CA2325055A1

CA2325055A1 - Vaccines containing recombinant pilin against neisseria gonorrhoeae or neisseria meningitidis

Info

Publication number: CA2325055A1
Application number: CA002325055A
Authority: CA
Inventors: Thomas N. Metcalf, Iii; Robert J. Zagursky; Peggy Ooi
Original assignee: Individual
Current assignee: Wyeth Holdings LLC
Priority date: 1998-04-29
Filing date: 1999-04-29
Publication date: 1999-11-04
Also published as: EP1073748A2; AU2003204738B2; WO1999055875A3; WO1999055875A2; KR20060118629A; KR20060118628A; CN1245512C; IL139311A0; KR20010043105A; BR9910005A; JP2002527041A; CN1298446A; AU3968599A; KR20060118630A

Abstract

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

Description

VACCINES CONTAINING RECOMBINANT PILIN AGAINST
NEISSERIA GONORRH!OEAE OR NEISSERIA MENINGITIDIS
Field of the Invention This invention relates to the use of recombinant pilin proteins in vaccines to protect against disease caused by Neisseria gonorrhoeae or Neisseria meningltld.is.
Background of the Invention Neisser3a gonorrhoeae (N. gonorrhoeae) and Neisseria meningitidis (N. meningitidis) are Gram-negative cocci. N. g~onorrhoeae and N. meningit3dis are genetically very closely related, but the clinical manifestations of the diseases they produce are very different. N. gonorrhoeae causes gonorrhea, while N.
meniagftidis causes meningococcal meningitis. These bacteria of the genus Nelsseria inhabit mucosal surfaces of the body.
Type IV pili are nonflagellar hairlike structures on the surface of numerous Gram-negative bacteria, including D3chelobacter (formerly Bactero3des) nodous, E~kenella corrodens, Kingella denitr.if3cans, Moraxella bovis, M. lacunata, M.
nonliquefaciens, N. g~onorrhoeae, N. meningitfdis, and Pseudomonas aerug~,aosa (Bibliography Entries 1,2). The toxin co-regulated pili from Vibrio cholerae and the bundle forming pili of enteropathogenic Escherchia coli exhibit a limited number of similarities to type IV
pill and are considered to be more distantly related (1,2). For both N. g~onorrhoeae and N. meaingitidis, piliated bacteria adhere to a variety of epithelial cells of human origin much more avidly than do nonpiliated cells, and thus the pili are thought to act as virulence factors by anchoring the organisms to mucosal infection sites.
Type IV pili are 5-7 nM in width and up to 5 ACM in length. The pilin protein subunits are linked in tandem to form long, thin polymers. In the case of the pathogenic Neisser:ia, the pili are apparently homopolymeric in nature, being comprised of a single structural subunit" the pilin protein. The pilin 1.0 protein has a molecular weight of 13,000 to 22,000 daltons (1,2,3) .
In nature, the pilin protein is assembled into a helical structure called a pilus (plural: pili) at the bacterial outer membrane that has a molecular l5 weight of approximately 10' daltons. When purified pili are dialyzed against pH 12 phosphate buffer, the intact pili are irreversibly dissociated into aggregates of the pilin protein which are called pilin oligomers (4). These pilin oligomer aggregates 2'.0 (molecular weight of approximately 600,000 daltons) are much smaller in size than the intact pilus.
N. gonor=:hoeae expresses a single pilin protein which was first isolated and sequenced by Schoolnik and co-workers (5). The gonococcal pilin 25 protein consists of: three regions: (a) the highly conserved amino terminal region (residues 1-53); (b) the middle third (residues 54-124) which exhibits a limited amount of ~oeguence variation and (c) the carboxy third of trae protein (residues 125-160) which 30 contains a highly variable disulfide loop.
During the course of natural infections, pili undergo high frequency phase and antigenic variation (3,6). The genetics of this variation are extraordinarily complex and have been extensively 35 studied. Each strain of Neisseria gonorrhoeae has the WO 99/55875 PCT/US99/094$6 ability to change t:he primary amino acid sequence of the pilin molecule, and thus the antigenic nature of its pili. The molecular mechanism responsible for this variation involves a nonreciprocal recombination event between the expression locus (pilE) and numerous (17 to 19), promoterless, silent (pilS) genes (6). Pilin sequences (or portions thereof) move from the pilS loci into an expression locus to generate new pilin variants, which in turn enables gonococci to express an extremely large number of pilin proteins.
In contrast to N. gonorrhoese, N.
meniagitidis expresses two distinct classes of pilin named class I and class II. As in the gonococci, the meningococcal class I pili undergo antigenic and phase 1:5 variation (3). Class I pilins have been shown to be similar to gonococcal pilin in terms of molecular weight (17-20 kd) and reactivity with a monoclonal antibody (SM1) which binds to a highly conserved epitope on the gonococcal pilin (3). A number of 21) class I pilins have been cloned and the amino acid sequences have been shown to have a high degree of similarity to the sequence of the gonococcal pilin (7).
In contrast, class II pilins do not react with the SM1 antibody and have a lower molecular weight (13-16 kd).
2'.i Several strains expressing class II pilin have been shown to react with a polyclonal antisera directed against gonococcal ;pili (3). Aho et al. (7) have recently determined the sequence of a class II pilin.
The first third of the neisserial pilins are 3(1 essentially identical. The class II pilin protein differs from the class I pilin and gonococcal pilin proteins in the hypervariable region of the protein where a large deletion has occurred. Achtman et al.
(8), using monoclonal antibodies specific for class I
3'~ or class II pilins, demonstrated that some serogroup A

isolates from Africa bound both antibodies. It has been demonstrated that strains expressing class II pili also have truncated, silent class I pilin genes (7).
Taken together, these data suggest the possibility that a single meningococcal cell might express both pilin proteins simultaneously.
Because pili are believed to mediate the initial contact with mucosal cells, there has been considerable interest in using these structures as vaccine antigens to prevent disease caused by piliated bacteria. Pili vaccines against travelers diarrhea and gonorrhea have been tested in human beings (9).
However, to date, they have been efficacious only against homologous strains. A number of pili-based vaccines have been reported for diseases affecting domestic livestock such as infectious keratoconjunctivitis (pinkeye) i.n cattle (1), footrot in sheep (1,10) anc~ diarrhea in piglets (9) or calves (9) . In each of these veterinary examples, the pili vaccine provided protection against challenge by strains expressing the homologous, but not heterologous, pili.
The earliest gonococcal vaccines contained whole organisms and provided little or no protection (11). Recent vaccine development against gonorrhea has focused on purifiedL surface components, in particular, pili (9,11) and they porin protein (P.I or Por) (11).
To date, however, only pili have been shown to protect humans from challenge, and this was limited to protection against the homologous strain (12). A
denatured form of gonococcal pili (4) has been shown to generate antibodies; in mice or guinea pigs which bind to heterologous pil,i in vitro. However, this has not been considered a commercially viable approach because 3.5 of the difficulty in growing piliated gonococci in liquid media (a necessity for commercial production) (1). Based on the success of the initial human challenge studies, the gonococcal piles vaccine was tested in a large, placebo-controlled double blind efficacy trial (12;). In this trial, the vaccine failed to protect male volunteers from gonococcal infections.
It was postulated ithat the most likely explanation for this was piles heterogeneity (12).
Indeed, the antigenic variability of the 1.0 pilin proteins, both gonococcal and meningococcal, has been repeatedly cited as a major obstacle in the development of pil:i-based vaccines (3,13,14,15,16,17).
It has been suggested that the dominant immune epitope on the assembled, :Lntact piles is the disulfide loop, which exhibits the greatest sequence variation (17,18).
This may account for the failure of the Korean field trial with formalin-treated intact pili from N.
goaorrhoeae strain Pgh3-2 (12). In addition, the literature contains a number of references in which f,0 antisera directed against purified pili, or pilin fragments, bound to denatured (Western blot) or isolated neisserial pili, but did not bind to heterologous pili an bacterial surfaces (16,17,19,20).
Whether this is due: to antigenic variation or concealment of the epitopes in the assembled piles has not been completely resolved. This is reinforced by several reports which demonstrated that the only monoclonal antibodies exhibiting functional activity in vitro were those which did not bind to heterologous pili (3). It has been shown for the intact pili from M. bovis (21) and Ia. nodosus that a protective immune response elicited by pili is possible (10). These vaccines, however, were only able to protect against challenge with bacteria expressing the homologous pili -- not heterologou~c pili (10,21). The consensus of the scientific community appears to be that pili-based vaccines, if possible, will protect only against bacteria expressing the homologous pili.
Recombinant expression of assembled pili has been described for a number of organisms, and is dependent on the presence of appropriate transport and assembly genes (22). In Neisseria, the genes encoding the proteins involved in pili assembly and export are not found in a single contiguous operon, so this :l0 approach was not feasible. Another alternative chosen in European Patent 202,260 Bl was to express type IV
pilin genes in a bacterial host which already possessed the proteins required for assembly of a different type IV pilus; e.g., Pseudomonas aeruginosa (23). But as l.5 reported by Hoyne et al., the expression and assembly of gonococcal pill at the outer membrane of Pseudomonas was unstable (24). When the recombinant strain was grown in liquid media in the presence of selective antibiotics, it was overgrown by wild-type, piliated c0 Pseudomonas. The authors stated:
" [T] he compatibility of foreign mePhe [N-methylphenylalanine] pilus production in host strains will depend on the extent of divergence of host and donor pilus assembly systems. The 25 observed instability of PAK/2PfS [Pseudomonas aeruginosa sts:ain K/2PfS] expressing gonococcal pilin ... may indicate that the limits of interspecies expression of mePhe pili are being approached in this instance." (24) 30 Because of this result, the expression of gonococcal pili in Pseudomonas was not viewed as a commercially viable approach.
Elleman, Egerton and co-workers described the development of a vaccine against ovine footrot using 35 intact pili from Di.chelobacter nodosus. Field trials had demonstrated that intact pili protected against D.
nodosus strains expressing the homologous pili. This meant that a commercial vaccine would need to contain eight or nine different pili in order to achieve S comprehensive protection. In an attempt to make this approach viable, the pilin gene of D. nodosus was cloned and expressed in E. coli (25). The recombinant pilin protein was found associated with the inner membrane. When a vaccine consisting of sonicated E.
coli cells expressing the recombinant pilin was tested in a challenge experiment, the recombinant E. coli cells generated an antibody titer similar to that seen for purified, native pili (25). However, the agglutination titer induced by the recombinant E. colf IS cell vaccine was significantly lower than that seen for the intact pili (6u0 vs. 47,800) and below the titer which correlated with protection (5,000-10,000) (25, 26) .
Following active challenge with D. nodosus, the recombinant pil.in vaccine failed to show any significant protective activity, in contrast to that seen for the intact: pili. Emery and co-workers had demonstrated that denaturation of intact pili abolished the ability of pili.n to induce protection in animals (27). In addition, pili dissociated with either detergent (octyl-~i-D-glucoside) or low pH (2.2) reduced the effectiveness of the protein to elicit protection against formation c~f severe lesions following challenge (28). This was despite the fact that the antibody titers were not significantly different between the groups. The authors stated that "there may be one or more epitopes associated with quaternary structure which are disturbed by the treatments". They further stated:

- g _ nThe failure of the E. coli expressed product as a vaccine ma.y result from its physical occlusion in the host cell membranes, although preliminary experiments indicated that this was not the major cause of its ineffectiveness (unpublished data). An alternative explanation for the failure of the E. colj expressed product as a vaccine, is that the monomeric prepilin units which are expressed are unable to associate into a native conformation for the appropriate presentation of important epitopes, possibly because of the presence of the leader sequence." (28).
Elleman and co-work:ers confirmed the importance of the presence of conformational epitopes on the recombinant pilin arid that they needed to increase the immune response by a better presentation of the antigen. They proposed two approaches: purification of the protein from the E. col~f membranes, or expression of protein in 2~~ P. aeruginosa so that it could assemble into filamentous pili on the surface of the cell. The latter approach was preferred because "[t]his should greatly improve the immunogenic properties and simplify the purification of the protein" (25).
Furthermore, Elleman and co-workers also viewed the use of pilin (the subunit protein of pili) as an inferior vaccine candidate to the mature fimbriae (intact pili) for D. nodosus:
"Mature fimbriae appear to provoke a more intense 30 and appropriate (i.e., K-agglutinating) immunological :reaction than the equivalent dose of fimbrial subun:it protein. A serological K-agglutination titre of about 5,000 is generally regarded as the minimum response commensurate with 3'' adequate protective immunity against infection 9 _ with a given strain of B. nodosus. This level of response (and up to an order of magnitude higher) is readily achieved upon vaccination with mature fimbriae, but not the isolated subunit protein, which elicits only poor levels of serum K-agglutinating antibodies" (23).
Additional data suggesting that the fimbrial (pilus) subunit protein is not a viable vaccine candidate was recently reported by Alves et al. (29). When mice were immunized with a polynucleotide vaccine encoding the E.
coli CFA/I fimbria7. adhesin protein (e.g., pilin), the antibodies induced were distinct from those induced by native, intact CFAfI fimbriae. Moreover, these antibodies against the recombinant protein did not exhibit any agglutination activities in contrast to antisera against the native protein.
Despite all the work described above, there is yet to be developed an effective pilus-based gonococcal or meningococcal vaccine. Meningococcal vaccines are limited to those possessing serotype A, C, Y, W135 capsules.
Accordingly, there is a need to identify components for inclusion in vaccines to protect against disease caused by N'. gonorrhoeae or all serotypes of N.
2:i meningi tidis .
Summary of the Invention Thus, it is an object of this invention to 3() identify suitable antigenic structures derived from N.
gonorrhoeae and N. :meniagitidis, respectively, which may constitute viable vaccine candidates against those bacteria. These candidates must induce antibodies which recognize and bind to diverse isolates of the 3'~ respective pathogenic neisserial organism.

These and other objects of the invention as discussed below are: achieved by the cloning and expression of the recombinant pilin protein (rpilin) of each of N. gonorrhaeae and N. meningit:idis.
This invention also relates to the construction of a plasmid which expresses a recombinant meningococcal chimeric class I pilin protein in which the amino-terminal region of the class I meningococcal pilin protein is replaced by the corresponding amino-1~~ terminal region of the gonococcal pilin protein. This plasmid expresses significantly higher amounts of the meningococcal chimeric class I rpilin protein than the class I meningococcal rpilin protein expressed from a full-length meningococcal pilE gene.
l:> In order to obtain expression of the meningococcal chimeric class I rpilin protein, the chimeric DNA sequence is first inserted into a suitable plasmid vector. A suitable host cell is then transformed or transfected with the plasmid. In an 20 embodiment of this ,invention, the host cell is an Escherichia coli strain. The host cell is then cultured under conditions which permit the expression of said chimeric class I rpilin protein by the host cell.
This invention further relates to the construction of a p:Lasmid which expresses a recombinant meningococcal chimeric class II pilin protein in which the carboxy-terminal region of the class II
meningococcal pilin protein is replaced by the 30 corresponding carboxy-terminal region of the gonococcal pilin protein.
In order t:o obtain expression of the meningococcal chimeric class II rpilin protein, the chimeric DNA sequence is first inserted into a suitable 35 plasmid vector. A suitable host cell is then transformed or transfected with the plasmid. In an embodiment of this invention, the host cell is an Escherichia coli strain. The host cell is then cultured under conditions which permit the expression of said chimeric c7.ass II rpilin protein by the host cell.
In another embodiment of this invention, the isolated and purified rpilin protein (either the gonococcal, the meningococcal or chimerics) is used to prepare a vaccine composition which elicits a protective immune response in a mammalian host. The vaccine composition may further comprise an adjuvant, diluent or carrier. Examples of such adjuvants include aluminum hydroxide, aluminum phosphate, MPL'"', Stimulon'""
QS-21, IL-12 and cholera toxin. The vaccine composition is administered to a mammalian host in an immunogenic amount sufficient to protect the host against disease caused by N. gonorrhoeae or N.
meniagi tidis .
2~7 Brief Description of the Fiaurea Figure 1 depicts transmission electron micrographs of piliated cells from N. gonorrhoese 2:i (strain I756 recA-) incubated with guinea pig antiaera directed against gonococcal rpilin (from strain Pgh3-1) (1:50 dilution for 15 minutes), followed by donkey anti-guinea pig IgG conjugated to 12 nm colloidal gold (1:5 dilution for 30 minutes and stained with NanoVan.
3C1 Figure 1A depicts anti-rpilin guinea pig immune sera (week 6); Figure 1B depicts normal guinea pig sera (week 0); Figure 2C depicts no primary antibody.
Figure 2 depicts the effect of guinea pig antiaera directed against gonococcal rpilin (from 35 strain Pgh3-1) on the attachment of piliated N.

gonorrhoeae cells (strain I756 recA-) to human cervical cells (ME180 cell line). Figure 2A depicts the inhibition of attachment by guinea pig antisera directed against rpilin (week 6); Figure 2B depicts the inability of normal guinea pig antisera to prevent attachment of piliated gonococcal cells to cervical cells (week 0). Representative sized clumps of bacteria bound to cervical cells are circled in each panel. Each panel shows four different views of the LO same experimental condition. The guinea pig antisera was diluted 1:10,000 for each panel.
Figure 3 depicts transmission electron micrographs of pil:iated cells from N. meningitidis (strain H355) incubated with guinea pig antisera directed against meningococcal chimeric class I rpilin (from strain H44/7!i) (1:60 dilution for 30 minutes), followed by donkey anti-guinea pig IgG conjugated to 12 nm colloidal gold (1:5 dilution for 30 minutes) and stained with NanoVan. Figure 3A depicts anti-rpilin guinea pig immune sera (week 6); Figure 3B depicts normal guinea pig sera (week 0); Figure 3C depicts no primary antibody. Cells were fixed before being incubated with antisera.
Detailed Description of the Invention This invention relates to vaccine compositions comprising a recombinant pilin protein of N. gonorrhoeae or ff. men~ngitidis. Notwithstanding the teachings of the art discussed above, it was decided to investigate the use of such recombinant pilin proteins expressed in E. coli. Surprisingly, these recombinant pilin proteins demonstrated characteristics of vaccine candidates.

