EP1073748A2 - Vaccines containing recombinant pilin against neisseria gonorrhoeae or neisseria meningitidis - Google Patents

Vaccines containing recombinant pilin against neisseria gonorrhoeae or neisseria meningitidis

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Publication number
EP1073748A2
EP1073748A2 EP99922760A EP99922760A EP1073748A2 EP 1073748 A2 EP1073748 A2 EP 1073748A2 EP 99922760 A EP99922760 A EP 99922760A EP 99922760 A EP99922760 A EP 99922760A EP 1073748 A2 EP1073748 A2 EP 1073748A2
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European Patent Office
Prior art keywords
protein
class
amino acid
rpilin
neisseria
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EP99922760A
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German (de)
English (en)
French (fr)
Inventor
Thomas N. Metcalf, Iii
Robert J. Zagursky
Peggy Ooi
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Wyeth Holdings LLC
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American Cyanamid Co
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Publication of EP1073748A2 publication Critical patent/EP1073748A2/en
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/22—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Neisseriaceae (F)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence

Definitions

  • This invention relates to the use of recombinant pilin proteins in vaccines to protect against disease caused by Neisseria gonorrhoeae or Neisseria meningi tidis .
  • Neisseria gonorrhoeae (N. gonorrhoeae) and Neisseria meningitidis (N. meningitidis) are Gra - negative cocci.
  • N. gonorrhoeae and N. meningi tidis are genetically very closely related, but the clinical manifestations of the diseases they produce are very different.
  • N. gonorrhoeae causes gonorrhea
  • N. meningi tidis causes meningococcal meningitis.
  • Type IV pili are nonflagellar hairlike structures on the surface of numerous Gram-negative bacteria, including Dichelobacter (formerly Bacteroides) nodous , Eikenella corrodens , Kingella deni trificans , Moraxella bovis , M. lacunata , M. nonliguefaciens , N. gonorrhoeae , N. meningitidis , and Pseudoz ⁇ ojnas aeruginosa (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 pili and are considered to be more distantly related (1,2) .
  • 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 ⁇ M in length.
  • the pilin protein subunits are linked in tandem to form long, thin polymers.
  • the pili are apparently homopolymeric in nature, being comprised of a single structural subunit, the pilin protein.
  • the pilin protein has a molecular weight of 13,000 to 22,000 daltons (1,2,3) .
  • the pilin protein is assembled into a helical structure called a pilus (plural: pili) at the bacterial outer membrane that has a molecular weight of approximately 10 7 daltons.
  • a pilus plural: pili
  • the intact pili are irreversibly dissociated into aggregates of the pilin protein which are called pilin oligomers (4) .
  • pilin oligomer aggregates molecular weight of approximately 600,000 daltons
  • N. gonorrhoeae expresses a single pilin protein which was first isolated and sequenced by Schoolnik and co-workers (5) .
  • the gonococcal pilin 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 sequence variation and (c) the carboxy third of the protein (residues 125-160) which contains a highly variable disulfide loop.
  • pili undergo high frequency phase and antigenic variation (3,6).
  • the genetics of this variation are extraordinarily complex and have been extensively studied.
  • Each strain of Neisseria gonorrhoeae has the ability to change the 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.
  • N. meningi tidis expresses two distinct classes of pilin named class I and class II.
  • 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 (SMI) which binds to a highly conserved epitope on the gonococcal pilin (3) .
  • SMI monoclonal antibody
  • 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) .
  • class II pilins do not react with the SMI antibody and have a lower molecular weight (13-16 kd) .
  • 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 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 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.
  • 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 traveler's 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) in cattle (1) , footrot in sheep (1,10) and diarrhea in piglets (9) or calves (9) .
  • the pili vaccine provided protection against challenge by strains expressing the homologous, but not heterologous, pili.
  • the gonococcal pilus vaccine was tested in a large, placebo-controlled double blind efficacy trial (12) .
  • the vaccine failed to protect male volunteers from gonococcal infections. It was postulated that the most likely explanation for this was pilus heterogeneity (12) .
