilin protein is a class II pilin protein of Neisseria meningitidis . 6. pilin protein, Neisseria gonorrhoeae (Neisseria having the amino acid sequence of amino acids 8-167 of SEQ ID NO: 2 after processing of or has the amino acid sequence of amino acids 1-167 of SEQ ID NO: 2 before processing, or to the mature protein
gonorrhoeae ) and class I meningitidis ( Neisseria meningitidis ) chimeric recombinant pilin proteins or their biologically equivalent amino acid sequences.
The vaccine composition according to claim 1. 7. The vaccine composition according to claim 1, further comprising an adjuvant, diluent or carrier. 8. The group wherein the adjuvant is aluminum hydroxide, aluminum phosphate, Stimulon ™ QS-21, 3-O-deacylated monophosphoryl lipid A, IL-12 and wild-type or mutant cholera toxin. The vaccine composition according to claim 7, which is selected from: 9. A method of immunizing against Neisseria gonorrhoeae , comprising administering to a human host an immunogenic amount of the vaccine composition of claim 2. 10. A method of immunizing against Neisseria gonorrhoeae comprising administering to a human host an immunogenic amount of the vaccine composition of claim 3. 11. A method of immunizing against Neisseria gonorrhoeae , comprising administering to a human host an immunogenic amount of the vaccine composition of claim 6. 12. A method of immunizing against Neisseria meningitidis comprising administering to a human host an immunogenic amount of the vaccine composition of claim 3. 13. A method of immunizing against Neisseria meningitidis comprising administering to a human host an immunogenic amount of the vaccine composition of claim 2. 14. A method of immunizing against Neisseria meningitidis comprising administering to a human host an immunogenic amount of the vaccine composition of claim 6. 15. A vaccine composition comprising isolated and purified recombinantly expressed pilin protein of Neisseria in an amount sufficient to induce a protective immune response in a human host. A method for preparing a vaccine composition comprising: 16. Under standard high stringency Southern hybridization conditions,
Has the amino acid sequence of amino acids 1-167 of SEQ ID NO: 2 before processing,
Or Neisseria gonorrhoeae and Class I meningococcus ( N. ) having the amino acid sequence of amino acids 8-167 of SEQ ID NO: 2 after processing to the mature protein.
eisseria meningitidis ) or a DNA hybridizing with a DNA sequence encoding their biologically equivalent amino acid sequence.
An isolated and purified DNA sequence comprising the A sequence. 17. The DNA sequence of claim 16, wherein said DNA sequence hybridizes under standard high stringency Southern hybridization conditions to a DNA sequence having a nucleotide sequence of nucleotides 1 to 501 or 22 to 501 of SEQ ID NO: 1. Isolated and purified DNA sequence. 18. It has the amino acid sequence of amino acids 1-167 of SEQ ID NO: 2 before processing, or has the amino acid sequence of SEQ ID NO: 2 after processing into the mature protein.
Neisseria gonorrhoeae and class I Neisseria meningitidis chimeric recombinant pilin proteins having the amino acid sequence of amino acids 8 to 167, or DNAs encoding their biologically equivalent amino acid sequences. An isolated and purified DNA sequence consisting of: 19. Under standard high stringency Southern hybridization conditions,
Has the amino acid sequence of amino acids 1-167 of SEQ ID NO: 2 before processing,
Or Neisseria gonorrhoeae and Class I meningococcus ( N. ) having the amino acid sequence of amino acids 8-167 of SEQ ID NO: 2 after processing to the mature protein.
eisseria meningitidis ) or a DNA hybridizing with a DNA sequence encoding their biologically equivalent amino acid sequence.
A plasmid comprising the isolated and purified DNA sequence comprising the A sequence. 20. The plasmid of claim 19, wherein the plasmid comprises a DNA sequence that hybridizes under standard high stringency Southern hybridization conditions to a DNA sequence having the nucleotide sequence of nucleotides 1 to 501 or 22 to 501 of SEQ ID NO: 1. The plasmid according to 1. 21. A Neisseria gonorrhoeae ( Neis) comprising the DNA sequence of claim 18.