The first report describing the cloning of the gonococcal pjlE gene in E. coli was in 1982 (30).
Since then, molecular characterization of pilE has been performed by numerous laboratories investigating the genetic factors controlling the expression of the pilin protein, transport of the pilin protein, variation in the pilin sequence and the host adherence properties of pili. However, none of the reports described the purification of recombinant pilin protein nor the 1.0 immune response of the recombinantly expressed pilin protein.
Cloning and expression of the pilE gene encoding the gonococcal recombinant pilin protein are described in Example 2 below. Expression was achieved by transforming an E. coli strain designated TOPlOF' with a plasmid containing the p.ilE gene. Successful cloning and express;ion was followed by the sequencing of the pilE gene to confirm identity with the native sequence. To assist in cloning, a Ncol site was introduced, which required modifying one base. As a result, the second amino acid in the seven amino acid long signal peptide was changed from asparagine to aspartic acid.
The plasmid containing the pilE gene in Example 2 (designated pPX2000) contains an ampicillin resistance (Amps) marker. As described in Example 3, another plasmid was constructed to contain a kanamycin resistance (Kana) marker instead of AmpR. This plasmid, designated. pPX2002, after transforming E. coli 3~D strain TOPlOF', expressed the gonococcal rpilin at a level similar to that obtained from pPX2000, which contains an AmpR marker.
As described in Example 4, a similar procedure was used to construct a plasmid, designated 3:> pPX2003, containing the class I pilE gene of N.

meningitidis. A NcoI site (CC ATG G) was introduced spanning 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 (the first residue remained methionine). An AmpR
marker was also included. This construct, after transforming E. co.Ii strain TOP10F', expressed class I
rpilin of N. meningitidis. However, the expression level was significantly lower than that for the :LO gonococcal rpilin .obtained from either pPX2000 or pPX2002. Without being bound by theory, this lower expression level may be due to a number of inverted repeats which are present in the recombinant class I
pilE.
In order to increase the expression of the meningococcal pilin, as described in Example 5, a chimeric plasmid was constructed. The DNA in pPX2003 encoding the first 60 amino acids of the meningococcal class I rpilin is replaced with the equivalent region 20 from the gonococca7L DNA in pPX2002. The resulting AmpR
piasmid, designated pPX2004, has the nucleotide sequence set forth in SEQ ID NO:1. The plasmid pPX2004 was used to transform an E. coli strain K12 designated TOPlOF'. Following induction, there was a significant 25 increase in expression of the chimeric rpilin compared to the amount of me:ningococcal rpilin expressed from pPX2003. The level of expression of the chimieric construct was comparable to the amount of gonococcal rpilin expressed from pPX2002. The chimeric class I
3~0 rpilin was 167 amina acids in length (including the signal) (SEQ ID N0:2), which is in accordance with the predicted size.
Samples o~f the E. coli strain K12 designated TOP10F' harboring the recombinant plasmid pPX2004 were 3:> deposited on January 27, 1998 by the Applicants with the American Type Culture Collection, 10801 University Boulevard, Manassas, Virginia 20110-2209, U.S.A., and have been assigned ATCC accession number ATCC 98637.
As described in Example 6, a chimeric plasmid was constructed wherein the 3' end of the class II p.ilE
gene of N. meningi~tidis was replaced with the corresponding region from N. gonorrhoeae.
Specifically, the DNA in pPX8001 encoding the disulfide loop (the last 22 aatino acids of the meningococcal :l0 class II pilin of N. meningitides strain FAM18) is zeplaced with a similiar (but larger) region plus additional portions of the carboxy-terminal region totalling 44 amino acids from the gonococcal (p~lE) DNA
from N. gonorrhoeae strain Pgh3-1 in pPX2000. The 1.5 resulting Amps plasmid, designated pPX8017, has the nucleotide sequence set forth in SEQ ID N0:3, in which nucleotides 1-378 are from N. men~ngjtidis class II and nucleotides 379-510 are from N. gonorrhoese. The plasmid pPX8017 waea used to transform the E. coli 20 strain R12 designated TOP10F'. Following induction, a chimeric class II ~:pilin was expressed which was 170 amino acids in length (including the seven amino acid long signal) (SEQ 7:D N0:4), in which amino acids 1-126 are from N. meningi:t~dis class II and amino acids 127-2',5 170 are from N. gonorrhoeae. This chimeric class II
rpilin was in accordance with the predicted size. A
NcoI site was introduced for cloning considerations, which changed the second amino acid in the signal sequence from lysine to glutamic acid. This change was 30 not expected to have any effect on antigenicity or immunogenicity.
Samples of the E. coli strain K12 designated TOP10F' harboring t:he recombinant plasmid pPX8017 were deposited on April 15, 1999 by the Applicants with the 35 American Type Culture Collection, 10801 University Boulevard, Manassas, Virginia 20110-2209, U.S.A., and have been assigned ATCC accession number ATCC 207199.
A variety of host cell-vector systems are suitable for use to express the gonococcal, meningococcal and chimeric rpilins used in the vaccines of this invention in addition to those detailed in Examples 2-6. The 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; yeast such as Pichia cells; insect cells such as Sf9 or Sf2l. cells; or mammalian cell lines such as Chinese hamster ovary cells; as well as other conventional organisms.
A variety of conventional transcriptional and translational elements can be used for the host cell-vector system. The: pilE DNA is inserted into an expression system and the promoter and other control elements are ligated into specific sites within the vector, so that when the plasmid vector is inserted into a host cell, the pilE DNA can be expressed by the host cell.
The plasmid is introduced into the host cell by transformation, transduction, transfection~or infection, depending on the host cell-vector system used. The host cell is then cultured under conditions which permit expression of the rpilin protein by the host cell.
This invention further relates to an isolated 3~~ and purified DNA sequence comprising a DNA sequence encoding the meningococcal chimeric class I rpilin protein whose amino-terminal region is from the gonococcal p~lE gene and whose central and carboxy-terminal regions are from the meningococcal pilE gene (SEQ ID NO: l). Nucleotides 1-501 in SEQ ID NO:l encode WO 99/55875 PC'T/US99/09486 the meningococcal chimeric class I rpilin protein prior to processing; nucleotides 22-501 encode the meningococcal chimeric class I rpilin protein after processing to a mature protein. The invention S additionally relates to the meningococcal chimeric class I rpilin protein having the amino acid sequence of amino acids 1-167 of SEQ ID N0:2 prior to processing or having the amino acid sequence of amino acids 8-167 of SEQ ID N0:2 after processing to a mature protein.
Approximately 10~ of the total protein produced by the gonococcal rpilin or the meningococcal chimeric class I
rpilin constructs 7Lacks the signal sequence, which has been removed by processing.
This invention further relates to an isolated and purified DNA sequence comprising a DNA sequence encoding the meningococcal chimeric class II rpilin protein whose carboxy-terminal region is from the gonococcal pflE gene and whose central and amino-terminal regions are from the meningococcal pilE gene (SEQ ID N0:3). Nucleotides 1-510 in SEQ ID N0:3 encode the meningococcal chimeric class TI rpilin protein prior to processing; nucleotides 22-5I0 encode the meningococcal chimeric class II rpilin protein after processing to a mature protein. The invention 2.5 additionally relates to the meningococcal chimeric class II rpilin protein having the amino acid sequence of amino acids 1-170 of SEQ ID N0:4 prior to processing or having the amino acid sequence of amino acids 8-170 of SEQ ID N0:4 after processing to a mature protein.
In addition to the chimeric DNA sequences contained in pPX2004 and pPX8017 which encode the meningococcal chimeric class I rpilin protein and the meningococcal chimeric class II rpilin protein, respectively, the present invention further comprises 3_'s DNA sequences which, by virtue of the redundancy of the _ lg _ genetic code, are biologically equivalent to the sequences which encode for the chimeric rpilin proteins, that is, these other DNA sequences are characterized by nucleotide sequences which differ from those set forth herein, but which encode a protein having the same amino acid sequence as that encoded by the DNA sequence in. SEQ ID NO:1 or SEQ ID N0:3.
In particular, the invention contemplates those DNA sequences which are sufficiently duplicative l~D of the sequence of SEQ ID N0:1 or SEQ ID N0:3 so as to permit hybridization therewith under standard high stringency Southern hybridization conditions, such as those described in Sambrook et al. (31).
This invention also comprises DNA sequences 1:~ which encode amino acid sequences which differ from those of the meningococcal chimeric class I or class II
rpilin proteins, but which are biologically equivalent to those described for one of these proteins (SEQ ID
N0:2 or SEQ ID N0:4). Such amino acid sequences may be 2l) said to be biologically equivalent to those of the chimeric rpilin protein if their sequences differ only by minor deletions from, insertions into or substitutions to the rpilin sequence, such that the tertiary configurations of the sequences are 2'.> essentially unchanged from those of the rpilin protein.
For example, a codon for the amino acid alanine, a hydrophobic amino acid, may be substituted by a codon encoding another less hydrophobic residue, such as glycine, or a more hydrophobic residue, such as 3(I valine, leucine, or isoleucine. Similarly, changes which result in substitution of one negatively charged residue for another, such as aspartic acid for glutamic acid, or one positively charged residue for another, such as lysine for arginine, as well as changes based 3'~ on similarities of residues in their hydropathic index, can also be expected to produce a biologically equivalent product. Nucleotide changes which result in alteration of the N-terminal or C-terminal portions of the protein molecu:Le would also not be expected to alter the activity of the protein.
Furthermore, changes in known variable regions are biologically equivalent where the tertiary configurations of t:he conserved regions are essentially unchanged from those of the rpilin protein. An alternative definition of a biologically equivalent sequence is one that is still capable of generating a cross-reactive immune response. In particular, the meningococcal chimeric class I and II recombinant pilins may be modified by lengthening or shortening the corresponding insertion from the gonococcal pilin, as long as the modified chimeric recombinant pilin is still capable of generating a cross-reactive immune response.
Each of t;he proposed modifications is well within the routine skill in the art, as is determi-nation of retention of structural and biological activity of the encoded products. Therefore, where the terms "meningococca.l chimeric class I rpilin protein"
or "meningococcal chimeric class II rpilin protein" are 2:i used in either the specification or the claims, it will be understood to encompass all such modifications and variations which result in the production of a biologically equivalent protein.
As described in Example 7, the gonococcal rpilin protein is associated with cellular membranes of the E. coli used to express it. A variety of detergents are able to selectively solubilize the rpilin protein from E. coli, including EmpigenTM BB, TritonT"~ X-100, reduced TritonTM X-100, octyl-~3-D-3'i glucopyranoside (OG), ZwittergentT"~ 3-10 or 3-14.

Following centrifugation, dialysis and fractionation on a column, the purified rpilin is obtained.
As described in Example 8, the chimeric class I rpilin was isolated and purified by disruption of E.
colt cells, clarification by centrifugation, filtration, and fractionation on two columns.
As described in Example 9, the meningococcal chimeric class II rpilin was isolated and purified by disruption of E. cola cells, clarification by centrifugation, dialysis, and fractionation on two columns.
The purified gonococcal rpilin was subjected to repeated N-terminal sequencing as described in Example 10. Sequencing of the 20-40 amino-terminal residues gave results which agreed with the amino acid sequence deduced from the DNA sequence. The molecular weight of the rpilin (with signal) was determined to be 18,006 daltons by mass spectrometry, which compares well to the predicted mass of 17,981 daltons based on f,0 the amino acid content. In contrast, an apparent molecular weight o3: 68,899 daltons was obtained when the rpilin was subjected to size exclusion column chromatography usirrg detergent. This suggested that the rpilin aggregated. Dialysis of the rpilin against PBS in an effort to remove detergent resulted in material having an apparent molecular weight of 452,349 daltons, as measured by gel filtration. This suggested that it had undergone further aggregation.
As detai7.ed in Example 11, immune sera are obtained by immunizing guinea pigs or mice with the purified gonococca3. rpilin. As set forth in Example 12, Western blot analysis showed that antisera against rpilin bound to whole cell lysates from piliated gonococcal cells, while there was no binding seen in non-piliated cell l.ysates. In contrast, antisera to the pilin oligomer bound to both piliated and nonpiliated cell l~~sates.
As detailed in Example 13, when analyzed by ELISA, this pooled antisera against gonococcal rpilin had high endpoint titers for binding to purified gonococcal rpilin protein. Example 13 also details the effects of various adjuvants. When the rpilin was adjuvanted with either MPL'"' alone, MPLT"' plus aluminum phosphate, or Stimu.lonT'" QS-21, good humoral immune responses in mice were obtained.
As set forth in Example 14, whole cell ELISA
showed that antisera against rpilin bound to piliated cells, but not to isogenic non-piliated cells of a particular gonococcal strain.
1:> As described in Example 15, mice were immunized intranasally with gonococcal rpilin with or without native cholera toxin. There was a significant immune response detected in the antigen ELISA from pooled sera generated after the mice were immunized 2() with rpilin in the absence of adjuvant; this response was enhanced by the addition of native cholera toxin.
The pooled sera had a low ELISA titer for binding to intact, piliated gonococcal cells; this binding was greatly enhanced when the mice were also immunized with 2_'. native cholera toxin.
As described in Example 16, immunoelectron microscopy demonstrated that antibodies against rpilin were bound along the length of the pili filaments on the surface of gonococci. This suggested that the 3Ci antibodies bound to epitopes which would be present on the surface of the bacteria in v.~vo.
Example 1'1 demonstrates the higher titers obtained for rpilin antisera binding to heterologous piliated bacterial :isolates as compared to that 35 obtained for antisera to recombinant pilin oligomer.