  • the literature contains a number of references in which antisera directed against purified pili, or pilin fragments, bound to denatured (Western blot) or isolated neisserial pili, but did not bind to heterologous pili on bacterial surfaces (16,17,19,20). Whether this is due to antigenic variation or concealment of the epitopes in the assembled pilus has not been completely resolved. This is reinforced by several reports which demonstrated that the only monoclonal antibodies exhibiting functional activity in vi tro were those which did not bind to heterologous pili (3) . It has been shown for the intact pili from M. bovis (21) and D.
  • the recombinant 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) .
  • the agglutination titer induced by the recombinant E. coli cell vaccine was significantly lower than that seen for the intact pili (690 vs. 47,800) and below the titer which correlated with protection (5,000-10,000) (25,26) .
  • K-agglutinating a more intense and appropriate immunological reaction than the equivalent dose of fimbrial subunit protein.
  • a serological K- agglutination titre of about 5,000 is generally regarded as the minimum response commensurate with adequate protective immunity against infection 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) .
  • Meningococcal vaccines are limited to those possessing serotype A, C, Y, W135 capsules.
  • N. gonorrhoeae and N. meningi tidis are suitable antigenic structures derived from N. gonorrhoeae and N. meningi tidis , respectively, which may constitute viable vaccine candidates against those bacteria. These candidates must induce antibodies which recognize and bind to diverse isolates of the respective pathogenic neisserial organism.
  • 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- 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.
  • the chimeric DNA sequence is first inserted into a suitable plasmid vector.
  • a suitable host cell is then transformed or transfected with the plasmid.
  • 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 plasmid 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 corresponding carboxy-terminal region of the gonococcal pilin 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.
  • the host cell is an Escherichia coli strain.
  • the host cell is then cultured under conditions which permit the expression of said chimeric class II rpilin protein by the host cell .
  • 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, MPLTM, 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. meningi tidis .
  • Figure 1 depicts transmission electron micrographs of piliated cells from N. gonorrhoeae (strain 1756 recA-) incubated with guinea pig antisera 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.
  • Figure 1A depicts anti-rpilin guinea pig immune sera (week 6)
  • Figure IB depicts normal guinea pig sera (week 0)
  • Figure 2C depicts no primary antibody.
  • Figure 2 depicts the effect of guinea pig antisera directed against gonococcal rpilin (from strain Pgh.3-1) on the attachment of piliated N. gonorrhoeae cells (strain 1756 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 same experimental condition. The guinea pig antisera was diluted 1:10,000 for each panel.
  • Figure 3 depicts transmission electron micrographs of piliated cells from N. meningi tidis (strain H355) incubated with guinea pig antisera directed against meningococcal chimeric class I rpilin (from strain H44/76) (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.
  • This invention relates to vaccine compositions comprising a recombinant pilin protein of N. gonorrhoeae or N. meningi tidis .
  • a recombinant pilin protein of N. gonorrhoeae or N. meningi tidis was decided to investigate the use of such recombinant pilin proteins expressed in E. coli .
  • these recombinant pilin proteins demonstrated characteristics of vaccine candidates .
  • the first report describing the cloning of the gonococcal pilE gene in E. coli was in 1982 (30) .
  • Example 2 Cloning and expression of the piIE gene encoding the gonococcal recombinant pilin protein are described in Example 2 below. Expression was achieved by transforming an E. coli strain designated TOP10F' with a plasmid containing the pilE gene. Successful cloning and expression 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 (Amp R ) marker.
  • Amp R ampicillin resistance
  • another plasmid was constructed to contain a kanamycin resistance (Kan E ) marker instead of Amp R .
  • This plasmid, designated pPX2002 after transforming E. coli strain TOP10F' , expressed the gonococcal rpilin at a level similar to that obtained from pPX2000, which contains an Amp R marker.
  • a similar procedure was used to construct a plasmid, designated pPX2003, containing the class I pilE gene of N. meningi tidis .
  • a Ncol 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 Amp R marker was also included. This construct, after transforming E. coli strain TOPIOF' , expressed class I rpilin of N. meningi tidis .
  • the expression level was significantly lower than that for the 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.
  • 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 from the gonococcal DNA in pPX2002.
  • the resulting Amp R plasmid designated pPX2004, has the nucleotide sequence set forth in SEQ ID NO:l.
  • the plasmid pPX2004 was used to transform an E. coli strain K12 designated TOPIOF' .