seria gonorrhoeae ) and plasmids containing isolated and purified DNA sequences encoding chimeric recombinant pilin proteins of class I Neisseria meningitidis . 22. The plasmid according to claim 21, wherein the plasmid was named pPX2004 (ATCC 98637). 23. A host cell transformed with the plasmid of claim 19. 24. The host cell according to claim 23, wherein the host cell is an E. coli strain. 25. The host cell according to claim 24, wherein the plasmid is named pPX2004 (ATCC 98637). 26. Transforming or transfecting a host cell with the plasmid of claim 19 and culturing the host cell under conditions that allow expression of the chimeric recombinant pilin protein by the host cell. Consists of Neisseria gonorrhoeae
ria gonorrhoeae ) and a class I Neisseria meningitidis chimeric recombinant pilin protein production method. 27 has the amino acid sequence of amino acids 1-167 of SEQ ID NO: 2 before processing, or has the amino acid sequence of SEQ ID NO: 2 after processing into the mature protein
Isolated and purified chimeric recombinant pilin proteins of Neisseria gonorrhoeae and Class I Neisseria meningitidis having the amino acid sequence of amino acids 8 to 167, or their biologically equivalent amino acids Array. 28. A gonococcus ( Neisser ) wherein the pilin protein has the amino acid sequence of amino acids 1-170 of SEQ ID NO: 4 before processing or has the amino acid sequence of amino acids 8-170 of SEQ ID NO: 4 after processing into the mature protein.
The vaccine composition according to claim 1, which is a chimeric recombinant pilin protein of C. ia gonorrhoeae ) and class I meningitidis ( Neisseria meningitidis ), or a biologically equivalent amino acid sequence thereof. 29. A method of immunizing against Neisseria gonorrhoeae comprising administering to a human host an immunogenic amount of the vaccine composition of claim 28. 30. A method of immunizing against Neisseria meningitidis comprising administering to a human host an immunogenic amount of the vaccine composition of claim 28. 31. Under standard high stringency Southern hybridization conditions,
Has the amino acid sequence of amino acids 1-170 of SEQ ID NO: 4 before processing,
Or Neisseria gonorrhoeae and Class II meningococci having the amino acid sequence of amino acids 8-170 of SEQ ID NO: 4 after processing to the mature protein
Neisseria meningitidis ) or a DNA hybridizing with a DNA sequence encoding their biologically equivalent amino acid sequence.
An isolated and purified DNA sequence comprising an NA sequence. 32. The method of claim 31, wherein said DNA sequence hybridizes under standard high stringency Southern hybridization conditions to a DNA sequence having a nucleotide sequence of nucleotides 1 to 510 or 22 to 510 of SEQ ID NO: 3. Isolated and purified DNA sequence. 33. It has the amino acid sequence of amino acids 1-170 of SEQ ID NO: 4 before processing, or has the amino acid sequence of SEQ ID NO: 4 after processing into the mature protein.
Chimeric recombinant pilin proteins Neisseria gonorrhoeae having the amino acid sequence of amino acids 8~170 (Neisseria gonorrhoeae) and Class II meningococcal (Neisseria meningitidis), or the DNA sequences encoding them biologically equivalent amino acid sequence An isolated and purified DNA sequence comprising: 34. Under standard high stringency Southern hybridization conditions,
Has the amino acid sequence of amino acids 1-170 of SEQ ID NO: 4 before processing,
Or Neisseria gonorrhoeae and Class II meningococci having the amino acid sequence of amino acids 8-170 of SEQ ID NO: 4 after processing to the mature protein
Neisseria meningitidis ) or a DNA hybridizing with a DNA sequence encoding their biologically equivalent amino acid sequence.
A plasmid comprising the isolated and purified DNA sequence comprising an NA sequence. 35. The plasmid according to claim 34, wherein the plasmid comprises a DNA sequence that hybridizes under standard high stringency Southern hybridization conditions to a DNA sequence having the nucleotide sequence of nucleotides 1 to 510 or 22 to 510 of SEQ ID NO: 3. The plasmid according to 1. 36. A Neisseria gonorrhoeae ( Neis) comprising the DNA sequence of claim 33.
seria gonorrhoeae ) and plasmids containing isolated and purified DNA sequences encoding chimeric recombinant pilin proteins of class II Neisseria meningitidis . 37. The plasmid according to claim 36, wherein the plasmid is named pPX8017 (ATCC 207199). 38. A host cell transformed with the plasmid of claim 34. 39. The host cell according to claim 38, wherein the host cell is an E. coli strain. 40. The host cell according to claim 39, wherein the plasmid is named pPX8017 (ATCC 207199). 41. Transforming or transfecting a host cell with the plasmid of claim 34 and culturing the host cell under conditions that permit expression of the chimeric recombinant pilin protein by the host cell. Consists of Neisseria gonorrhoeae
ria gonorrhoeae ) and a class II Neisseria meningitidis chimeric recombinant pilin protein production method. 42. It has the amino acid sequence of amino acids 1-170 of SEQ ID NO: 4 before processing, or the sequence of SEQ ID NO: 4 after processing into the mature protein.
An isolated Neisseria gonorrhoeae having the amino acid sequence of amino acids 8~170 (Neisseria gonorrhoeae) and Class II meningococcal (Neisseria meningitidis), and purified chimeric recombinant pilin proteins, or their biologically equivalent Amino acid sequence.