The rpilin is converted to rpilin oligomer by dialysis of the rpilin against pH 12 phosphate buffer.
Pili mediate the initial binding of N.
gonorrhoeae to human mucosal cells. Therefore, if an antigen is able to elicit antibodies which inhibit the attachment of these bacteria to those cells, this would provide evidence that such an antigen is a vaccine candidate.
As discussed in Example 18, guinea pig 1~.0 antisera to rpilin significantly inhibited the binding of gonococci expressing heterologous pili to human cervical epithelia:L cells. Piliation of gonococci correlates with the infectivity of this bacterium (2,3,32).
These data indicate that the recombinant pilin was able to generate antibodies which bind to diverse pili on intact gonococcal cells and that the antisera exhibits a functional activity (inhibition of bacterial adherence:) which would protect immunized 20 human beings against gonococcal colonization and infection (32,33). It has been previously reported that immunization with E. coli cells which expressed recombinant pilin from D. nodosus were immunogenic (23,25,28), but not: protective against challenge.
25 Because of these results, these researchers turned away from the use of recombinant subunit pilin in favor of the assembled pilus. Yet, the data described herein suggest that, following purification, the recombinantly expressed pilin protein induces an immune response 30 which should correlate with protection of humans from gonococcal colonization. Thus, these data support the view that rpilin is a viable vaccine candidate against N. gonorrhoese.
As described in Example 19, the meningococcal 3:i chimeric class I rpilin protein was subjected to N-terminal sequencing. Sequencing of the 35 amino-terminal residues gave results which agreed with the amino acid sequence deduced from the DNA sequence. The molecular weight of the chimeric rpilin (with signal) was determined to be 17,659 daltons by mass spectrometry, which compares well to the predicted mass of 17,676 daltons based on the amino acid content. In contrast, an apparent molecular weight of 69,480 daltons was obtained when the meningococcal chimeric class I rpilin protein was subjected to size exclusion column chromatography using detergent. As with the gonococcal rpilin, this suggested that the meningococcal chimeric class I rpilin protein aggregated.
As detailed in Example 20, when analyzed by ELISA, pooled antisera against the meningococcal chimeric class I rpilin protein had high endpoint titers to both meni.ngococcal class I rpilin protein and to piliated meningococcal cells. As also detailed in Example 20, adjuvants, in particular Stimulon'"" QS-21, generated significant responses for the binding of antisera against the meningococcal chimeric class I
rpilin protein to both meningococcal class I rpilin protein and to piliated meningococcal cells.
As described in Example 21, immunoelectron microscopy demonstrated that antibodies against the meningococcal chimeric class I rpilin protein were bound along the length of the pili of meningococci.
This suggested that the antibodies bound to epitopes which would be present on the surface of the bacteria in vivo .
In Example 4, it was shown that antisera directed against gonococcal rpilin recognized and bound to piliated meningococcal cells. In Example 22, it was 3_'i shown that antisera raised against meningococcal _ 24 _ chimeric class I rpilin protein bound to piliated gonococcal cells.
As descra.bed in Example 23, passive immunization of infant rats with guinea pig antisera against meningococc:al chimeric class I rpilin protein can help prevent meningococcal bacteremia in vivo.
There was a significant decrease in the level of colonization in anj'.mals who received this immune sera.
Furthermore, the immune response generated using recombinantly expreassed pilin can protect, in vivo, against meningococc:i which express a heterologous pilin protein.
As described in Example 24, mice were immunized intranasally with meningococcal chimeric class I rpilin with or without cholera toxin, where the cholera toxin is in a mutant form wherein the glutamic acid at amino acid position 29 is replaced by a histidine (CT-CRM, E29H). There was a significant immune response detected in the antigen ELISA from pooled sera generated after the mice were immunized with rpilin in the absence of adjuvant; this response was enhanced by the addition of mutant CT-CRM, E29H
cholera toxin.
As described in Example 25, the inhibition of 2:S colonization of mouse nasopharynx by a class I strain of N. men~ngitidis was demonstrated in mice immunized subcutaneously with meningococcal chimeric class I
rpilin adjuvanted with MPLT"~.
As described in Example 26, Western blot 3!) analysis showed that antisera obtained from guinea pigs immunized with meni;ngococcal chimeric class II rpilin bound to whole cell lysates from piliated meningococcal cells which expressed either class I or class II pilin.
As described in Example 27, antisera elicited 3_'i against partially purified meningococcal chimeric class II rpilin bound to meningococcal cells from the homologous bacterial strain.
Taken together, these data support the view that rpilin, in particular the meningococcal chimeric S class I and class :II rpilin proteins, are viable vaccine candidates against N. menjngitidis.
The gonococcal rpilin protein is useful in the preparation of vaccines to confer protection to mammals against disease caused by N. gonorrhoeae. The i10 meningococcal rpilin protein, the meningococcal chimeric class I rpilin protein and the meningococcal chimeric class II rpilin protein are useful in the preparation of vaccines to confer protection to mammals against disease caused by N. mening~tidjs.
15 In addition, cross-protection against a different Neisseria species is afforded by immunizing with a vaccine containing the gonococcal rpilin protein to confer protection to mammals against disease caused by N. meningttidis or by immunizing with a vaccine f,0 containing the menj.ngococcal rpilin protein, the meningococcal chimeric class I rpilin protein or the meningococcal chimeric class II rpilin protein to confer protection t:o mammals against disease caused by N. gonorrhoese.
25 These vaccine compositions comprise an isolated and purified rpilin protein, wherein the vaccine composition elicits a protective immune response in a mammalian host.
Vaccines containing a rpilin protein may be 30 mixed with immunolGgically acceptable diluents or carriers in a conventional manner to prepare injectable liquid solutions or suspensions. The level of antibodies elicited) by the vaccine may be improved by using certain adjuvants such as Stimulont"" QS-21 (Aquila 35 Biopharmaceuticals, Inc., Framingham, MA), MPL'"" (3-O-deacylated monophosphoryl lipid A; RIBI ImmunoChem Research, Inc., Hamilton, MT), aluminum phosphate, aluminum hydroxide, IL-12 (Genetics Institute, Cambridge, MA) and cholera toxin (either in a wild-type or mutant form, fo:r example wherein the glutamic acid at amino acid position 29 is replaced by another amino acid, preferably a histidine, in accordance with U.S.
Provisional Patent Application Number 60/102,430).
The vaccines of this invention are 1.0 administered by injection in a conventional manner, such as subcutaneous, intraperitoneal or intramuscular injection into humans, as well as by oral, mucosal, intranasal or vaginal administration, to induce an active immune response for protection against disease caused by N. gonors:hoeae or N. meningit~fdis. The dosage to be administered is determined by means known to those skilled in the art. Protection may be conferred by a single dose of vaccine, or may require the administration of several booster doses.
In order that this invention may be better understood, the fo7.lowing examples are set forth. The examples are for the purpose of illustration only and are not to be construed as limiting the scope of the invention.
Examples Standard molecular biology techniques are utilized according to the protocols described in Sambrook et al. (31).
3:5 Example 1 Bacteria and Cell Cultures Bacteria and Culture Conditions The gonoc:occal isolates were obtained from Tampa, FL; Ottawa, Canada; Washington, D.C.; Seattle, WA; Rochester, NY; Chapel Hill, NC; and Evanston, IL.
The meningococcal isolates were obtained from Chapel Hill, NC; and Bilthoven, Netherlands.
The bacteria were stored in lyophilized form or frozen at -70°C until required. When grown on solid media, the agar plates were incubated in an incubator at 37°C containing a humidified atmosphere and 5~ (v/v) CO2. N. gonorrhoeae and N. men~ngjtidis were grown on l:i GC medium base (Difco Laboratories, Detroit, MI) without hemoglobin, but supplemented with dextrose (400 mg/L), glutamine (10 mg/L), cocarboxylase (20 ~,g/L) and ferric nitrate (500 ~,g/L). Liquid suspension cultures of N. men~ngitidis 'were grown in the same media which lacked agar in a shaking incubator (70 RPM) at 37°C.
In experiments involving culturing of meningococci from mouse nasal tissue :homogenates, the bacteria were grown on GC media described above with the following mixture of antibiotics {Difco): colistin sulfate (75 ~g/mL), 2'~ nystatin (125 ~g/mL), vancomycin (30 ~,g/mL) and trimethoprim lactate (50 ~,g/mL). Piliated gonococci were identified by colony morphology and individual colonies passaged daily in order to maintain the phenotype. The piliation state of meningococcal cells was assessed by transmission electron microscopic examination of samples stained with NanoVan stain (Nanoprobes, Stony l3rook, NY) at pH 8 for 30 seconds.
E. colj were grown on SOB agar which consists of 20 g/L
Bacto tryptone (Difc:o), 5 g/L yeast extract (Difco), 0.6 g/L NaCl, 0.2 g/L RCl and l~ (w/v) agar (pH 7.5) or in SOB broth to which the agar is not added. For some experiments, the Bacto tryptone was replaced with an equivalent amount of HySoyT"' (Sheffield Products, Norwich, NY) .
S
Epithelial cell cultures ME-180 cell line (ATCC, Beltsville, 1~) is an epidermoid carcinoma which was originally derived from a cervical carcinoma. The cells were grown in RPMI
1640 (Gibco BRL, Gaithersburg, MD) supplemented with 10% (v/v) 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-glutamine in a humidified atmosphere of 5~ (v/v) COZ at 37°C.
The cells were split every three to four days.
Example 2 Clonincr and Expression of Gonococcal pllE in E coli A frozen sample of piliated N. gonorrhoese strain Pgh3-1 was used as the source of the pilE DNA in a PCR reaction. Th.e pilE gene was amplified using the following primers which recognized the 3' and 5' ends of the complete pilin protein (including the leader sequence) : 5' CCC C:GC GCC ATG GAT ACC CTT CAA AAA GGC
3' (PILEFWD) (SEQ II) N0:5) and 5' GGG CCT GGA TCC GTG
GGA AAT CAC TTA CCG 3' (PILEREV) (SEQ ID N0:6). The resulting PCR product contained a NcoI site at the 3() beginning of the p.ilE coding region and a BamHI site at the end. The NcoI site was introduced into the gene because of cloning considerations. This resulted in a change of the second amino acid in the signal sequence from asparagine (AAT) to aspartic acid (GAT). Because 3'~ amino acid 2 is part of the signal peptide which is WO 99/55875 PC'T/US99/09486 cleaved during normal processing of the mature protein, this change was not: expected to have any effect on antigenicity or immunogenicity. The PCR product was cloned into a pCRT""7:I TA cloning vector (Invitrogen, S Carlsbad, CA), ligated, and transformed into E. cola TOP10F' (Invitroger~). Colonies were selected on 100 ug/mL ampicillin containing plates or on 50 ~.g/mL
kanamycin containing plates. The plasmid DNA was isolated from overnight cultures of these transformants and analyzed by restriction digests using the enzymes EcoRI and NotI.
Four clones containing an insert of the correct size were submitted for DNA sequence analysis in order to verify the presence of pilE PCR DNA
1:5 fragment. Clone #17, designated pPX1999, was used as the source of the pilE gene. Plasmid DNAs from pPX1999 and pTrcHisA (Invitrogen) were digested with NcoI and BamHI restriction enzymes, the DNA fragments gel isolated, ligated arid transformed into E. coli TOP10F'.
Ampicillin resistant colonies were selected, the plasmid DNA of the .new transformant isolated, and a DNA
restriction analysis done using BamHI and NcoI
restriction enzymes. Two clones with the correct restriction pattern were submitted for DNA sequence 2.'i analysis. Both clones had the correct DNA sequence and were designated pPX2000.
To test for expression of the recombinant pilin, cultures containing these clones were grown in either shake flasks or a fermentor in SOB plus 100 ~.g/mL ampicillin and 12 ~,g/mL tetracycline.
then shake flasks were used, E. coli were grown in 1L
of media until an AE;oo= 0.9-1.0 was obtained. The expression of the recombinant pilin was induced by the addition of isopropyl-~i-D-thiogalactopyranoside (IPTG) to a final concentration of 1 mM and allowing growth to continue for 1-4 hours, at which point the cells were collected by centrifugation (13,689 x g for 20 minutes at 4°C) and stored at -20°C. For the fermentor, an overnight culture from a plate was used to inoculate a flask containing 500 mL media which was again grown overnight. This liquid culture was then used to inoculate a Biostat B Fermentor (Braun Biotech, Allentown, PA) containing 8.9 L of media. Enhanced growth of the bacteria in the fermentor was obtained l~~ when HySoy1""-containing media was supplemented with dextrose at a final concentration of 1~ (w/v). When the culture reached. A6oo = 1.0, IPTG was added to a final concentration of 1 mM and the cells were allowed to grow for another 1-4 hours before being harvested by 1:~ centrifugation (13,689 x g for 20 minutes at 4°C). The media was discarded and the cell pellet stored at -20°C. Upon induction with IPTG, expression of rpilin protein increased significantly and reached maximal levels at three to four hours post induction.
21) Samples of the induced cultures were analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE). The recombinant pilin was visualized using Coomassie blue staining and its identity confirmed by Western blot with a 2:i monoclonal antibody specific for gonococcal pili (clone #A33020023, Biospacific, Emeryville, CA).
Example 3 Construction of Gonococcal Recombinant p~lE Plasmid 30 Containing Kanamvcin Resistance Marker A plasmid was constructed where the AmpR
marker was replaced with a KanR marker. Except as noted below, the procedures of Example 2 were used. A
3' PCR reaction was performed on pTrcHisA plasmid DNA

using TrcFXba primer, 5' GGC TCT AGA CTG TCA GAC CAA
GTT TAC TC 3' (SEQ ID NO:7), and TrcRXba primer, 5' GGC
TCT AGA TTG AAG CAT TTA TCA GGG 3' (SEQ ID N0:8). The underlined sequences code for an XbaI restriction site.
:> The approximate 3.5 kb PCR product contained the pTrcHisA DNA minus the ampicillin coding region.
Another PCR reaction was performed on pACYC177 plasmid DNA (New England Biolabs, Beverly, MA) using KanFXba primer, 5' GGC TCT AGA TAA ACA GTA ATA CAA GGG G 3' 11) (SEQ ID N0:9), and 'KanRXba primer, 5' GGC TCT AGA TTA
GAA AA.A CTC ATC GAG C 3' (SEQ ID NO:10). Again, the underlined sequences code for an Xbal restriction site.
The approximate 860 by PCR DNA product contains the kanamycin resistance gene. Both PCR products were 1.'> purified from an agarose gel, the DNAs Were digested with the XbaI restriction enzyme, extracted and ligated together. An aliquot of the ligation reaction was transformed into E. coli TOP10F' and an aliquot of the transformation mix ;plated on SOH plates containing 30 2U ~.g/mL of kanamycin .
A total of 48 KanR colonies were streaked in duplicate onto SOB plates containing either ampicillin or kanamycin. As anticipated, all KanR colonies were ampicillin sensitive. Since the cloning design was 2_'i symmetrical, both orientations of the kanamycin insert were isolated. The kanamycin insert in the same clockwise orientation as the original ampicillin gene was selected for future studies and called pZ564. The DNA region containing the laclq gene, trc promoter and 3C1 the multiple cloning site in pZ564 was then replaced with the similar region from the pPX2000 plasmid (which also contained pilE;1 in the following manner: Both pZ564 and pPX2000 were digested with SphI and XmnI
restriction enzymes. The approximate 2.2 kb DNA
3~~ fragment from pPX20ID0 and the approximate 2.6 kb DNA

fragment from pZ564 were gel purified, ligated together and transformed into E. coli TOPlOF~. The resulting correct plasmid was called pPX2002.
A similar time course of induction and level of recombinant pilin protein expression was seen when the selection antibiotic was changed to kanamycin from ampicillin.
Example 4 ll) Cloning and Expression of Meningococcal Class I nilE in E. coli Because of the highly homologous nature of the DNA and amino acid sequences of gonococcal and meningococcal class I pilins (7), the ability of gonococcal rpilin antisera to bind to pili expressed on meningococcal cells was assessed using the whole cell ELISA. This antisera exhibited a titer of 151,100 for binding to N. meningft~dis piliated cells from strain 21) H3 5 5 .
This binding appeared to be directed against the pilin protein, since a Western blot of the whole cell lysate from this strain showed that only a single band co-migrating with pilin bound the antisera (data 2.'i not shown). A number of other meningococcal strains exhibited lower titers in the whole cell ELISA, but the presence of pill was not confirmed by transmission electron microscopy. Based on these data, it was decided to clone and recombinantly express the class I
30 pilin from N. meningitidis. Initially, the same strategy was followed as with the pilE from N.
gonorrhoeae. Except as noted below, the procedures of Example 2 were used.
The class I pilE was amplified from the 3-'i genomic DNA of N. meningiti.dis strain H44/76 using the following primers: 5' CCC CGC GCC ATG GAC ACC CTT CAA
AAA GGT TTT ACC 3' (NMFPILE) (SEQ ID N0:11), and 5' GGG
CCT GGA TCC GAG TG(s CCG TGG AAA ATC ACT TAC CGC 3' (NMRPILE) (SEQ ID N0:12). As anticipated, a PCR
product of approximately 600 by DNA was obtained. An aliquot of the PCR reaction product was digested with BamHI and NcoI restriction enzymes for insertion into pTrcHisA. The digested DNAs were electrophoresed on an agarose gel and the: DNA fragments gel purified. The DNA fragments were then ligated together and transformed into E.. cola TOP10F'. Miniplasmid prep analysis of ampici7Llin resistant clones Was performed using BamHI and NcoI restriction enzymes. Clones expressing the correct restriction digest pattern were called RZ1142 and t:he plasmid was called pPX2003.
Following induction, the presence of a Coomassie blue stained polypeptide with a molecular weight of approximately 15,000 daltons was observed when a whole cell lysate was analyzed by SDS-PAGE.
2.0 Analysis of whole cell lysates from four of these clones by SDS-PAGE and Western blot demonstrated the presence of a protein of appropriate mobility and reactivity with polyclonal antisera against the intact pili from N. gonors;hoeae strain LB2. The purified class I rpilin was 167 amino acids in length. However, the level of expression of this recombinant pilin was significantly lower- than that obtained with either pPX2000 or pPX2002 grown under the same conditions. An analysis of the DNA sequence in the recombinant class I
pilE showed that there were a number of inverted repeats which might: explain the low level of expression of this protein in E. coli.

Example 5 Construction arad Expression of a Gonococcal and Meninaococcal ~~lass I Chimeric pilE in E coli In order to increase the expression of the meningococcal pilin protein, the DNA encoding the first 60 amino acids in pPX2003 (the meningococcal class I
p3,lE construct described in Example 4) was replaced with the equivalent. region from pPX2002 (the gonococcal 1~ pilE construct described in Example 3, including the seven amino acid signal peptide) (SEQ ID N0:4). Except as noted below, the: procedures of Example 2 ware followed.
The conserved 5' terminal region of the meningococcal p~lE gene was replaced by the same region from N. gonorrhoeae strain Pgh3-1 in the following manner. A BsmBI site was introduced into the meningococcal pilE gene as follows: DNA was PCR
amplified from pPX2003 using the following primers: 5' 2n CCG GCG CGT CTC TCA CGG CGA ATG GCC CGG C 3' (CL-lESPF) (SEQ ID N0:13) and 5' GGG CCT GGA TCC GAG TGG CCG TGG
ATC ACT TAC CGC 3' (NMRPILE) (SEQ ID N0:14) and Taq DNA polymerase. The expected PCR DNA product was cloned directly into pCR2.l (Invitrogen) and 2:> transformed into TOPlOF' cells and the resulting plasmid was designated pZ578. A BsmBI site was then introduced into the gonococcal p~lE by the following method. Using the ,primers 5' GCA TAA TTC GTG TCG CTC
AAG GCG C 3' (TRCUPFy~T) (SEQ ID N0:15) and 5' GCC GCG CGT
3() CTC CCG TGA TTC AGG TAA TAC TCG G 3' (PILEESPR) (SEQ ID
N0:16) and Pfu DNA ;polymerase, the 5' end of the p.ilE
gene from pPX2000 was PCR amplified. The resulting gonococcal PCR product and pZ578 were then digested with BsmBI and ligated together. The ligated DNAs were then PCR amplified using 5' GCA TAA TTC GTG TCG CTC AAG
GCG C 3' (TRCUPFHT} (SEQ ID N0:17) and 5' GGG CCT GGA TCC
GAG TGG CCG TGG AAA ATC ACT TAC CGC 3' (NMRPILE) (SEQ
ID N0:18) primers.
The DNA pCR product was of the predicted size (approximately 850 bp) and was digested with Ncol and BamHI to yield an approximately 600 by fragment. This fragment was gel isolated and cloned directly into NcoI- and BamHI-cut ppX2000 vector, replacing the l0 gonococcal p~lE gene. The resulting plasmid, which was ampicillin resistant, was labeled pPX2004 and used to transform TOPlOF~. Analysis of this transformant demonstrated the presence of the desired chimeric p3lE
DNA. Following induction with IPTG, there was a significant increase in the amount of the meningococcal chimeric class I rpilin construct expressed compared to the amount of meningococcal rpilin expressed from pPX2003. Using the extraction with 1~ octyl-~i-D-glucopyranoside (O(~) and purification protocol (TMAE
2,0 FractogelT"" column in 10 mM TrisT~~, pH 8.5 with 0.1~
(w/v) ZwittergentT"~ 3-14) described in Example 7 for the recombinant gonococcal pilin, highly purified meningococcal chimE:ric class I rpilin protein was obtained (yield approximately 5 mg/gram cell wet weight). This material was greater than 90~ pure when analyzed by SDS-PAGE and laser densitometry. SDS-PAGE
demonstrated the presence of a major band of approximately 15,000 daltons in size. The meningococcal chimeric class I rpilin protein was also 167 amino acids in length, and includes the signal sequence of seven amino acids as demonstrated by sequencing of the amino-terminal 36 residues of the purified protein.