  • TOPIOF' E. coli strain K12
  • the level of expression of the chimieric construct was comparable to the amount of gonococcal rpilin expressed from pPX2002.
  • the chimeric class I rpilin was 167 amino acids in length (including the signal) (SEQ ID NO:2) , which is in accordance with the predicted size.
  • a chimeric plasmid was constructed wherein the 3' end of the class II pilE gene of N. meningi tidis was replaced with the corresponding region from N. gonorrhoeae .
  • the DNA in pPX8001 encoding the disulfide loop (the last 22 amino acids of the meningococcal class II pilin of N. meningi tidis strain FAM18) is replaced with a similiar (but larger) region plus additional portions of the carboxy-terminal region totalling 44 amino acids from the gonococcal (pilE) DNA from N. gonorrhoeae strain Pgh3-1 in pPX2000.
  • the resulting Amp R plasmid designated pPX8017, has the nucleotide sequence set forth in SEQ ID NO: 3, in which nucleotides 1-378 are from N. meningi tidis class II and nucleotides 379-510 are from N. gonorrhoeae .
  • the plasmid pPX8017 was used to transform the E. coli strain K12 designated TOPIOF' . Following induction, a chimeric class II rpilin was expressed which was 170 amino acids in length (including the seven amino acid long signal) (SEQ ID NO:4), in which amino acids 1-126 are from N. meningi tidis class II and amino acids 127- 170 are from iV.
  • gonorrhoeae This chimeric class II rpilin was in accordance with the predicted size.
  • a iVcol site was introduced for cloning considerations, which changed the second amino acid in the signal sequence from lysine to glutamic acid. This change was not expected to have any effect on antigenicity or immunogenicity.
  • 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 Sf21 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 and purified DNA sequence comprising a DNA sequence encoding the meningococcal chimeric class I rpilin protein whose amino-terminal region is from the gonococcal pilE 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 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 additionally relates to the meningococcal chimeric class I rpilin protein 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.
  • Approximately 10% of the total protein produced by the gonococcal rpilin or the meningococcal chimeric class I rpilin constructs lacks 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 pilE gene and whose central and amino- terminal regions are from the meningococcal piIE gene (SEQ ID NO:3) .
  • Nucleotides 1-510 in SEQ ID NO:3 encode the meningococcal chimeric class II rpilin protein prior to processing; nucleotides 22-510 encode the meningococcal chimeric class II rpilin protein after processing to a mature protein.
  • the invention additionally relates to the meningococcal chimeric class II rpilin protein 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.
  • the present invention further comprises DNA sequences which, by virtue of the redundancy of the 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 N0:1 or SEQ ID NO: 3.
  • the invention contemplates those DNA sequences which are sufficiently duplicative of the sequence of SEQ ID NO:l or SEQ ID NO: 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 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 NO: 2 or SEQ ID NO: 4) .
  • amino acid sequences may be 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 essentially unchanged from those of the rpilin protein.
  • 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 valine, leucine, or isoleucine.
  • a codon encoding another less hydrophobic residue such as glycine
  • a more hydrophobic residue such as valine, leucine, or isoleucine.
  • 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 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 molecule would also not be expected to alter the activity of the protein.
  • changes in known variable regions are biologically equivalent where the tertiary configurations of the 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.
  • 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.
  • 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, TritonTM X-100, reduced TritonTM X-100, octyl- ⁇ -D- glucopyranoside (OG) , ZwittergentTM 3-10 or 3-14. Following centrifugation, dialysis and fractionation on a column, the purified rpilin is obtained.
  • the chimeric class I rpilin was isolated and purified by disruption of E. coli cells, clarification by centrifugation, filtration, and fractionation on two columns.
  • meningococcal chimeric class II rpilin was isolated and purified by disruption of E. coli 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 the amino acid content.
  • an apparent molecular weight of 68,899 daltons was obtained when the rpilin was subjected to size exclusion column chromatography using 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.
  • Example 11 immune sera are obtained by immunizing guinea pigs or mice with the purified gonococcal rpilin.
  • 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 lysates.
  • antisera to the pilin oligomer bound to both piliated and nonpiliated cell lysates are examples of the pilin oligomer bound to both piliated and nonpiliated cell lysates.
  • 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 MPLTM alone, MPLTM plus aluminum phosphate, or StimulonTM QS-21, good humoral immune responses in mice were obtained.
  • mice were immunized intranasally with gonococcal rpilin with or without 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 native cholera toxin.
  • Example 17 demonstrates the higher titers obtained for rpilin antisera binding to heterologous piliated bacterial isolates as compared to that obtained for antisera to recombinant pilin oligomer.
  • the rpilin is converted to rpilin oligomer by dialysis of the rpilin against pH 12 phosphate buffer.
  • guinea pig antisera to rpilin significantly inhibited the binding of gonococci expressing heterologous pili to human cervical epithelial cells. Piliation of gonococci correlates with the infectivity of this bacterium (2,3,32) .
  • 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 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. 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 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. gonorrhoeae .
  • the meningococcal 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.
  • 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.
  • Example 20 when analyzed by
  • Example 4 it was shown that antisera directed against gonococcal rpilin recognized and bound to piliated meningococcal cells.
  • Example 22 it was shown that antisera raised against meningococcal chimeric class I rpilin protein bound to piliated gonococcal cells.
  • 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) .
  • CT-CRM histidine
  • Example 25 the inhibition of colonization of mouse nasopharynx by a class I strain of N. meningitidis was demonstrated in mice immunized subcutaneously with meningococcal chimeric class I rpilin adjuvanted with MPLTM.
  • rpilin in particular the meningococcal chimeric class I and class II rpilin proteins, are viable vaccine candidates against N. meningi tidis .
  • the gonococcal rpilin protein is useful in the preparation of vaccines to confer protection to mammals against disease caused by N. gonorrhoeae .
  • the 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. meningi tidis .
  • 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.
  • meningi tidis or by immunizing with a vaccine containing the meningococcal rpilin protein, the meningococcal chimeric class I rpilin protein or the meningococcal chimeric class II rpilin protein to confer protection to mammals against disease caused by N. gonorrhoeae .
  • 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 mixed with immunologically 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 StimulonTM QS-21 (Aquila Biopharmaceuticals, Inc., Framingham, MA) , MPLTM (3-0- 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, for example wherein the glutamic acid at amino acid position 29 is replaced by another amino acid, preferably a histidine, in accordance with U.S.
  • the vaccines of this invention are 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. gonorrhoeae or N. meningi tidis .
  • 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.
  • the following 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.
  • meningitidis were grown on 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. meningi tidis were grown in the same media which lacked agar in a shaking incubator (70 RPM) at 37 °C.
  • E. coli were grown on SOB agar which consists of 20 g/L Bacto tryptone (Difco) , 5 g/L yeast extract (Difco) , 0.6 g/L NaCl, 0.2 g/L KC1 and 1% (w/v) agar (pH 7.5) or in SOB broth to which the agar is not added.
  • Bacto tryptone was replaced with an equivalent amount of HySoyTM (Sheffield Products, Norwich, NY) .
  • ME-180 cell line (ATCC, Beltsville, MD) 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) C0 2 at 37°C. The cells were split every three to four days.
  • a frozen sample of piliated N. gonorrhoeae strain Pgh3-1 was used as the source of the pilE DNA in a PCR reaction.
  • the 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 CGC GCC ATG GAT ACC CTT CAA AAA GGC 3' (PILEFWD) (SEQ ID NO: 5) and 5' GGG CCT GGA TCC GTG GGA AAT CAC TTA CCG 3' (PILEREV) (SEQ ID NO: 6).
  • the resulting PCR product contained a Ncol site at the beginning of the pilE coding region and a BamHI site at the end.
  • the Ncol 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 amino acid 2 is part of the signal peptide which is 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 pCRTMII TA cloning vector (Invitrogen, Carlsbad, CA) , ligated, and transformed into E. coli TOPIOF' (Invitrogen) .
  • Colonies were selected on 100 ⁇ g/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 BcoRI and Notl .
  • 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.
  • a plasmid was constructed where the Amp R marker was replaced with a Kan R marker. Except as noted below, the procedures of Example 2 were used.
  • a 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 NO: 8) .
  • the underlined sequences code for an Xbal restriction site.
  • the approximate 3.5 kb PCR product contained the pTrcHisA DNA minus the ampicillin coding region.