Example 6 Construction and Expression of a Gonococcal and Meninaococcal Class II Chimeric pilE in E. coli The initial cloning of the meningococcal class II pilE involved isolation of chromosomal DNA
from piliated N. meningitidis strain FAM18 cells and amplifying the classII pilE DNA in a PCR reaction. The class II pilE gene was amplified using the following primers which recognized the 3' and 5' ends of the complete pilin protein (including the leader sequence):
5' GCG GCC GCC ATG GAA GCA ATC CAA AAA GGT TTC ACC C 3' (PILE2FWD) (SEQ ID N0:19) and 5' GCG GCC GGA TCC GGT
CAT TGT CCT TAT TTG'~ GTG CGG C 3' (PILE2REV) (SEQ ID
N0:23). In a similar strategy as in Example 2, the resulting PCR product contained an NcoI site at the beginning of the pilE coding region and a BamHI site at the end. The NcoI site was introduced into the gene because of cloning considerations. This resulted in a change of the secan.d amino acid in the signal sequence from lysine (AAA) to glutamic acid (GAA). As stated previously, this change was not expected to have any effect on antigenicity or immunogenicity. The PCR
product was cloned into a pCR2.l cloning vector (Invitrogen), ligated, and transformed into E. coif TOP10F'. Colonies were selected on 100 ~Cg/mL
ampicillin-containing plates or 50 ~,g/ml kanamycin plates. The plasmid DNA was isolated from overnight cultures of these t,ransformants and analyzed by 3D restriction digests using the enzyme EcoRI.
Clone #8, designated pPX8001, was used as the source of the pilE gene. Plasmid DNAs from pPX8001 and pTrcHisC (Invitrogen) were each digested with NcoI and BamHI restriction enzymes, and the resulting DNA
fragments were gel isolated, ligated and transformed into E. coli TOP10H". Following selection of ampicillin resistant colonies, the plasmid DNA of the new transformants were isolated, and a DNA restriction analysis performed using BamHI and NcoI restriction enzymes. Two clones with the correct restriction pattern were submitaed for DNA sequence analysis. Both clones had the correct DNA sequence, designated as pPX8002.
To test for expression of the recombinant class II pilin, 10 mL cultures containing these clones, pPX8002, were grown in 50 mL tubes in SOB containing 100 ~Cg/mL ampicillin and 12 ~g/mL tetracycline, to an Asoo = 1Ø Expression of the recombinant protein was induced by adding 7:PTG to final concentration of 1 mM
and the culture was continued for three hours. i~hen a whole cell lysate of the induced cells was separated by SDS-PAGE and stained with Coomassie blue, no new (induced) band was detected. This suggested that the FAM18 p3IE gene product was expressed at levels lower than those discernable with Coomassie blue. When the FAM18 pilE was cloned into the pETl7b plasmid and transformed into E. cold BL21(DE3)pLysS with or without the p3lE signal sequence, no significant expression of recombinant protein was detected. Similar results were obtained when the class II pjlE gene from two other strains of N. meningitides (NmB, 2996) were cloned into the same pTrcHis pl.asmid and TOP10F' expression system.
Specifically, the strains N1~ and 2996 were also determined to be expressing the class II pilin, based on PCR and sequencing data. The pilE gene was amplified from several N. meningitides strains using a class I set of primers (NMFPILE and NMRPILE) and a class II set of primers (PILE2FWD and PILE2REV) in separate reactions with either chromosomal DNA or cells as the template. F~CR products were cloned into pTrcHisC and sequer.~ced, or were sequenced directly.
Alignments of sequences were carried out; sequences similar to those from the H44/76 strain were classified as class I, while sequences similar to those from the FAM18 strain were classified as class II. Sequences were not obtained f:or all the strains amplified. A
preliminary classification was also made based on PCR
data. Class I strains were those which gave a correct size PCR product wj.th class I primers but riot with class II primers, while class II strains were those which gave a correct size PCR product with class II
primers but not with class I primers.
Based on the experience with the meningococcal class I pilin, the region encoding the first 60 amino acids (the conserved amino-terminal region) of the class II pilE Were replaced with the corresponding region from N. gonorrhoeae strain Pgh3-1.
Expression of the resulting chimeric pilE was investigated in a number of E. coli expression strains using a variety of promoters. The strains studied included: PR13 (Rnase deficient), BL21 (protease deficient), KS474 (deficient in periplasmic protease), AD494 (Novagen, which allows disulfide bond formation in cytoplasm) and three strains of TOPP (Stratagene, non-K12 strains useful for hard to express proteins).
In all cases, no recombinant protein was detected in Coomassie blue- stained SDS-PAGE. An alternative expression plasmid pETl7b (which includes a T7 promoter) was investigated with similar results.
It shouldl be noted that the native class II
pilE gene sequence in pPX8002 ends at base 447. The DNA sequence found downstream (3') from the native meningococcal classII pilE termination site, nucleotides 447 to 519, contains an inverted repeat which might form a stem and loop structure. Because stem and loop structures can be effective terminators of transcription, it was postulated that the omission of this additional 3' sequence (74 bases) in pPX8002 might affect the transcription of the chimeric class II
pilE message in E. coli. Therefore, all subsequent cloning restored this downstream 3' end sequence.
A systematic replacement of various portions of the class II pilE gene from the N. meningit~dis strain with the corresponding regions of the pilE gene lid from N. gonorrhoeae strain Pgh3-1 was undertaken in order to identify regions that inhibited expression.
Replacement regions started at the 5' or 3' ends and were made progressively larger. Double replacements of 5' and 3' ends were also constructed until an internal 1:5 region of only 84 nucleotides from the native pilE
class II remained. This region was also replaced, resulting in the reconstruction of the rGC and, as expected, this clone expressed rpilin at similar Coomassie blue stained levels as pPX2000. The 20 following regions (listed by nucleotide numbers) of the FAM18 pflE gene were replaced with the corresponding regions from Pgh3-1 (listed in parentheses): single region replacements were 1-108 (1-108), 1-181 (1-181), 1-294 (1-282), 439-499 (478-553), 379-519 (367-553), 25 295-519 (283-553), 295-378 (283-366); double region replacements were 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 (478-553) .
When these constructs were expressed in E.
31) coli TOPlOF' using the pTrcHis expression system, two constructs produced recombinant protein at levels detectable by Coomassie blue: the first containing the replacement of nucleotides 379-519 (which comprises the disulfide loop and 3' extension); and the second 3;> containing the replacement of both nucleotides 1-181 (which comprises the conserved 5' region) & 379-519 (which comprises the disulfide loop and 3' extension).
Because replacement. of the 5' region alone did not lead to expression of recombinant protein and because the first construct retained most of the native meningococcal pilin, sequence, this construct (nucleotides 379-519) was selected for further investigation. While the amino acid sequence of the meningococcaal and gonococcal proteins are 1~~ significantly different in this region, it is well documented (17,18) that the disulfide loop undergoes significant antigenic variation. Therefore, any immune response directed against this region (e.g., the disulfide loop) would exhibit minimal cross-reactivity l:i among meningococcal strains. Lastly, because the gonococcal insert is nearly twice the size of the meningococcal disulfide loop (39 residues versus 18 residues), the resulting chimeric protein migrates on an SDS-PAGE gel with an apparent molecular weight of 20 approximately 19,000 daltons.
The construction of this chimeric gene was carried out in the following manner. The 5' fragment was obtained by amplifying pPX8002 (FAM18 class II
pilE) with the following primers: 5' GCG GCC GCC ATG
2-'s GAA GCA ATC CAA AAA GGT TTC ACC C 3' (PILE2FWD) (SEQ ID
N0:19) and 5' GCC GCG CGT CTC CGA ACC GGA GTT TTG TTT
GCC 3' {REV-CYS) (S;EQ ID N0:20). The gonococcal disulfide loop (i.e., the 3' end of the gonococcal gene) was amplified from pPX2000 using primers 5' CCG
3(I GGC CGT CTC GGT TCG GTA AAA TGG TTC TGC 3' (FWD-CYS) (SEQ ID N0:21) and !5' GGG CCT GGA TCC GTG GGA AAT CAC
TTA CCG 3' (PILEREV) (SEQ ID N0:22). The resulting PCR
products were each purified, digested separately with restriction enzyme ,lgsmBI, then ligated to form the full 3~~ length chimeric pilE, which was amplified using primers PILE2FWD and PILEREV. This PCR product was digested with restriction enzymes NcoI and BamHI, ligated into a similarly restricted pTrcHisC vector and transformed into TOP10F' competent cells.
Transformants were cultured and analyzed using the restriction enzymes NcoI and BamHI. Four clones with the right sized insert were analyzed with restriction enzyme Stul. Of these, three provided the correct restriction map. Two of the three clones with the correct restriction pattern were sequenced. Clone #5 had the correct DNA sequence and was designated as pPX8017. This clone contains the nucleotide sequence set forth in SEQ II) N0:3, in which nucleotides 1-378 are from N. meningj:tid3s and nucleotides 379-510 are from N. gonorrhoeae.
Expression was checked with lOmL cultures in 50mL tubes. Cells were grown in SOB supplemented with 100 ~.g/ml ampicillin and 12 ~,g/ml tetracycline until Asoo aPProximately 1Ø A culture was induced by the addition of IPTG to a final concentration of 1 mM.
Growth was allowed to continue for 3-4 hours, at which point the cells were collected by centrifugation (13,689 x g for 20 minutes at 4°C) and stored at -20°C.
For the fermentor, an overnight culture from a plate or frozen stock was used to inoculate a flask containing 500 mL media which was again grown overnight. This liquid culture was then used to inoculate a Biostat B
Fermentor (Braun Biotech, Allentown, PA) containing 8.9 L of media. Enhanced growth of the bacteria in the fermentor was obtained when HySoy~"-containing media was supplemented with dextrose at a final concentration of 1% (w/v). When the culture reached A6oo = 4.0-6.0, IPTG
was added to a final concentration of 1 mM and the cells were allowed to grow for another 2-4 hours before being harvested by centrifugation (13,689 x g for 20 minutes at 4°C). The media was discarded and the cell pellet stored at -20°C. Upon induction with IPTG, expression of chimeric class II rpilin protein increased significantly.
Samples of the induced cultures ware analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE). The recombinant meningococcal chimeric class II pilin was visualized using Coomassie blue staining (apparent molecular weight of approximately 19,000 daltons) and its identity confirmed by Western blot with a polyclonal antisera directed against a gonococcal peptide (Glu Ala Ile Leu Leu Ala Glu Gly Gln Lys Ser Ala Val Thr Glu Tyr Tyr Leu Asn His Gly Lys) (SEQ ID N0:24), which is located in the conserved region of the amino terminus of the class II pilin protein. The meningococcal chimeric class II x-pilin was 170 amino acids in length (including the sigrial) (SEQ ID N0:4), in which amino acids 1-126 are from N. men~(ngit3dis and amino acids 127-170 are from N. gonorrhoeae.
Example 7 Isolation and Purification of Recombinant Gonococcal Pilin from E. coif The follawing procedure was used to purify the recombinant gonococcal pilin obtained in Examples 2 and 3 above. This procedure is also used to purify the meningococcal recombinant pilin obtained in Example 4, and was used initially to purify the meningococcal chimeric class I rpilin protein obtained in Example 5, above. Subsequently, the isolation procedure for the meningococcal chimeric class I rpilin protein was modified as described in Example 8 below.

Sub-cellular fractionation of the E. coli expressing rpilin demonstrated that the protein was associated with the cellular membranes, most likely the inner membrane, based on the ability of 1% (v/v) TritonT"' X-100 to solubilize this protein. When an attempt was made to remove contaminating E. coli proteins in the presence of 0.05-0.1% (v/v) TritonTM X-100, it was discovered that, below pH 9.5, the rpilin did not bind consistently to an ion exchange column.
In Therefore, the ability of a number of detergents to selectively solubilize the rpilin protein from E. coli membrane preparations was examined.
The cell pellet (approx. 5 g wet weight) from 1 L of culture was thawed by adding 30 mL of 10 mM
1:S Hepes (pH 7.2) (Research Organics, Cleveland, OH), 1 mM
EDTA and the cells broken using a Microfluidizer cell homogenizer (Microfluidics International Corp., Newton, MA). The lysate was clarified by centrifugation (12,000 x g for 10 minutes) and the membranes pelleted 2n (288,652 x g for one hour). The membranes were resuspended in 33 mL of 10 mM Hepes (pH 7.4), 1 mM
MgCl2 and extracted with one of the following detergents: (a) TritonTM X-100 (TX100) (Calbiochem-Novabiochem International, San Diego, CA), (b) reduced 25 TritonT"~ X-100 (Calbiochem) , (c) octyl-(3-D-glucopyranoside (OG) (Calbiochem) , (d) ZwittergentT"" 3-8 (Z3-8) (Calbiochem), (e) ZwittergentT"" 3-10 (Z3-10) ( Calbiochem) , ( f ) Zwi ttergentT'" 3 -12 ( Z3 -12 ) (Calbiochem) , (g) ZwittergentT"~ 3-14 (Z3-14) 3n (Calbiochem), (h) Empigen BBTM (Calbiochem) or (i) TweenTM 80 (ICN, Cleveland, OH) for one hour at room temperature.
EmpigenT"~ BB (1% v/v) , ZwittergentT"" 3-10 (1%
w/v), reduced TritonTM X-100 (1% v/v), octyl glucoside 3:S (1% w/v) with ZwittergentTM 3-10 (1% w/v) or 3-14 (0.1%

w/v) each selectively extracted the recombinant pilin protein with minimal contamination with the E. coli proteins. ZwittergentT"" 3-12, even at 0.1~ (w/v), solubilized both th.e recombinant protein and a significant number of E. coli proteins. TweenT"~ 80 did not extract the recombinant protein at any tested concentration (0.1-1~ v/v).
The solubilized proteins were separated from insoluble membrane material by centrifugation (288,652 x g for one hour). The supernatant (containing rpilin) was dialyzed overnight at 4°C against 10 mM Tris"'~ (pH
8.5) containing one of the following non-ionic detergents: (a) 0.1~ (w/v) ZwittergentT"" 3-14, (b) l~
(w/v) ZwittergentT~ 3-10 or (c) l~ (w/v) OG. The dialyzed material was fractionated on a FractogelT"" E1~
TMAE-650(S) (EM Separations Technology, Wakefield, RI) column equilibrated, in 10 mM TrisT"" (pH 8.5) and the respective detergent. The bound protein was eluted with a linear gradient of 0 to 0.2 M NaCl in 10 mM
TrisTM (pH 8.5) containing the appropriate detergent.
Fractions containing rpilin were pooled, analyzed for purity and protein content. Occasionally, to increase the purity of the rpilin, the pooled material was dialyzed against th.e starting buffer and fractionated a second time on the TMAE column.
The rpilin, which was selectively eluted from the column, was highly purified, as judged by laser densitometric analysis of a Coomassie blue stained SDS-PAGE (>90~ homogeneous). Similar results were obtained 3~0 when the extraction. and column chromatography were done with 1$ (w/v) ZwittergentT"" 3-10, l~ (w/v) OG or 0.1~
(w/v) ZwittergentTM 3-14. The yield of rpilin, which was a significant proportion of the total E. coli protein, was approximately 10 mg/L of culture grown in 3.5 1.5 L shake flasks with SOB media. When the recombinant E. coli (containing pPX2002) were grown in a fermentor using HySoyTM based media, the yield of purified rpilin increased to approximately 30 mg/L of culture, which corresponds to seven mg rpilin per gram of cell mass. When. 1~ dextrose was included in the fermentor, the overall yield of rpilin increased to approximately 100 mg/L.
The purified rpilin was dialyzed against 10 mM sodium phosphate:, 140 mM NaCl (pH 7) (PBS) containing 0.05 (w/v) Z3-14, sterile filtered and stored at 4°C or frozen at -20°C.
Example 8 Isolation and Purification of Meningococcal Chimeric Class I rpilin from E. cola Large scale cultures of E. coli cells containing pPX2004 were grown in a Hiostat B Fermentor as described in Example 2. Bacterial cells (approximately 88 grams wet weight of E. coli pPX2004) were resuspended in. 440 mL of 10 mM Hepes, 1 mM EDTA
(pH 7.5) and disrupted using a Microfluidizer Model 110Y (Microfluidics. Corp., Newton, MA). The disrupted cells were clarified by centrifugation at 6,084 x g for 20 minutes at 10°C. The supernatant was collected and the membrane fraction isolated by centrifugation at 205,471 x g for 1 hour at 10°C. The pellet was resuspended by homogenization in 220 mL of lOmM Hepes, 1 mM MgCl2, 1~ (w/v) octyl-~i-D-glucopyranoside (pH 7.5) 3~D and stirred for 90 minutes at room temperature. The suspension was centrifuged at 205,471 x g for one hour at 10°C. Following centrifugation, the supernatant, which contained the: solubilized chimeric class I
rpilin, was filtered through a 0.22. Nalgene vacuum filter and stored a.t 4°C. The pH of the octylglucoside extract was adjusted to pH 8.5 with concentrated NaOH
and subsequently loaded onto a 200 mL TMAE FractogelT"' column (EM Separations Technology, Gibbstown, NJ) equilibrated with 25 mM TrisTM, 0.1~ (w/v) ZwittergentTM
S 3-14 (pH 8.5). Unbound protein was washed through the column with an additional 400 mL of the equilibration buffer. The rpilin, was eluted using a linear NaCl gradient (0-0.2 M NaCl) in 25 mM TrisTM, 0.1~ (w/v) ZwittergentTM 3-14 (pH 8.5) over 10 column volumes at a l~D flow rate of 10.0 mL/minute. Fractions containing the chimeric class I rpilin were pooled and diluted 1:1 with dHzO and loaded onto a 100 mL 40 E.~m ceramic hydroxyapatite column (Bio-Rad, Hercules, CA) equilibrated with 10 mM NaP09, 0.1~ (w/v) ZwittergentTM
15 3-14 (pH 6). Unbound protein was washed through the column with an additional 200 mL of equilibration buffer. The chimeric class I rpilin was eluted using a linear NaP04 gradient (10-150 mM NaP04) containing 0.1~
(w/v) ZwittergentTM 3-14 over 10 column volumes at a 2~D flow rate of 5.0 mL~/minute. Fractions were screened by SDS-PAGE analysis a.nd those containing the chimeric class I rpilin were pooled. The purified material was at least 95~ pure, as determined by laser densitometry of Coomassie blue-stained gels. The yield of purified 2:5 chimeric class I rpiiin was approximately 35 mg/g wet weight cells.
Example 9 Isolation and Purification of Meningococcal 3n Chimeric Class IT rpilin from E. coli All steps. were performed at room temperature unless specified. Frozen E. coli cells were resuspended in 10 ml of 10 mM Hepes (pH 7.2), 1 mM EDTA