  • Kan R colonies were streaked in duplicate onto SOB plates containing either ampicillin or kanamycin. As anticipated, all Kan R colonies were ampicillin sensitive. Since the cloning design was 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 the multiple cloning site in pZ564 was then replaced with the similar region from the pPX2000 plasmid (which also contained pilE) in the following manner: Both pZ564 and pPX2000 were digested with Sp l and .Xmnl restriction enzymes.
  • the approximate 2.2 kb DNA fragment from pPX2000 and the approximate 2.6 kb DNA fragment from pZ564 were gel purified, ligated together and transformed into E. coli TOPIOF' .
  • the resulting correct plasmid was called pPX2002.
  • the class I pilE was amplified from the genomic DNA of N. meningi tidis strain H44/76 using the following primers: 5' CCC CGC GCC ATG GAC ACC CTT CAA AAA GGT TTT ACC 3' (NMFPILE) (SEQ ID NO:ll), and 5' GGG CCT GGA TCC GAG TGG CCG TGG AAA ATC ACT TAC CGC 3' (NMRPILE) (SEQ ID NO: 12). As anticipated, a PCR product of approximately 600 bp DNA was obtained. An aliquot of the PCR reaction product was digested with BamHI and Ncol 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. coli TOPIOF' . Miniplasmid prep analysis of ampicillin resistant clones was performed using BamHI and Ncol restriction enzymes. Clones expressing the correct restriction digest pattern were called RZ1142 and the plasmid was called pPX2003.
  • the DNA encoding the first 60 amino acids in pPX2003 (the meningococcal class I pilE construct described in Example 4) was replaced with the equivalent region from pPX2002 (the gonococcal pilE construct described in Example 3, including the seven amino acid signal peptide) (SEQ ID NO: 4) . Except as noted below, the procedures of Example 2 were followed.
  • the conserved 5' terminal region of the meningococcal pilE 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' CCG GCG CGT CTC TCA CGG CGA ATG GCC CGG C 3' (CL-1ESPF) (SEQ ID NO: 13) and 5' GGG CCT GGA TCC GAG TGG CCG TGG AAA ATC ACT TAC CGC 3' (NMRPILE) (SEQ ID NO: 14) and Taq DNA polymerase.
  • the expected PCR DNA product was cloned directly into pCR2.1 (Invitrogen) and transformed into TOPIOF' cells and the resulting plasmid was designated pZ578.
  • a BsmBI site was then introduced into the gonococcal pilE by the following method. Using the primers 5' GCA TAA TTC GTG TCG CTC AAG GCG C 3' (TRCUPFW) (SEQ ID NO: 15) and 5' GCC GCG CGT CTC CCG TGA TTC AGG TAA TAC TCG G 3' (PILEESPR) (SEQ ID NO: 16) and Pfu DNA polymerase, the 5' end of the pilE 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' (TRCUPFW) (SEQ ID NO: 17) and 5' GGG CCT GGA TCC GAG TGG CCG TGG AAA ATC ACT TAC CGC 3' (NMRPILE) (SEQ ID NO: 18) primers.
  • the DNA PCR product was of the predicted size
  • 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 Meningococcal Class II Chimeric pilE in E. coli
  • the initial cloning of the meningococcal class II pilE involved isolation of chromosomal DNA from piliated N. meningi tidis strain FAM18 cells and amplifying the classll 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 NO: 19) and 5' GCG GCC GGA TCC GGT CAT TGT CCT TAT TTG GTG CGG C 3' (PILE2REV) (SEQ ID NO: 23) .
  • the resulting PCR product contained an Ncol site at the beginning of the pilE coding region and a BamHI site at the end. The Ncol site was introduced into the gene because of cloning considerations.
  • the PCR product was cloned into a pCR2.1 cloning vector (Invitrogen) , ligated, and transformed into E. coli TOPIOF' . Colonies were selected on 100 ⁇ g/mL ampicillin-containing plates or 50 ⁇ g/ml kanamycin plates .
  • the plasmid D ⁇ A was isolated from overnight cultures of these transformants and analyzed by restriction digests using the enzyme JSJcoRI .
  • Plasmid D ⁇ As from pPX8001 and pTrcHisC were each digested with Ncol and BamHI restriction enzymes, and the resulting D ⁇ A fragments were gel isolated, ligated and transformed into E. coli TOPIOF' .
  • the plasmid DNA of the new transformants were isolated, and a DNA restriction analysis performed using BamHI and Ncol restriction enzymes. Two clones with the correct restriction pattern were submitted for D ⁇ A sequence analysis. Both clones had the correct D ⁇ A sequence, designated as pPX8002.
  • the strains ⁇ MB 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. meningi tidis 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.
  • PCR products were cloned into pTrcHisC and sequenced, 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.
  • 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 following regions (listed by nucleotide numbers) of the FAM18 pilE 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), 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).
  • 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 among meningococcal strains.
  • 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 approximately 19,000 daltons.
  • 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 GAA GCA ATC CAA AAA GGT TTC ACC C 3' (PILE2FWD) (SEQ ID NO: 19) and 5' GCC GCG CGT CTC CGA ACC GGA GTT TTG TTT GCC 3' (REV-CYS) (SEQ ID NO:20).
  • the gonococcal disulfide loop (i.e., the 3' end of the gonococcal gene) was amplified from pPX2000 using primers 5' CCG GGC CGT CTC GGT TCG GTA AAA TGG TTC TGC 3' (FWD-CYS) (SEQ ID NO: 21) and 5' GGG CCT GGA TCC GTG GGA AAT CAC TTA CCG 3' (PILEREV) (SEQ ID NO: 22) .
  • the resulting PCR products were each purified, digested separately with restriction enzyme BsmBI, then ligated to form the full length chimeric pilE, which was amplified using primers PILE2FWD and PILEREV.
  • This PCR product was digested with restriction enzymes Ncol and BamHI, ligated into a similarly restricted pTrcHisC vector and transformed into TOPIOF' competent cells. Transformants were cultured and analyzed using the restriction enzymes Ncol 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 D ⁇ A sequence and was designated as pPX8017. This clone contains the nucleotide sequence set forth in SEQ ID NO: 3, in which nucleotides 1-378 are from N. meningi tidis and nucleotides 379-510 are from N. gonorrhoeae .
  • IPTG 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 were 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 lie Leu Leu Ala Glu Gly Gin Lys Ser Ala Val Thr Glu Tyr Tyr Leu Asn His Gly Lys) (SEQ ID NO:24), which is located in the conserved region of the amino terminus of the class II pilin protein.
  • the meningococcal chimeric class II rpilin was 170 amino acids in length (including the signal) (SEQ ID NO:4), in which amino acids 1-126 are from N. meningi tidis and amino acids 127-170 are from N. gonorrhoeae .
  • the following 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) TritonTM X-100 to solubilize this protein.
  • the cell pellet (approx. 5 g wet weight) from 1 L of culture was thawed by adding 30 mL of 10 mM 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 (288,652 x g for one hour).
  • the membranes were resuspended in 33 mL of 10 mM Hepes (pH 7.4), 1 mM MgCl 2 and extracted with one of the following detergents: (a) TritonTM X-100 (TX100) (Calbiochem- Novabiochem International, San Diego, CA) , (b) reduced TritonTM X-100 (Calbioche ) , (c) octyl- ⁇ -D- glucopyranoside (OG) (Calbiochem), (d) ZwittergentTM 3- 8 (Z3-8) (Calbiochem), (e) ZwittergentTM 3-10 (Z3-10) (Calbiochem), (f) ZwittergentTM 3-12 (Z3-12) (Calbiochem), (g) ZwittergentTM 3-14 (Z3-14) (Calbiochem) , (h) Empigen BBTM (Calbiochem) or (i)
  • TweenTM 80 (ICN, Cleveland, OH) for one hour at room temperature.
  • 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 TrisTM (pH 8.5) containing one of the following non-ionic detergents: (a) 0.1% (w/v) ZwittergentTM 3-14, (b) 1% (w/v) ZwittergentTM 3-10 or (c) 1% (w/v) OG.
  • the dialyzed material was fractionated on a FractogelTM EMD TMAE-650(S) (EM Separations Technology, Wakefield, RI) column equilibrated in 10 mM TrisTM (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 the 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 when the extraction and column chromatography were done with 1% (w/v) ZwittergentTM 3-10, 1% (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 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.