per gram of cells and homogenized using a Microfluidizer cell. homogenizer to disrupt the cells.
The cell lysate was clarified by centrifugation at 13,689 x g for 30 minutes. The resulting supernantant was then centrifuged at 388,024 x g for 30 minutes at 4°C. The supernatant was discarded and the pellet containing the membranes was frozen at -20°C overnight.
The membrane pellet: was resuspended in 9 mL/tube of 10 mM Hepes (pH 7.2), 1 mM MgCl2 and extracted with 1~
(w/v) ZwittergentTM 3-16 (Calbiochem) for one hour.
The suspension was centrifuged at 388,024 x g for 30 minutes and the resulting pellet was extracted again with ZwittergentTM 3-16 as described above. Following centrifugation (388,024 x g for 30 minutes), the pellet was resuspended into 9 mL of 50 mM TrisTM (pH 8.0), 5 mM EDTA and extracted with 1% (w/v) N-laurylsarcosyl (Sigma) with gentle; agitation overnight at room temperature. This resulted in the solubilzation of the meningococcal chimeric class II rpilin. The insoluble material was removed by centrifugation (388,024 x g for minutes) and discarded. ZwittergentTM 3-14 was added to the supernantant, which contained the meningococcal chimeric class II rpilin, to a final concentration of l~s (w/v) and the material was dialyzed 25 overnight against 50 mM TrisTM (pH 8.0), 10 mM EDTA, 1~
ZwittergentTM 3-14. An aliquot (1 mL, l.3mg protein) of the dialyzed material was then passed over a Mono-QTM (Pharmacia, Piscataway, NJ) column (5x10 mm) which was equilibrated in 50 mM TrisTM (pH 8.0), 10 mM EDTA, 30 10 (w/v) ZwittergentTM 3-14 at a flow rate of 0.5 mL/minute. The unbound material containing the chimeric class II x~pilin was pooled and dialyzed overnight against 1.0 mM NaP04 (pH 6.8), 1~ (w/v) ZwittergentTM 3-14. This material was approximately 80~ pure and was used in the studies described in Examples 26 and 27 below. Further purification of this material was obtained by passing it over a 1 mL
hydroxyapatite column (Bio-Rad) which was equilibrated in 10 mM NaP04 (pH 6.8), l~ (w/v) ZwittergentTM 3-14.
The purified meningococcal chimeric class II protein was eluted with a linear gradient of 0-0.5M NaPO, containing 1$ (w/v) ZwittergentTM 3-14. Fractions were screened for chimeric class II rpilin by SDS-PAGE, using gels which were stained with either Coomassie blue or silver. Both analyses demonstrated the presence of a sing7Le polypeptide band which had a molecular weight oi: approximately 19,000 daltons. This material was shown to be greater than 95~ pure by laser densitometric analysis of the polyacrylamide gels.
Example 10 Analytical Methods for Gonococcal rpilin c;0 Protein content was determined by the bicinchoninic acid protein assay (Pierce, Rockford, IL) using HSA as the si~andard. The purity of protein preparations was determined by Coomassie brilliant blue stained polyacrylamide gel electrophoresis in the ~5 presence of SDS (SDS-PAGE) and analyzed by laser denistometry with a Personal Densitometer SI (Molecular Devices). The identity of pilin in the preparations was confirmed by Western blotting using the monoclonal antibody described in Example 2, which is raised against purified pili from N. gonorrhoeae strain P9 (Biospacific). The N-terminal sequences of the pilin proteins were determined using an Applied Hiosystems 477A Protein Sequencer. Two sequences were often detected when the purified rpilin was submitted for N-~~5 terminal sequencing. The major sequence represented the complete pilin protein, including the seven amino acid leader sequence. The minor sequence, comprising 10-20~ of the sample, was rpilin protein in which the leader sequence was missing and the sequence started at phenylalanine, the N-terminal residue of the mature gonococcal pilin protein. For both rpilin protein forms, sequencing of the amino-terminal residues gave results which agreed with the sequence deduced from the DNA sequence.
The mass of recombinant pilin was determined by matrix assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometry using a Finnagan MAT LasermatT"" 2000 (San Jose, CA). The instrument was calibrated with horse myoglobin to within 0.01 of its expected mass of lEi,951.5 daltons. Recombinant pilin was mixed with an equal volume of a cyano-4-hydroxycinnamic acid matrix (10 mg/mL in 70:30 acetonitrile . 0.19's (v/v) trifluoroacetic acid/water).
An aliquot (1 ~,L) of this mixture was deposited on a sample stage, allowed to air dry, and subjected to MALDI-TOF mass spe<:trometry analysis. Data from 15 runs (each run representing a sum of 10 shots) were averaged to determine the mass of rpilin. The molecular weight of the rpilin (with signal) was ~!5 determined to 18,001 daltons, which compares well to the predicted mass of 17,981 daltons based on the amino acid content. A manor peak with a mass of 17,232 daltons (average) was detected in each lot. The difference in molecular weights of the two forms of the .CO recombinant pilin (769 daltons) is ascribed to the loss of the first six amino acids of the leader sequence (Met Asp Thr Leu G:Ln Lys) (SEQ ID N0:2, amino acids 1-6) which has a mass of 774 daltons.
A very different apparent molecular weight of ;SS the rpilin was obtained by size exclusion column chromatography using an analytical SuperoseT"" 12 column (Pharmacia, Piscataway, NJ) equilibrated in PBS
containing 0.05 (w/v) Zwittergentl"" 3-14. Under these conditions, the protein eluted at a position corresponding to a molecular weight of 68,899. This suggested that the recombinant protein aggregated.
However, the elution of a protein from a size exclusion column can be greatly influenced by the shape of the protein. Results from velocity sedimentation lU centrifugation experiments demonstrated that the recombinant pilin had a molecular weight in solution of approximately 45,000 daltons. In an attempt to remove the detergent ( Zwi t tergent'"" 3 -14 ) , the recombinant protein was dialyzed extensively against PBS alone.
1_'~ The dialyzed recombinant protein appeared soluble and was not pelleted by high speed centrifugation (122,000 x g for one hour). No attempt was made to verify the complete removal of the detergent from the recombinant protein. Analysis of this material by gel filtration 20' in PBS indicated that the protein had an apparent molecular weight of 452,349 daltons. This suggested that it had undergone further aggregation. The number of subunits in eithesr aggregate has not been determined.
25 The value of 452,349 must be considered an estimate, because the protein may still be in a micelle, as it is unknown if the detergent was completely removed from the sample. Given the fact that rpilin is diluted approximately 15-30 fold when 30 formulated as a vaccine, it appears likely that the rpilin in the vaccine will have an apparent molecular weight of approximately 450 kD.

Example 11 Preparation of Immune Sera from rpilin Immunogenicity studies were performed using guinea pigs (female:, 200 g) immunized subcutaneously (s. c.) with 20 ~g of purified gonococcal rpilin protein mixed with an adjuvant. The adjuvants studied were:
(a) Stimulon'"" QS-21. (25 ~,g/dose) in PBS (pH 6) ; (b) aluminum phosphate (Lederle Laboratories, Pearl River, NY, 100 ~Cg/dose) in PBS (pH 7) ; or (c) PBS (pH 7) only.
Initially, the animals were immunized on weeks 0, 4 and 8 and sera were obtained on weeks 0, 4, 6 and 10.
Analysis of the time course of the immune response demonstrated that giving a third vaccination at week 8 did not boost the immune response and, therefore, later studies with the recombinant pilin were terminated at the week 6 bleed.
In order to investigate the ability of adjuvants to modulate the immune response of gonococcal 2n rpilin, mice (female, 8 weeks old, 5 or 10 animals per group) were immunized subcutaneously with I-10 ~Cg of purified protein on weeks 0, 4 and 6 and sera were obtained on weeks 0, 4, 6 and 8. Vaginal lavages were done at week 8 by instilling RPMI 1640 (75 ~,L) into the 2.'i vagina and aspirating 3-4 times. The lavage fluids from each group were pooled together and 50 ~,L of fetal bovine serum was added to each pool.
For the meningococcal chimeric class I rpilin protein, the mice received immunizations on week 0 and 3() 4 only and sera was obtained on weeks 0, 4 and 6. For all rpilin parenteral immunogenicity studies in mice, the following adjuvants were studied: (a) Stimulonl""
QS-21 (25 ~Cg/dose) .in PBS (pH 6) ; (b) aluminum phosphate (100 ~Cg/dose) in PBS (pH 7) ; (c) MPL'"" (50 3'~ ~.g/dose) in PBS (pH 7) ; (d) aluminum phosphate (100 ~.g/dose) and MPL'"' (50 ~.g/dose) in PBS (pH 7) ; or PBS
(pH 7) only.
For the meningococcal chimeric class II
rpilin protein, the guinea pigs received immunizations S of 20 ~,g protein adjuvanted with StimulonT"" QS-21 (25 ~,g/dose) in PBS (pl:I 6) on week 0 and 4 only and sera was obtained on weeks 0, 4 and 6.
The ability of the recombinantly expressed pilins (either gonococcal or meningococcal chimeric 1.0 class I) to induce a mucosal immune response was assessed by immunizing mice intranaeally with (a) 1 or ~g of gonococcal rpilin in 2.5 ~L of saline with or without 1 ~,g of native cholera toxin, or (b) 5 ~g of chimeric class I rpilin diluted in 10 ~,L of PBS (pH 7), with or without 1 ~,~g of mutant CT-CRM, E29H cholera toxin. The immunizations were given on weeks 0, 2 and 3.
Exam.,ple 12 Western Blot Analysis of the Immune Response Acrainst Gonocoecal rpilin The purified gonococcal rpilin was used to immunize guinea pigs following the protocol described in Example 11. The: antisera derived from guinea pigs immunized with gonococcal rpilin were analyzed first by Western blots (data. not shown). These blots demonstrated that the antisera against gonococcal rpilin recognized a band corresponding to pilin in whole cell lysate from piliated gonococcal cells; there was no staining seen in non-piliated cell lysate from the same gonococcal strain.
Comparative data were obtained from antisera from guinea pigs immunized with the gonococcal pilin 3:i oligomer. Pilin oligomer was obtained by dissociation of intact pili as previously described (4). Briefly, this involved dialysis of intact pili against 37 mM
sodium phosphate (pH 12) for 48 hours at 4°C, followed by dialysis against 50 mM TrisT"", 145 mM NaCl (pH 8.0).
S The pilin oligomers were then clarified by centrifugation (100,000 x g far one hour). Following centrifugation, the pilin oligomers remained in the supernatant. In comparison to antisera against gonococcal rpilin, pilin oligomer antisera, while binding to pilin in the piliated cell lysate, also bound to a number of other bands in the lysates from both piliated and non-piliated cells (data not shown).
These bands represent contaminants in the pilin oligomer preparation and are presumed to be not associated with pi7.i.
Example 13 Recombinant Gonococcal Pilin ELISA
The endpoint titers against purified proteins or bacterial cells were determined by ELISA. In all ELISA procedures, incubations were for one hour at room temperature, unless. otherwise specified. Endpoint titers were defined as the extrapolated dilution at 2:5 which the optical absorbance was 0.10 greater than that of the blank wells (which do not contain primary antibody). For the analysis of guinea pig antisera, purified recombinant pilin was diluted in 0.1 M TrisT"' (pH 8) to a final concentration of 1 ~,g/mL. Aliquots 31) (100 ~,L) were added to the wells of a microtiter plate (Immulon II, Nunc, Naperville IL) and incubated overnight at 4°C. The plates were washed five times with PBS containing 0.05$ (v/v) TweenT"~-20 (PBS-T) using a Skanwash 300 plate washer (Skatron Instruments, 3_'> Alexandria, VA) . The wells were blocked using 200 ~,L

of 1$ (w/v) HSA in PBS-T, washed and aliquots of antisera (diluted in 0.1$ (w/v) BSA in PBS-T) were added to the wells,. The plate was then washed and the bound primary antibodies were detected using 100 ~L of S alkaline phosphatase conjugated to rabbit anti-guinea pig IgG (heavy & light chains) (Zymed Laboratories, South San Francisco, CA) diluted 1:2000 dilution in 0.1$ (w/v) BSA in _°~0 mM TrisT"~ (pH 8) . The plates were washed and the color developed using 100 JCL per well of p-nitrophenol phosphate (Sigma) (2 mg/mL in 0.5 M
diethanolamine, 0.2.5 mM MgCl2, pH 9.8) . After 30 minutes, the reaction was stopped by adding 50 ~,L of 3 N NaOH. The absorbance was read in a Thermomax ELISA
plate reader (Molec:ular Devices, Sunnyvale, CA) at 405 nm , ' All the animals immunized with rpilin responded very well, as demonstrated by the antigen ELISAs shown in Table 1.
Table 1 Endpoint Titers for the Binding of Pooled Guinea Pig Antisera Against Gonococcal rpilin to Purified Recombinant Gonococcal Pilin Protein*
Endpoint Prep Immunogen Titers Week 0 Week 4 Week 1 r Pgh3-1 pilin X100 51,345 494,805 (Prep 1) ____2 ____~_ pg~3 _l- p'ilin~____.______54__3 ~ 594, 298 ____ _ ~ 237 (Prep 2) 3 r Pgh3-1 pilin 5100 24,830 546,682 (Prep 3) 2 c.

* Three different lots of rpilin were used as immunogen. Guinea pigs were immunized (s.c.) on weeks 0 and 4 and bled on weeks 0, 4 and 6. Analyses were done on pooled sera.
Effect of Adiuvants on Immune Response The effect of the following adjuvants upon the immune response against gonococcal rpilin was studied in mice: (;a) StimulonT'" QS-21 in PBS (pH 6) ;
(b) aluminum phosphate in PBS (pH 7) ; (c) MPLT"" in PBS
(pH 7) ; (d) aluminum phosphate and MPLT"" in PBS (pH 7) ;
or PBS (pH 7) only. For the analysis of the mouse antisera, the antigen ELISA protocol was modified as follows. The microtiter plates (Costar EIA/RIA, Corning Costar, Cambridge, MA) were coated with 100 ~,L
of 1 ~.g/mL rpilin in PBS overnight at 37°C. The plate IS was washed five times using PHS containing 0.1% (v/v) TweenT~"-20 using a Skantron 300 plate washer. The wells were blocked with PBS containing 0.1% (w/v) gelatin and 0.02% (w/v) NaN3. The primary antibody was diluted in PBS containing 0.1% (w/v) gelatin, 0.05%
(v/v) TweenTM-,20 (PBS-TG) and 0.02% (w/v) NaN3 and 100 ~.L aliquots were incubated in the microtiter plate for 2 hours. After washing, the bound primary antibody was detected using biotinylated rabbit anti-mouse IgG (Fc region) (Brookwood Biomedical, Birmingham, AL) diluted 2:i 1:8000 in PBS-TG and 0.02% (w/v) NaN3. The plate was washed and the secondary antibody was detected, in turn, using streptavidin conjugated horseradish peroxidase diluted ;1:5000 (Zymed Laboratories) in PBS-TG and 0.02% (w/v) NaN3 (30 minute incubation). The plate was washed and the color was developed using 0.5 mg/mL 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) in 0.1 M citrate (pH 4.2) containing 0.03%
(v/v) hydrogen peroxide for 30 minutes and monitored at 405 nm using an SLT 340 ATTC microplate reader (SLT
3~~ Labinstruneents, Research Triangle Park, NC). The data were plotted using a log-log plot and the endpoint titers were determined as previously described.
in'flaen the rpilin was adjuvanted with MPL'"", a humoral immune response in mice was obtained, which was S similar in magnitude to that seen with Stimulon'"" QS-21 (Table 2A). Analysis of vaginal washes from the same animals also revealed a demonstrable IgG titer in the vaginal washes of these animals (Table 2B).