  • E. coli cells containing pPX2004 were grown in a Biostat 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 HOY (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 MgCl 2 , 1% (w/v) octyl- ⁇ -D-glucopyranoside (pH 7.5) 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 at 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 FractogelTM column (EM Separations Technology, Gibbstown, NJ) equilibrated with 25 mM TrisTM, 0.1% (w/v) ZwittergentTM 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 flow rate of 10.0 mL/minute.
  • Fractions containing the chimeric class I rpilin were pooled and diluted 1:1 with dH 2 0 and loaded onto a 100 mL 40 ⁇ m ceramic hydroxyapatite column (Bio-Rad, Hercules, CA) equilibrated with 10 mM NaP0 4 , 0.1% (w/v) ZwittergentTM 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 NaP0 4 gradient (10-150 mM NaP04) containing 0.1% (w/v) ZwittergentTM 3-14 over 10 column volumes at a flow rate of 5.0 mL/minute. Fractions were screened by SDS-PAGE analysis and 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 chimeric class I rpilin was approximately 35 mg/g wet weight cells.
  • 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 MgCl 2 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 30 minutes) and discarded.
  • ZwittergentTM 3-14 was added to the supernantant, which contained the meningococcal chimeric class II rpilin, to a final concentration of 1% (w/v) and the material was dialyzed overnight against 50 mM TrisTM (pH 8.0), 10 mM EDTA, 1% ZwittergentTM 3-14.
  • Protein content was determined by the bicinchoninic acid protein assay (Pierce, Rockford, IL) using BSA as the standard.
  • the purity of protein preparations was determined by Coomassie brilliant blue stained polyacrylamide gel electrophoresis in the 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
  • the ⁇ -terminal sequences of the pilin proteins were determined using an Applied Biosystems 477A Protein Sequencer. Two sequences were often detected when the purified rpilin was submitted for ⁇ - 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 LasermatTM 2000 (San Jose, CA) .
  • the instrument was calibrated with horse myoglobin to within 0.01% of its expected mass of 16,951.5 daltons.
  • Recombinant pilin was mixed with an equal volume of a cyano-4- hydroxycinnamic acid matrix (10 mg/mL in 70:30 acetonitrile : 0.1% (v/v) trifluoroacetic acid/water).
  • the difference in molecular weights of the two forms of the recombinant pilin (769 daltons) is ascribed to the loss of the first six amino acids of the leader sequence (Met Asp Thr Leu Gin Lys) (SEQ ID NO: 2, amino acids 1- 6) which has a mass of 774 daltons.
  • mice In order to investigate the ability of adjuvants to modulate the immune response of gonococcal rpilin, mice (female, 8 weeks old, 5 or 10 animals per group) were immunized subcutaneously with 1-10 ⁇ g 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 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 .
  • mice received immunizations on week 0 and 4 only and sera was obtained on weeks 0, 4 and 6.
  • adjuvants were studied: (a) Stimulon TI QS-21 (25 ⁇ g/dose) in PBS (pH 6) ; (b) aluminum phosphate (100 ⁇ g/dose) in PBS (pH 7) ; (c) MPLTM (50 ⁇ g/dose) in PBS (pH 7) ; (d) aluminum phosphate (100 ⁇ g/dose) and MPLTM (50 ⁇ g/dose) in PBS (pH 7); or PBS
  • the guinea pigs received immunizations of 20 ⁇ g protein adjuvanted with StimulonTM QS-21 (25 ⁇ g/dose) in PBS (pH 6) on week 0 and 4 only and sera was obtained on weeks 0 , 4 and 6.
  • 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.
  • 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 TrisTM, 145 mM NaCl (pH 8.0). The pilin oligomers were then clarified by centrifugation (100,000 x g for one hour). Following centrifugation, the pilin oligomers remained in the supernatant.
  • 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 pili.
  • 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 which the optical absorbance was 0.10 greater than that of the blank wells (which do not contain primary antibody) .
  • purified recombinant pilin was diluted in 0.1 M TrisTM (pH 8) to a final concentration of 1 ⁇ g/mL. Aliquots (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) TweenTM-20 (PBS-T) using a Skanwash 300 plate washer (Skatron Instruments, Alexandria, VA) .