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* o m * * w Example 14 Gonococcal Whole Cell ELISA
The expression by the rpilin of a significant number of the cross-reactive epitopes found on the intact, assembled ;pili was subsequently verified in the whole cell ELISA, 'where antisera against purified rpilin exhibited high titers against numerous gonococcal strains expressing heterologous pili. The l0 ability of guinea pig antisera to bind to live gonococcal cells was done using the following protocol.
Microtiter plates (Iumnunolon II) were incubated overnight with 100 ~,L of 0.01 (w/v) poly-L-lysine (Sigma) in PBS at ~4°C. Overnight agar cultures of l5 bacteria were harvested into PBS with a Dacron swab and the turbidity of the suspension adjusted to an A6oo =
1Ø The poly-lysine solution was discarded from the microtiter plate and 100 JCL aliquots of the bacterial suspension added to the wells. The plates were washed :'0 five times with PB:3 using a hand-held Nunc plate washer and blocked with 200 ~.L of PBS containing 1~ (w/v) BSA
(PHS-H). The plates were then washed five times with PBS and 100 JCL aliquots of antisera diluted in 0.1$
(w/v) BSA in PBS were added to the wells. After ~!5 washing five times with PBS, the bound antisera was detected using 100 JCL of the appropriate alkaline phosphatase conjugates listed above at a 1:2000 dilution in 0.1~ (w/v) HSA in PBS. The plates were washed, the color developed and the absorbance read (at ?.0 30 minutes development) as described above.
When ana7Lyzed by the whole cell ELISA, the guinea pig antisera against rpilin bound to piliated isolates from diverse geographical locations, but not to the corresponding non-piliated cells of the same 35 gonococcal strain (Table 3).

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O p, tia b ~na * --w w The whole cell ELISA analysis of mouse antisera against rpilin (Table 2) was done using microtiter plates :in which the bacteria were dried down in the wells by the following protocol. Overnight agar S cultures of bacteria were harvested into PBS with a Dacron swab and the turbidity of the suspension adjusted to an Asoo = 0.1. Aliquots (100 ~L) of the bacterial suspension were added to the wells and the plate was air dried at 37°C. After evaporation of all 110 the liquid, the plates were sealed and stored at 4°C
until used. The remainder of the assay was done following the protocol previously described for the antigen ELISA of mouse antisera. The data from the whole cell ELISAs (Tables 2 and 3) suggest that rpilin 15 induces antibodies which bind to conserved epitopes on the surface of piliated gonococci.
Examt~le 15 Induction of a Mucosal Immune Response 2.0 A,g~ai.nst Gonococcal rpilin Because gonorrhea is a disease of the genital mucosae, it was of interest to examine the ability of mucosal immunization to induce a mucosal immune 25 response. This wasp accomplished in the following manner. Mice were immunized intranasally with gonococcal rpilin 3.n saline (1 or 10 ~,g in 10 ~L) with or without 1 ~,g of native cholera toxin on weeks 0, 1 and 2. Groups of five Swiss-Webster mice were 30 immunized intranasa,lly with rpilin with or without cholera toxin on weeks 0, 1 and 2. Analyses were conducted on pooled, sera. Endpoints titers for week 0 were <50.

As shown in the following Table 4, there was a significant immune response detected in the antigen ELISA when the animals were immunized with rpilin in the absence of adjuvant (the week 0 titers were <300).
This response was enhanced by the addition of native cholera toxin.
Table 4 Endpoint Titers for the Binding of Pooled Mouse .l0 Antisera Against Gonococcal rpilin to Purified Recombinant Gonococcal Pilin Protein Antigen Adjuvant Day 22 Day 36 Day 50 (dose fig) (dose fig) rpilin (1) none 1,684 1,287 2,291 rpilin (10) none 29,046 20,658 71,067 rpilin (1) cholera 4,673 7,526 3,273 toxin ( 1 ) rpilin (10) cholera 107,011 321,714 280,079 toxin (1) None cholera <300 <300 <300 toxin (1) Next, these sera were examined for their ability to bind to intact, piliated gonococcal cells by an ELISA performed against cells from N. gonorrhoeae strain FA1090. ThE: cells were dried down onto the microtiter plate as described previously. As shown in t.0 Table 5, a low titE:r was detected for rpilin alone (the week 0 titers were <300). The binding to piliated cells was greatly enhanced by addition of cholera toxin.
2.5 Table 5 Endpoint Titers for the Binding of Pooled Mouse Antisera Against Gonococcal rpilin to Whole, Piliated Gonococcal cells Antigen Adjuvant Day 22 Day 36 Day 50 (dose ~,g) (dose fig) rpilin (1) none 1,009 852 729 rpilin (10) none 6,009 6,252 5,564 rpilin (1) cholera 1,133 1,456 1,048 toxin (1) rpilin (10) cholera 209,522 57,767 38,127 toxin ( 1 ) None cholera 239 <500 <500 toxin ( 1 ) Example 16 Gonococcal Immunoelectron Microscopy Visualization of the binding of antisera to piliated bacteria was conducted using the following protocol. Gold coated grids were spotted with an aliquot from a late log phase liquid culture of recA-, piliated N. gonorrhaeae for five minutes and the excess fluid was removed with a piece of filter paper. The grids were blocked with PBS-B for five minutes, followed by 1$ (w/v) fish gelatin (Fluka, Ronkonkoma, NY) in PBS for 10 minutes. The grids were then incubated with polyclonal antisera diluted in PBS-B for 1-60 minutes at room temperature. Unbound antibodies were removed by floating the grid on droplets of PHS-B
(4 x 30 seconds). The bound primary antibodies were detected by floating the grid on a drop of 12 nm 2> colloidal gold bound to donkey anti-guinea pig IgG
(Jackson Research Labs, West Grove, PA) diluted 1:5 in PBS-B for 30 minutes. The grids were then washed five times on droplets of PBS-B as described above. The sample was then stabilized with 1% (v/v) glutaraldehyde (Electron Microscopy Sciences, Fort Washington, PA) in PBS for three minutes, then rinsed 5 x 1 minute in distilled water and lightly stained using NanoVan stain (NanoProbes, Stony Brook, NY) (pH 8) for 30 seconds.
All liquid was removed by touching the grid to a piece of filter paper and examined on a Zeiss lOC
transmission electron microscope at 15-75,OOOX using an acceleration voltage of 80 kv.
As shown by immunoelectron microscopy (Figure lA), antibodies against the recombinant pilin were bound along the length of the heterologous pili filaments on the surface of gonococci. This suggested :l5 that the antibodies would bind to these epitopes which would be present on the surface of the bacteria jn vi vo .
Example 17 Whole Cell ELISA of Gonococcal rvilin Oliaomer In order to distinguish the biochemical and immunological properties of rpilin from those of intact pili (or pilin oligomer), purified rpilin was converted ~S to rpilin oligomer by dialysis against pH 12 phosphate buffer. Antisera induced by this material (rpilin oligomer) was examined for its ability to bind to live, piliated gonococci using the whole cell ELISA. As shown in Table 6, antisera against rpilin oligomer had 30 significantly lowez- endpoint titers for binding to diverse piliated gonococcal cells as compared to antisera induced by untreated rpilin. This suggests that rpilin oligome:r had lost a significant number of the cross-reactive epitopes normally present on the 35 rpilin protein.

Table 6 Effect of pH 12 ("oligomerization") on the Endpoint Titers for rpilin Guinea Pig Antisera Binding to Piliated N. gonorrhoeae Cells*
Strain rpilin rpilin Oligomer**

I-756 927,564 16,903 FA-19 107,100 ~ 1,982 __-FA109p ____________________________________ -__ (2-57-U17) 721,786 6,737 LB2 905,711 4,205 #11 ~ 225,602 6,999 ______#4___..____288,12p-___._8, 104 -_ 3138IC~~ 106, 315 5, 429 _____T _ -'4 9 __ 4 2 ~
~ __..__ 7 , 9 1 6 2 -_ 8 7 '_ 1948 ~~ 166,864 8,616 J474B~ 576,640 25,002 * Guinea pigs (4 per group) were immunized (s. c.) with 20 ~,g of rpilin antigen adjuvanted with 25 ~,g of StimulonT"" QS-21 on weeks 0 and 4. Pooled sera from week 6 were analyzed.
**Purified rpilin was dialyzed against 37 mM sodium phosphate (pH 12) f:or 48 hours at 4°C, followed by dialysis against PBS for 24 hours.
Example 18 Inhibition of Adherence to Human Cervical Cells Because pili mediate the initial binding of N. gonorrhoeae to human mucosal cells, the ability of rpilin antibodies to inhibit the attachment of these WO 99/55875 PC'T/US99/09486 bacteria to epithelial cells was investigated. ME-180 cells, which were derived from a cervical carcinoma, ware selected. In addition, to minimize the clumping of the piliated bacteria in these experiments, they were grown in liqu:fd suspension cultures. This required using recA' derivatives of gonococcal strains Pgh3-1 or 1756 in order to maintain pili expression.
These recA' strains did not show significant clumping during the 4-5 hours growth in liquid culture, which made it easier to interpret the results.
In these experiments, an eight well chamber slide (Nunc) was seeded with ME-180 cells such that the cells were 80-90% confluent on the day of the experiment. An ovex-night culture of recA' gonococcal cells was swabbed into P8S (warmed to 37°C) and used to inoculate a flask of liquid GC media supplemented with 0 .4% (w/v) NaHC03 to a final Asoo = 0 ~ 2 ~ The cell culture was incubated with shaking at 37°C at 120 RPM
for approximately four hours, at which point the culture reached an A6oo= 0.8. The bacterial cell suspension was diluted 1:8 in RPMI 1640. The wells of a second 8 well chamber were incubated for at least one hour with 300 JCL of RPMI 1640 and fetal calf serum.
The RPMI 1640 block; was discarded and 40 ~,L of antisera or RPMT 1640 (without calf serum) added, followed by 260 ~,L of the diluted bacterial suspension {~gxl0' CFU) and incubated for one hour at 37°C/5% (v/v)CO2. The chamber well slide with the ME180 cells was washed once with RPMI 1640 devoid of antibiotics. Then, pre-3I~ incubated mixtures of bacteria and antisera were added onto the ME180 cells and the slides incubated for 30 minutes at 37°C. The media containing unbound bacterial cells was removed from the cervical cell monolayer and the wells washed gently three times with 3.'> RPMI 1640. After t;he last wash, the chamber wells were removed from the slide, the cells fixed in methanol for 30-60 seconds and stained with Wright-Giemsa stain (VWR
Scientific, West Chester, PA). After destaining in water, coverslips were mounted over each well.
The slides were examined by light microscopy using oil immersion and pictures of representative views were taken by a person blinded as to the test sera. The resulting pictures were analyzed by persons who were also blinded as to the identity of the 1'.0 samples. In addition, because the piliated gonococci bound the epithelial monolayers in clumps, the effect of antisera on the binding of the gonococcal cells was quantitated by counting clumps of bacteria instead of individual bacteria. The numbers of clumps of piliated 15 bacteria observed in ten random scans across each well were determined. The percent difference between wells containing immune and normal sera was determined.
Again, this analysis was done independently by researchers blinded as to the samples that they were f.0 analyz ing .
Initial x-esults demonstrated that piliated and non-piliated cells had different binding patterns to the confluent monolayers of ME180 cells. Hoth piliated strains of: gonococci typically bound as 25 bacterial clumps to selected epithelial cells within the confluent monolayer. In contrast, the corresponding isoge:nic non-piliated bacteria either did not bind (Pgh3-1) or showed a low level, disperse binding (I756) to the epithelial monolayer. Thus, the 30 aggregation of the bacteria on the epithelial monolayer correlated with the: presence of pili.
Next, the: ability of guinea pig antisera against Pgh3-1 rpil.in to inhibit the binding of piliated cells of strain I-756 to ME-180 cells was 35 tested. Analysis of representative pictures (compare Figure 2A (week 6) to Figure 2B (week 0)) demonstrated that antibodies against rpilin significantly inhibited the binding of pil:iated gonococci to cervical epithelial cells. In contrast, the rpilin antisera had no effect on the binding of non-piliated cells from the same strain when compared to normal guinea pig sera (data not shown). While the number of bacteria bound under these conditions could not be determined, adherence was quantitated by counting the bacterial clumps bound in the presence of either normal or immune sera. Using this method, it was determined that antisera against rpilin resulted in ~60~ decrease in the bacterial clumps bound to epithelial cells as compared with normal guinea pig serum.
While this assay did not yield readily quantifiable data, the estimates obtained by counting bacterial clumps probably resulted in an underestimation of the effectiveness of the antisera.
This is because of the binding mediated by other cell surface components (e.g., Opa proteins) which would not be expected to be overcome with antisera against rpilin alone.
Example 19 Analytic Methods for Meningococcal Chimeric Class I x~ilin The analytic methods described in Example 10 were used for the chimeric meningococcal class I
rpilin. As determined by MALDI-TOF mass spectroscopy, the subunit molecular weight of the meningococcal chimeric class I rpilin protein is 17,659 daltons, which is very similar to the anticipated mass of 17,676 daltons based on the amino acid content. When this 3:i protein was analyzed by size exclusion chromatography using a SuperoseT"" 12 column equilibrated in PBS
containing 0.05 (w/v) ZwittergentT"' 3-14, the chimeric class I rpilin has an apparent molecular weight of 69,480 daltons. This is essentially identical to the apparent molecular weight of gonococcal rpilin (68,899 daltons) analyzed under the same conditions.
The N-ternninal sequence of the purified meningococcal class I rpilin protein was determined by Edman degradation and the results (from three different samples) agreed with the predicted protein sequence.
At least 35 residues were determined for all sequences.
Example 20 Meningococcal Chimeric Class I rpilin ELISA
The endpoint titers against purified proteins or bacterial cells were determined by ELISA using the methods described i.n Examples 13 and 14. The ELISAs were performed using pooled sera from the respective bleeds. Whole cell. ELISA was done on meningococcal cells which had been heat killed (56°C for 60 minutes) or dried down directly onto the microtiter plates. The cell suspension was diluted to an absorbance of 0.1 at 620 nm and 100 ~,L aliquots were placed into the wells of microtiter plates. Each plate was dried at 37°C or at room temperature, sealed and stored at 4°C until used. The protocol for the whole cell ELISA was modified as follows: (1) primary and secondary antisera were diluted in PHS containing 0.1~ (v/v) TweenTM-20 317 and 0.1~ (w/v) BSA; and (2) the plates were washed five times with PHS containing 0.05 (v/v) TweenT~~-20 using a Skanwash 300 plate washer.
All the guinea pigs immunized with the chimeric class I rpilin responded very well, as 3'_i demonstrated by the antigen ELISAs shown in Table 7.

Table 7 Endpoint Titers for the Binding of Meningococcal Chimeric Class I rpilin Antisera to Purified Meningococcal Chimeric Class I rpilin*
Adjuvant:
Bleed: StimulonTM A1P04 None Week 0 23 12 32 Week 4~ 26,607 12,067 4,829 Week 6~i 1,519,956 372,539 302,911 * Guinea pigs (four per group) were immunized (s.c.) on weeks 0 and 4 with 20 ~,g of chimeric class I rpilin adjuvanted with (a) 25 ~,g of StimulonTM QS-22 in PBS
(pH6) ; (b) 100 ~,g of A1P09 in saline; or (c) PBS (pH7) only. The animals were bled on weeks 0, 4 and 6.
Pooled sera was used for all analyses.
1:5 Significant responses were also seen with the ELISAs to piliated cells, as shown in Table 8.
Table 8 Endpoint Titers for the Binding of Meningococcal Chimeric Class I rpilin Antisera to Piliated Cells from 1V. men~:ngitidis (strain H355) Adjuvant:
Bleed: StimulonT"~A1P04 None Week 0 28 19 53 Week 4~ 1, 293 1., 052 381 Week 6 61,497 ~ 25,477 16,467 * Cells were heat killed.
Effects of Adiuvants on Immune Response S
The effect of adjuvants upon the immune response against the meningococcal chimeric class I
rpilin was studied in mice using the methods described in Example 13. As shown in Table 9, the most significant response for the binding of antisera with the meningococcal c:himeric class I rpilin was achieved with the addition of StimulonT"' QS-21.
Table 9 Endpoint Titers for the Binding of Meningococcal Chimeric Class I rpilin Antisera to Purified Meningococcal Chimeric Class I rpilin*
Adjuvant:
Bleed: StimulonT""A1P09 MPLTM A1P04/MPLT"" None Week_ ___ <50 44 <50 31 <50 0 _ _______________._________________._______ __.______ ____ ___ ____________________________________ _Week__4___ 44,_011____ 29,_925__ 40 110, <250 ,146 093 Week 707,084 103,437 _ _____ 115,022 6 284,455 _ ____ 137,686 * Mice (ten per group) were immunized (s.c.) on weeks 0 and 4 with 10 ~,g of meningococcal chimeric class I
rpilin adjuvanted with (a) 25 ~g of StimulonTM QS-21 in PBS (pH 6) ; (b) 100 ~,g of A1P04 in saline; (c) 50 ~g 2:5 MPLT"" in PBS (pH 7) ,; (d) 100 beg of A1P04 and 50 ~,g MPLT""
in saline; or (e) PHS (pH 7) only. The animals were bled on weeks 0, 4 and 6. Pooled sera was used for all analyses.

As shown in Table 10, the most significant response for the binding of antisera to piliated cells from N. meningitidis was also achieved with the addition of StimulonT~'t QS-21.
S
Table 10 Endpoint Titers for the Binding of Meningococcal Chimeric Class I rpilin Antisera to Piliated Cells from N. merxingitidis (strain H355) Adjuvant Bleed: StimulonT"" A1P0,, MPLT"~ A1P04/MPLT~~ None Week 172 146 153 160 <50 Week _______ S ___2'_31p'____ 4_~ ______-5. _______31i 4' ~ 08$ _._ 205-_ 64'7'____ __ Week 171,718y 25,135 52,053 17,039 16,617 * Cells were heat killed.
IS Further analyses demonstrated that this antisera against the chimerj.c class I rpilin bound to meningococcal cells which expressed either class I
pilin or, in some eases, class TI pilin. The results, shown in Table 11, evidence this partial cross-reactivity.

Endpoint Titers for the Binding of Meningococcal Chimeric Class I rpilin Antisera to Piliated Cells from N. meniagi tf di s Ad juvant Strain Class Day 0 StimulonTM ALPO, MPLTM

Pilin QS-21 expressed H355 I 409 127,383 41,190 102,987 M982 I 217 >36,540 >36,540 >36,540 CDC1521 II 988 2,602 1,345 1,768 FAM18 II 3,518 >36,540 26,513 >36,540 ~ ~ ~

* Cells were dried down (at room temperature) directly onto microtiter p:Lates without being heat killed.
Examt~le 21 Meningococcal Chimeric Class I rpilin Immunoelectron Microscopy Visualization of the binding of chimeric meningococcal class I rpilin antisera to piliated cells from N. meniagitidis strain H355 was conducted using transmission-electron microscopy as follows. A colony from an overnight culture of N. meningitidis was carefully picked up using a sterile loop and placed in a microfuge tube containing 0.5-1.0 mL of modified Franz media [1.3 g/L glutamic acid, 20 mg/L cysteine, 10 g/L Na2HP04 ~ 7Hz0, 9 0 mg/L KC1, 6 g/L NaCl , 2 g/L
yeast dialysate anal supplemented with dextrose (4 g/L), glutamic acid (100 mg/L), cocarboxylase (200 ~,g/L) and ferric nitrate (5 mg/L)). Gold coated grids were spotted with an aliquot of the cell suspension for five SUBSTIT17TE SHEET (RULE 26) spotted with an al:Lquot of the cell suspension for five minutes and the 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. The grids were incubated, in sequence, with (a) PBS-B for five minutes, (b) 1$ (w/v) fish gelatin in PBS for 10 minutes, and (c) PBS containing 0.2 M glycine for five minutes. The blocked grids were then probed with antisera against meningococcal chimeric class I rpilin protein as described in Example 16. As shown in Figure 3A), the antibodies against the meningococcal chime:ric class I rpilin protein bound along the length of: the pili. In contrast, normal serum (week 0) did not show any binding to the pili (Figure 3H).
Example 22 Cross-reactivity of Meningococcal Chimeric Class I
2'~ rt~ilin Antisera with Gonococcal Piliated Cells Based on the sequence similarity of meaingococcal class I pilin and gonococcal pilin, it was shown in Example 4 above that antisera directed 2:i against gonococcal rpilin recognized and bound to piliated meningococcal cells. In this example, it is demonstrated that antisera raised against meningococcal chimeric class I rpilin binds to piliated gonococcal cells. The data from the mouse and guinea pig 30 experiments are summarized in Tables 12 and 13, respectively.
3 <~

Table 12 Endpoint Titers for the Binding of Mouse Antisera Against Meningococcal Chimeric Class I rpilin to Piliai~ed N. gonorrhoeae Cells Antigen / Adjuvant GC

GC Nm rclass rpilin I pilin Strain + MPLT"~

StimulonT~" A1P04 MPLT~~ A1P04/ None QS-21 MpLTnn I756 4,527,943 79,927 114,958 56,627 57,426 356,936 FA1090 531,627 40,406 97,224 31,267 38,122 219,600 Table 13 Endpoint Titers fo:r the Binding of Guinea Pig Antisera Against Meningococcal Chimeric Class I rpilin to Piliat.ed N. goaorrhoeae Cells Antigen / Adjuvant GC Nm rclass GC rpilin +
Strain I pilin StimulonTM

StimulonrM A1P04 None QS-21 I756 301,969 122,714 78,111 46,424 FA1090 322,311 ~ 262,422 170,842 108,094 GC - N. gonorrrhoeae; Nm - N. meningit3dis - 77 _ Example 23 Passive Protection Against Meningococcal Hacteremia by Meninsococcal Chimeric Class I. rpilin Antisera An accepted animal model for evaluating the ability of vaccines to protect against meningococcal bacteremia is the infant rat model originally described by Saukkonen and L~einonen (34). The ability of guinea l0 pig antisera against meningococcal chimeric class I
rpilin (adjuvanted with StimulonT"" QS-21) to protect against bacteremia caused by a meningococcal strain, which expresses a pilin, was tested. On day 0, Sprague-Dawley infant rats (4-5 days old) were :l5 passively immunized (i.p.) with 0.1 mL of guinea pig antiserum (week 6) against chimeric class I rpilin diluted 1:5, 1:10 or 1:20 in PBS. The control group received 0.1 mL in;jection of normal guinea pig serum (week 0) diluted 1:5 in PBS. Twenty-four hours later, a!0 the animals were challenged i.p. with approximately 5 x 105 colony forming units (cfu) in 0.1 mL of piliated N.
meningit~dis (stra:Ln H355) . Three hours after challenge, the animals were sacrificed and aliquots of cardiac blood were diluted and plated onto GC agar c5 plates. The plates were then incubated for 18-24 hours at 37°C with 5~ COz. The bacterial colonies were counted and the level of bacteremia was then determined. One way analysis of variance t test was used to compare groups receiving immune serum (week 6) 30 with the control g~°aup receiving normal serum (week 0).
As shown in Table 7!4 below, animals passively immunized with guinea pig antiserum specific for meningococcal chimeric class I rpilin showed more than a log reduction in the level of bacteremia as compared to 35 those animals iamnuxiized with normal guinea pig serum.

WO 99/55875 PC'T/US99/09486 _ 78 -This difference was statistically significant, with a p value of <0.05.
Table 14 Ability of Guinea Pig Antisera Against Meningococcal Chimeric Class I rpilin Protein to Prevent Bacteremia in Infant Rats Challenged with Piliated N'. meningitid.is (Strain H355) Bleed Dilution Mean cfu t std Week 0 1:5 4.87 t 0.18 Week 6 1:5 3.55 t 0.48**

Week 6 1:I0 3.63 t 0.36**

Week 6 1:20 3.98 t 0.75**

* Antisera against meningococcal chimeric class I
rpilin protein was obtained from guinea pigs as described in Table 7.
** p <0.05.
cfu t std = colony forming units f standard deviation.
Example 24 Induction of a Mucosal Immune Response Against Meninctococcal Chimeric Class I rpilin ?0 Mice were immunized intranasally with meningococcal chimeric class I rpilin in saline (5 ~,g in 10 ~,L) with or without 1 ~.g of a cross-reactive mutant form of cholera toxin (CT-CRM, E29H) on weeks 0, :>.5 1 and 2. As shown in the following Table 15, there was a significant immune response detected in the antigen ELISA when the animals were immunized with rpilin in the absence of adjuvant. This response was enhanced by the addition of cholera toxin.
a0 _ 79 _ Table 15 Endpoint Titers for the Binding of Pooled Mouse Antisera Against Meningococcal Chimeric Class I rpilin to Purified Recombinant Meningococcal Chimeric Class I
rpilin Protein*
rpilin, no rpilin plus adjuvant CT-CRM

Sera IgG 6,168 1,181,871 IgA 490 3,940 Bronchial wash**

IgG <10 580 IgA <10 19 Nasal wash**

IgG <10 98 IgA 12 236 Vaainal wash**

IgG 174 70 IgA 1S 687 * Pooled sera from week 4 were analyzed. Endpoint titers for pooled sera from week 0 for IgG and IgA were <50.
** Washes were perf;armed as follows:
Bronchial: Lungs were washed 5 times with 1 mL
RPMI 1640, then 50 ~,L fetal bovine serum (FBS) was then added to the sample:, which was clarified by centrifugation (12,000 x g x 5 minutes) and stored at -20°C.
Nasal: The nasal passages were washed once with 0.5 mL of RPMI 164iD and 20 ~L of FHS was then added to the sample before storage at -20°C.
Vaginal: Vaginas were washed 5 times with 0.075 mL
of RPMI 1640 and 10 ~,L of FBS was then added to the sample before storage at -20°C.
Example 25 Active Protection. Against Meningococcal Colonization by Meningococca:L Chimeric Class I rpilin Antisera The init:Lal step in meningococcal disease in 1.5 human beings is the colonization of the nasopharynx by the bacteria. In i=his process, pili are believed to play a major role Ln mediating the inital attachement to the epithelial c:elle. A number of researchers have described procedures for colonizing the nasophayrnx in neonatal animals, but no one has investigated this as a model for testing t:he efficacy of meningococcal vaccines (35). In order to assess the invention described herein, a nasal colonization model for N.
mening~tidis using adult outbred mice has been 2,5 developed. Swiss-Webster mice were immunized with meningococcal chimearic class I rpilin adjuvanted with MPLT"~ subcutaneously on weeks 0, 4 and 8. At week 10, the animals received an intraperitonal injection of 2 mg iron dextran (Sigma) and were challenged 30 intranasally with approximately 1 x 10' cfu of mid-log phase piliated meningococci in a volume of 10 ~,L which also contained 40 ~,g of iron dextran. On day 1 after challenge, half then animals received an additional intraperitonal injection of 2 mg iron dextran. The number of viable bacteria in the nose were determined on days 1 and 2 after challenge by plating nasal tissue homogenates on GC agar plates containing selective antibiotics. The results are shown in Table I6.
Table 16 Number of Viable Bacteria (cfu) Recovered from Nasal Homogenates of Mice Challenged with Piliated N.
meni:ngit~dis Strain H355*
cfu per nose Antigen (Dose ~g)* Day 1** Day 2**

H355 Whole cell (25) 1,165 67 Class I rpilin (10) 6,866 63 Saline 17,943 3,406 * All vaccines were formulated with 100 ~,g of MPLT~~
per dose. Each group consisted of five mice.
** Days after intranasal challenge.
1:~
Example 26 Western Blot Analysis of the Immune Response Against Menincrococcal Class II Chimeric rpilin The purified meningococcal class II chimeric rpilin was used to immunize guinea pigs following the protocol described .in Example 11. The antisera derived from guinea pigs immunized with meningococcal class II
chimeric rpilin were analyzed first by Western blots 2_'~ (data not shown). These blots demonstrated that the antisera against meningococcal class II chimeric rpilin recognized a band corresponding to pilin in whole cell lysate from piliated meningococcal cells which expressed either class II pilin (FAM18) or class I
3G~ pilin (H355). In contrast, antisera directed against an extract from E. coli containing the pTrcHis vector only did not react with either pilin band in these Western blots.
Example 27 Binding of Antisera against Meningococcal Chimeric Class II rpilin to Piliated Meningococcal Cells Antisera elicted against partially purified meningococcal chime:ric class II rpilin was shown to bind to meningococcal cells from the homologous strain, FAM18 with a titer of >36,450 (the week 0 titer was 473 ) .
1.5 Bibliog~ra~phy 1. Tennent, J.M., and Mattick, J.S., pages 127-146 of Fimbriae. Adhesion, genetics, biogenesis and vaccines, P. Kleaun, Ed. (CRC Press, Boca Raton, FL
1994) .
2. Strain, M.S., and Lory, S., Ann. Rev.
Microbiol., 47, 565.-596 (1993).
3. Heck:els, J.E., Clin. Microbiol. Rev., 2 (Suppl. ) , S66-73 (1.989) .
4. U.S. Patent Number 4,702,911.
5. Scho~olnik, G.K., J. Exp. Med., 159, 1351-1370 (1984).
6. Davies, J.K., et al., pages 147-155 of Fimbriae. Adhesion, genetics, biogenesis and vaccines, P. Klemm, Ed. (CRC Press, Boca Raton, FL 1994).
7. Aho, E.L., et al., Infect. Immun., 65, 2613-2620 (1997).
8. Achtman, M., et al., J. Inf. Dis., 165, 53-68 (1992).

9. Levine, M.M., et al., pages 255-270 of Fimbriae. Adhesion..genetics, biogenesis and vaccines, P. Klemm, Ed. (CRC Press, Boca Raton, FL 1994).

10. Stewart, D.J., et al., Aust. Vet. J., 62, 153-159 (1985).

11. Tramont, E.C., Clin. Microbiol. Rev., 2 (Suppl.Z, S74-7 (1989).

12. Boslego, J.W., et al., Vaccine, 9, 154-162 (1991).

13. Rothbard, J.B., and Schoolnik, G.K., Adv. Exp. Med. Biol_, 185, 247-73 (1985).

14. Olaf~son, R.W. , et al. , Infect. Iamnun. , 48, 336-342 (1985) .

15. Seifert, H.S., et al., Vaccine, 6, 107-109 (1988).

16. Spa;rling, P.F., et al., Proc. Natl.
Acad. Sci. USA, 91, 2456-2463 (1994).

17. Rothbard, J.B., et al., Proc. Natl.
Acad. Sci. USA, 82, 915-919 (I985).

18. Forest, K.T., and Tainer, J.A., pages 167-173 of Vaccines 97. Molecular Approaches to the Control of Infectious Diseases, F. Brown, et al., Eds.
(Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY 1997).

19. Roba.nson, E.N.J., et al., Mol.
Microbiol., 3, 57-!i4 (1989) .

20. Eisentein, H.I., et al., pages 89-113 of Recombinant DNA vaccines: rationale and strataQies, R.E. Isaacson, Ed.,. (Marcel Dekker, Inc., New York, NY
1992) .

21. Lepper, A.W., Aust. Yet. J., 65, 310-316 (1988) .

22. Hull., R.A., et al., Infec. Immun., 33, 933-938 (1981) .

23. Granted European Patent Number 202,260 B1.

24. Hoyne, P.A., et al., J. Bact., 174, 7321-7327 (1992).

25. Elleman, T.C., et al., Infect. Immun., 51, 187-192 (1986).

26. O'Meara, T.J., et al., Immun. Cell.
Biol., 71, 473-488 (1993).

27. Emery, D.L., et al., Aust. Vet. J., 61, 237-8 (1984).

28. Stewart, D.J., et al., Vet. Microbiol., 27, 283-93 (1991).

29. Aloes, A.M.B., et al., American Society for Microbioloav, Abstract E-79, page 253 (1997).

30. Meyer, T.F., et al., Cell, 30, 45-52 (1982) .

CA 02325055 2000-10-10 ' 31. Sambrook, J., et al., Molecular CloninQ~
A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989).

32. Stephens, D.S., Clin. Microbiol. Rev., 2 (Suppl.), S104-111 (1989).

33. Tramont, E.C., et al., J. Clin. Invest., 68, 881-888 (1981),.

34. Saukkonen, K. and Leinonen, M., pages 815-820 of Gonococci and Menincrococci, J. T. Poolman, Ed. (Kluwer Academic Publications, Dordrecht, Netherlands, 1986)..

35. Sali.t, I.E., et al., Can. J. Microbiol., 30, 1022-1029 (1984).

:>EQUENCE LISTING
<110> American Cyanamid Company <120> Vaccines Containing Recombinant Pilin Against Neisseria Gonorrhoeae or Ne~isseria Meningitidis <130> 33377-00/PCT
<140>
<141>
<160> 29 <170> PatentIn Ver. 2,0 <210> 1 <211> 509 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence:Chimeric of Neisseria Meningitidis Class I and Neisseria Gonorrhoeae <220>
<221> CDS
<222> (1)..(501) <900> 1 atg gat acc ctt caa aaa ggc ttt acc ctt atc gag ctg atg att gtg 98 Met Asp Thr Leu Gln Lys Gly Phe Thr Leu Ile Glu Leu Met Ile Val atc gcc atc gtc ggc att ttg gcg gca gtc gcc ctt ccc gcc tac caa 96 Ile Ala Ile Val Gly Ile Leu Ala Ala Val Ala Leu Pro Ala Tyr Gln gac tac acc gcc cgc gcg caa gtt tcc gaa gcc atc ctt ttg gcc gaa 144 Asp Tyr Thr Ala Arg Ala Gln Val Ser Glu Ala Ile Leu Leu Ala Glu 35 40 . 45 ggt caa aaa tca gcc gtt acc gag tat tac ctg aat cac ggc gaa tgg 192 Gly Gln Lys Ser Ala Val Thr Glu Tyr Tyr Leu Asn His Gly Glu Trp ccc ggc aac aac act tct gcc ggc gtg gca tct tct tca aca atc aaa 240 SUBSTITUTE SHEET (RULE 26) Pro Gly Asn Asn Thr Ser Ala Gly Val Ala Ser Ser Ser Thr Ile Lys ggc aaa tat gtt aag gaa gtt aca gtc gca aac ggc gtc att acc gcc 288 Gly Lys Tyr Val Lys Glu Val Thr Val Ala Asn Gly Val Ile Thr Ala aca atg ctt tca agc ggc gta aac aaa gaa atc caa ggc aaa aaa ctc 336 Thr Met Leu Ser Ser Gly Val Asn Lys Glu Ile Gln Gly Lys Lys Leu tcc ctg tgg gcc aag cgt caa gac ggt tcg gta aaa tgg ttc tgc gga 389 Ser Leu Trp Ala Lys Arg Gln Asp Gly Ser Val Lys Trp Phe Cys Gly cag ccg gtt acg cgc acc gac gcc aaa gcc gac acc gtc gcc gcc gcc 432 Gln Pro Val Thr Arg Thr Asp Ala Lys Ala Asp Thr Val Ala Ala Ala gcc aag acc gcc gac aac atc aac acc aag cac ctg ccg tca acc tgc 480 Ala Lys Thr Ala Asp Asn Ile Asn Thr Lys His Leu Pro Ser Thr Cys cgc gac gca agt gat gcc agc taa 504 Arg Asp Ala Ser Asp Ala Ser <210> 2 <211> 167 <212> PRT
<213> Artificial Sequence <400> 2 Met Asp Thr Leu Gln Ly,s Gly Phe Thr Leu Ile Glu Leu Met Ile Val Ile Ala Ile Val Gly Ile Leu Ala Ala Val Ala Leu Pro Ala Tyr Gln Asp Tyr Thr Ala Arg Ala Gln Val Ser Glu Ala Ile Leu Leu Ala Glu Gly Gln Lys Ser Ala Va:l Thr Glu Tyr Tyr Leu Asn His Gly Glu Trp Pro Gly Asn Asn Thr Ser Ala Gly Val Ala Ser Ser Ser Thr Ile Lys SUBSTITUTE SHEET (RULE 26) GlyLysTyrValLys Gl.uValThr ValAla AsnGlyVal IleThrAla ThrMetLeuSerSer Gl.yValAsn LysGlu IleGlnGly LysLysLeu SerLeuTrpAlaLys ArgGlnAsp GlySer ValLysTrp PheCysGly GlnProValThrArg ThrAspAla LysAla AspThrVal AlaAlaAla Ala Lys Thr Ala Asp Asn Ile Asn Thr Lys His Leu Pro Ser Thr Cys Arg Asp Ala Ser Asp Ala Ser <210> 3 <211> 5I0 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence:Chimeric of Neisseria Mening:itidis Class II and Neisseria Gonorrhoeae <220>
<221> CDS
<222> cl)..t510) <400> 3 atg gaa gca atc caa aaa ggt ttc acc ctg atc gag ctg atg atc gtc 98 Met Glu Ala Ile Gln Lys Gly Phe Thr Leu Ile Glu Leu Met Ile Val atc gcc atc gtc ggt atc: ttg gca gcc gtc gcc ctg ccc gca tac caa 96 Ile Ala Ile Val Gly Ile: Leu Ala Ala Val Ala Leu Pro Ala Tyr Gln 20 25 ~ 30 gac tac acc gcg cgc gcc: caa atg tcc gaa gcc ctg act ttg gca gaa 144 Asp Tyr Thr Ala Arg Ala Gln Met Ser Glu Ala Leu Thr Leu Ala Glu ggt caa aaa tcc gca gtc~ atc gag tat tat tcc gac aac ggc aca ttc 192 SUBSTITUTE SHEET (R ULE 26) WO 99/55875 PC"T/US99/09486 Gly Gln Lys Ser Ala Val Ile Glu Tyr Tyr Ser Asp Asn Gly Thr Phe ccg aac agc aat act tcc gca ggt att get gcc tct aac gag att aaa 240 Pro Asn Ser Asn Thr Se:r Ala Gly Ile Ala Ala Ser Asn Glu Ile Lys 65 7~0 75 80 ggt aag tat gtg gca tcg gtt aag gtt gaa ggt aat gcc tct gtt get 288 Gly Lys Tyr Val Ala Se:r Val Lys Val Glu Gly Asn Ala Ser Val Ala tct att acc get acc atg aac tct agt aat gtg aat aag gac atc aaa 336 Ser Ile Thr Ala Thr Me't Asn Ser Ser Asn Val Asn Lys Asp Ile Lys ggt aaa acc ttg gta ctc gtc ggc aaa caa aac tcc ggt tcg gta aaa 389 Gly Lys Thr Leu Val Leu Val Gly Lys Gln Asn Ser Gly Ser Val Lys tgg ttc tgc gga cag ccg gtt acg cgc gac aac gcc gac aac gac gac 432 Trp Phe Cys Gly Gln Pro Val Thr Arg Asp Asn Ala Asp Asn Asp Asp gtc aaa gac gcc ggc aac aac ggc atc gaa acc aag cac ctg ccg tca 480 Val Lys Asp Ala Gly Asn Asn Gly Ile Glu Thr Lys His Leu Pro 5er acc tgc cgc gat acg tca tct gat gcc aaa 510 Thr Cys Arg Asp Thr Ser Ser Asp Ala Lys <210> 9 <211> 170 <212> PRT
<213> Artificial Sequence <400> 4 Met Glu Ala Ile Gln Lys Gly Phe Thr Leu Ile Glu Leu Met Ile Val Ile Ala Ile Val Gly Ile>. Leu Ala Ala Val Ala Leu Pro Ala Tyr Gln Asp Tyr Thr Ala Arg Ala Gln Met Ser Glu Ala Leu Thr Leu Ala Glu Gly Gln Lys Ser Ala Val. Ile Glu Tyr Tyr Ser Asp Asn Gly Thr Phe SUBSTITUTE SHEET (RULE 26) Pro Asn Ser Asn Thr Ser Ala Gly Ile Ala Ala Ser Asn Glu Ile Lys Gly Lys Tyr Val Ala Ser Val Lys Val Glu Gly Asn Ala Ser Val Ala Ser Ile Thr Ala Thr Met Asn Ser Ser Asn Val Asn Lys Asp Ile Lys Gly Lys Thr Leu Val L~eu Val Gly Lys Gln Asn Ser Gly Ser Val Lys Trp Phe Cys Gly Gln P:ro Val Thr Arg Asp Asn Ala Asp Asn Asp Asp Val Lys Asp Ala Gly Aan Asn Gly Ile Glu Thr Lys His Leu Pro Ser 145 1!50 155 160 Thr Cys Arg Asp Thr Ser Ser Asp Ala Lys <210> 5 <211> 30 <212> DNA
<213> Neisseria gonorrhoeae <400> 5 ccccgcgcca tggataccct tcaaaaaggc 30 <210> 6 <211> 30 <212> DNA
<213> Neisseria gonorrhoeae <900> 6 gggcctggat ccgtgggaaa tcacttaccg 30 <210> 7 <211> 29 <212> DNA
<213> Neisseria gonorrhoeae <400> 7 SUBSTITD'TE SHEET (RULE 26) WO 99/55875 PC'T/US99/09486 ggctctagac tgtcagacca agtttactc 2g <210> 8 <211> 27 <212> DNA
<213> Neisseria gonorrhoeae <400> 8 ggctctagat tgaagcattt atcaggg 27 <210> 9 <211> 28 <212> DNA
<213> Neisseria gonorrhoeae <900> 9 ggctctagat aaacagtaat acaagggg 2g <210> 10 <211> 28 <212> DNA
<213> Neisseria gonorrhoeae <900> 10 ggctctagat tagaaaaact catcgagc 28 <210> 11 <211> 36 <212> DNA
<213> Neisseria Mening~itidis Class I
<900> 11 ccccgcgcca tggacaccct tcaaaaaggt tttacc 36 <210> 12 <211> 39 <212> DNA
<213> Neisseria Meningitidis Class I
<400> 12 gggcctggat ccgagtggcc gtggaaaatc acttaccgc 39 SUBSTITUTE SHEET (RULE 26) <210> 13 <211> 31 <212> DNA
<213> Neisseria Mening~itidis Class I
<400> 13 ccggcgcgtc tctcacggcg aatggcccgg c <210> 14 <211> 39 <212> DNA
<213> Neisseria Meningitidis Class I
<400> 19 gggcctggat ccgagtggcc gtggaaaatc acttaccgc 3g <210> 15 <211> 25 <212> DNA
<213> Neisseria gonorrhoeae <900> 15 gcataattcg tgtcgctcaa ggcgc 25 <210> 16 <211> 34 <212> DNA
<213> Neisseria gonorrlzoeae <400> 16 gccgcgcgtc tcccgtgatt caggtaatac tcgg 34 <210> 17 <211> 25 <212> DNA
<213> Neisseria Mening:itidis Class I
<400> 17 gcataattcg tgtcgctcaa ggcgc 25 <210> 18 <211> 39 <212> DNA

SUBSTITUTE SHEET (RULE 26) <213> Neisseria Meningitidis Class I
<400> 18 gggcctggat ccgagtggcc: gtggaaaatc acttaccgc 3g <210> 19 <211> 37 <212> DNA
<213> Neisseria Meningitidis Class II
<400> 19 gcggccgcca tggaagcaat ccaaaaaggt ttcaccc 37 <210> 20 <211> 33 <212> DNA
<213> Neisseria Meningitidis Class II
<400> 20 gccgcgcgtc tccgaaccgg agttttgttt gcc 33 <210> 21 <211> 33 <212> DNA
<213> Neisseria gonorrhoeae <400> 21 ccgggccgtc tcggttcggt aaaatggttc tgc 33 <210> 22 <211> 30 <212> DNA
<213> Neisseria gonorrhoeae <400> 22 gggcctggat ccgtgggaaa tcacttaccg 30 <210> 23 <211> 37 <212> DNA
<213> Neisseria Meningitidis Class II
<400> 23 SUBSTITUTE SHEET (RULE 26) WO 99/55875 PC'TNS99/09486 gcggccggat ccggtcattg tccttatttg gtgcggc <210> 29 <2I1> 22 <212> PRT
<213> Neisseria gonorrhoeae <400> 24 Glu Ala Ile Leu Leu Ala Glu Gly Gln Lys Ser Ala Val Thr Glu Tyr Tyr Leu Asn His Gly Lys SUBSTITUTE SHEET (RULE 26)

Claims

What is claimed is:

1. A vaccine composition comprising an isolated and purified recombinantly-expressed pilin protein of the genus Neisseria, wherein said vaccine composition eliciteo a protective immune response in a human host.

2. The vaccine composition of Claim 1 where the pilin protein is from the species Neisseria gonorrhoeae.

3. The vaccine composition of Claim 1 where the piiin protein is from the species Neisseria meningitidis.

4. The vaccine composition of Claim 3 where the pilin protein is the class I pilin protein of Neisseria meningitidis.

5. The vaccine composition of Claim 3 where the pilin protein is the class II pilin protein of Neisseria meningitidis.

6. The vaccine composition of Claim 1 where the pilin protein is a chimeric recombinant pilin protein of Neisseria gonorrhoeae and class I Neisseria meningitidis having the amino acid sequence of amino acids 1-167 of SEQ ID NO:2 prior to processing or having the amino acid sequence of amino acids 8-167 of SEQ ID N0:2 after processing to a mature protein, or a biologically equivalent amino acid sequence thereof.

7. The vaccine composition of Claim 1 which further comprises an adjuvant, diluent or carrier.

8. The vaccine composition of Claim 7 wherein the adjuvant is selected from the group consisting of aluminum hydroxide, aluminum phosphate, Stimulon TM QS-21, 3-O-deacylated monophosphoryl lipid A, IL-12 and wild-type, or mutant cholera toxin.

9. A method of immunizing against Neisseria gonorrhoeae which comprises administering to a human host an immunogenic amount of the vaccine composition of Claim 2.

10. A method of immunizing against Neisseria gonorrhoeae which comprises administering to a human host an immunogenic: amount of the vaccine composition of Claim 3.

11. A method of immunizing against Neisseria gonorrhoeae which comprises administering to a human host an immunogenic amount of the vaccine composition of Claim 6.

12. A method of immunizing against Neisseria meningitidis which comprises administering to a human host an immunogenic: amount of the vaccine composition of Claim 3.

13. A method of immunizing against Neisseria meningitidis which comprises administering to a human host an immnunogenic amount of the vaccine composition of Claim 2.

14. A method of immunizing against Neisseria meningitidis which comprises administering to a human host an immunogenic amount of the vaccine composition of Claim 6.

15. A method for preparing a vaccine composition which comprises including an isolated and purified recombinantly-expressed pilin protein of the genus Neisseria, in an amount sufficient such that said vaccine composition elicits a protective immune response in a human host.

16. An isolated and purified DNA sequence comprising a DNA sequence which hybridizes under standard high stringency Southern hybridization conditions with a DNA sequence encoding the chimeric recombinant pilin protein of Neisseria gonorrhoeae and class I Neisseria meningitidis having the amino acid sequence of amino acids 1-167 of SEQ ID NO:2 prior to processing or having the amino acid sequence of amino acids 8-167 of SEQ ID NO:2 after processing to a mature protein, or a biologically equivalent amino acid sequence thereof.

17. The isolated and purified DNA sequence of Claim 16, wherein said DNA sequence hybridizes under standard high stringency Southern hybridization conditions with a DNA sequence having the nucleotide sequence of nucleotides 1-501 or 22-501 of SEQ ID NO:1.

18. An isolated and purified DNA sequence.
comprising a DNA sequence encoding the chimeric recombinant pilin protein of Neisseria gonorrhoeae and class I Neisseria meningitidis having the amino acid sequence of amino acids 1-167 of SEQ ID NO:2 prior to processing or having the amino acid sequence of amino acids 8-167 of SEQ ID NO:2 after processing to a mature protein, or a biologically equivalent amino acid sequence thereof.

19. A plasmid containing an isolated and purified DNA sequence comprising a DNA sequence which hybridizes under standard high stringency Southern hybridization conditions with a DNA sequence encoding the chimeric recombinant pilin protein of Neisseria gonorrhoeae and class I Neisseria meningitidis having the amino acid sequence of amino acids 1-167 of SEQ ID
NO:2 prior to processing or having the amino acid sequence of amino acids 8-167 of SEQ ID NO:2 after processing to a manure protein, or a biologically equivalent amino acid sequence thereof.

20. The plasmid of Claim 19 wherein the plasmid contains a DNA sequence which hybridizes under standard high stringency Southern hybridization conditions with a DNA sequence having the nucleotide sequence of nucleotides 1-501 or 22-501 of SEQ ID NO: 1.

21. A plasmid containing an isolated and purified DNA sequence encoding a chimeric recombinant pilin protein of Neisseria gonorrhoeae and class I
Neisseria meningitidis comprising the DNA sequence of Claim 18.

22. The plasmid of Claim 21 wherein the plasmid is that designated pPX2004 (ATCC 98637).

23. A host cell transformed with the plasmid of Claim 19.

24. The host cell of Claim 23 wherein the host cell is an Escherichia coli strain.

25. The host cell of Claim 24 wherein the plasmid is that designated pPX2004 (ATCC 98637).

26. A method of producing a chimeric recombinant pilin protein of Neisseria gonorrhoeae and class I Neisseria meningitidis which comprises transforming or transfecting a host cell with the plasmid of Claim 19 and culturing the host cell under conditions which permit the expression of said chimeric recombinant pilin protein by the host cell.

27. An isolated and purified chimeric recombinant pilin protein of Neisseria gonorrhoeae and class I Neisseria meningitidis having the amino acid sequence of amino skids 1-167 of SEQ ID NO:2 prior to processing or having the amino acid sequence of amino acids 8-167 of SEQ ID NO:2 after processing to a mature protein, or a biologically equivalent amino acid sequence thereof.

28. The vaccine composition of Claim 1 where the pilin protein is a chimeric recombinant pilin protein of Neisseria gonorrhoeae and class II Neisseria meningitidis having the amino acid sequence of amino acids 1-170 of SEQ ID NO:4 prior to processing or having the amino acid sequence of amino acids 8-170 of SEQ ID NO:4 after processing to a mature protein, or a biologically equivalent amino acid sequence thereof.

29. A method of immunizing against Neisseria gonorrhoeae which comprises administering to a human host an immunogenic amount of the vaccine composition of Claim 28.

30. A method of immunizing against Neisseria meningitidis which comprises administering to a human host an immunogenic amount of the vaccine composition of Claim 28.

31. An isolated and purified DNA sequence comprising a DNA sequence which hybridizes under standard high stringency Southern hybridization conditions with a DNA sequence encoding the chimeric recombinant pilin protein of Neisseria gonorrhoeae and class II Neisseria mengitidis having the amino acid sequence of amino acids 1-170 of SEQ ID NO:4 prior to processing or having the amino acid sequence of amino acids 8-170 of SEQ ID NO:4 after processing to a mature protein, or a biologically equivalent amino acid sequence thereof.

32. The isolated and purified DNA sequence of Claim 31, wherein said DNA sequence hybridizes under standard high stringency Southern hybridization conditions with a DNA sequence having the nucleotide sequence of nucleotides 1-510 or 22-510 of SEQ ID NO:3.

33. An isolated and purified DNA sequence comprising a DNA sequence encoding the chimeric recombinant pilin protein of Neisseria gonorrhoeae and class II Neisseria meningitidis having the amino acid sequence of amino acids 1-170 of SEQ ID NO:4 prior to processing or having the amino acid sequence of amino acids 8-170 of SEQ ID NO:4 after processing to a mature protein, or a biologically equivalent amino acid sequence thereof.

34. A plasmid containing an isolated and purified DNA sequence comprising a DNA sequence which hybridizes under standard high stringency Southern hybridization conditions with a DNA sequence encoding the chimeric recombinant pilin protein of Neisseria gonorrhoeae and class II Neisseria meningitidis having the amino acid sequence of amino acids 1-170 of SEQ ID
NO:4 prior to processing or having the amino acid sequence of amino acids 8-170 of SEQ ID NO:4 after processing to a mature protein, or a biologically equivalent amino acid sequence thereof.

35. The plasmid of Claim 34 wherein the plasmid contains a DNA sequence which hybridizes under standard high stringency Southern hybridization conditions with a DNA sequence having the nucleotide sequence of nucleotides 1-510 or 22-510 of SEQ ID NO:3.

36. A plasmid containing an isolated and purified DNA sequence encoding a chimeric recombinant pilin protein of Neisseria gonorrhoeae and class II
Neisseria meningitides comprising the DNA sequence of Claim 33.

37. The plasmid of Claim 36 wherein the plasmid is that designated pPX8017 (ATCC 207199).

38. A host cell transformed with the plasmid of Claim 34.

39. The host cell of Claim 38 wherein the host cell is an Escherichia coli strain.

40. The host cell of Claim 39 wherein the plasmid is that designated pPX8017 (ATCC 207199).

41. A method of producing a chimeric recombinant pilin protein of Neisseria gonorrhoeae and class II Neisseria meningitides which comprises transforming or transfecting a host cell with the plasmid of Claim 34 and culturing the host cell under conditions which permit the expression of said chimeric recombinant pilin protein by the host cell.

42. An isolated and purified chimeric recombinant pilin protein of Neisseria gonorrhoeae and class II Neisseria meningitidis having the amino acid sequence of amino acids 1-170 of SEQ ID NO:4 prior to processing or having the amino acid sequence of amino acids 8-170 of SEQ ID NO:4 after processing to a mature protein, or a biologically equivalent amino acid sequence thereof.