  • the wells were blocked using 200 ⁇ L of 1% (w/v) BSA 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 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 50 mM TrisTM (pH 8).
  • the plates were washed and the color developed using 100 ⁇ L per well of p-nitrophenol phosphate (Sigma) (2 mg/mL in 0.5 M diethanolamine, 0.25 mM MgCl 2 , 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 (Molecular Devices, Sunnyvale, CA) at 405 nm.
  • Prep Immunogen Week 0 Week 4 Week 6 r Pgh3-1 pilin ⁇ 100 51,345 494,805 (Prep 1) r Pgh3-1 pilin 54 30,237 594,298 (Prep 2) r Pgh3-1 pilin ⁇ 100 24,830 546,682 (Prep 3)
  • mice (a) StimulonTM QS-21 in PBS (pH 6); (b) aluminum phosphate in PBS (pH 7) ; (c) MPLTM in PBS (pH 7); (d) aluminum phosphate and MPLTM in PBS (pH 7) ; or PBS (pH 7) only.
  • StimulonTM QS-21 in PBS (pH 6)
  • aluminum phosphate in PBS (pH 7) a) aluminum phosphate in PBS (pH 7)
  • MPLTM in PBS (pH 7)
  • aluminum phosphate and MPLTM in PBS (pH 7)
  • PBS PBS (pH 7) only.
  • the antigen ELISA protocol was modified as follows.
  • 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 was washed five times using PBS containing 0.1% (v/v) TweenTM-20 using a Skantron 300 plate washer. The wells were blocked with PBS containing 0.1% (w/v) gelatin and 0.02% (w/v) NaN 3 .
  • 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) NaN 3 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 1:8000 in PBS-TG and 0.02% (w/v) NaN 3 .
  • 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) NaN 3 (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 Labinstruments, Research Triangle Park, NC) .
  • the data were plotted using a log-log plot and the endpoint titers were determined as previously described.
  • mice (5 per group) were immunized with 10 ⁇ g of rpilin adjuvanted as described above on week 0, 4 and 6. The animals were bled on weeks 0, 4, 6 and 8. Analyses were done on pooled sera from week 8. Endpoint titers for week 0 were ⁇ 5,000.
  • P+ piliated cells.
  • P- non-piliated variants derived from piliated parental cells.
  • the bound antisera was detected using 100 ⁇ L of the appropriate alkaline phosphatase conjugates listed above at a 1:2000 dilution in 0.1% (w/v) BSA in PBS. The plates were washed, the color developed and the absorbance read (at 30 minutes development) as described above.
  • mice were immunized intranasally with gonococcal rpilin in 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 immunized intranasally 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.
  • the bound primary antibodies were detected by floating the grid on a drop of 12 nm 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 IOC transmission electron microscope at 15-75, 000X using an acceleration voltage of 80 kv.
  • rpilin oligomer In order to distinguish the biochemical and immunological properties of rpilin from those of intact pili (or pilin oligomer) , purified rpilin was converted 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 significantly lower endpoint titers for binding to diverse piliated gonococcal cells as compared to antisera induced by untreated rpilin. This suggests that rpilin oligomer had lost a significant number of the cross-reactive epitopes normally present on the rpilin protein. Table 6
  • Guinea pigs (4 per group) were immunized (s.c.) with 20 ⁇ g of rpilin antigen adjuvanted with 25 ⁇ g of StimulonTM QS-21 on weeks 0 and 4. Pooled sera from week 6 were analyzed.
  • 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.
  • 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 ⁇ L of RPMI 1640 and fetal calf serum.
  • the RPMI 1640 block was discarded and 40 ⁇ L of antisera or RPMI 1640 (without calf serum) added, followed by
  • 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 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 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 analyzing.
  • Example 10 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 protein was analyzed by size exclusion chromatography using a SuperoseTM 12 column equilibrated in PBS containing 0.05% (w/v) ZwittergentTM 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- terminal 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.
  • the endpoint titers against purified proteins or bacterial cells were determined by ELISA using the methods described in 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 PBS containing 0.1% (v/v) TweenTM-20 and 0.1% (w/v) BSA; and (2) the plates were washed five times with PBS containing 0.05% (v/v) TweenTM-20 using a Skanwash 300 plate washer.
  • Adjuvant Adjuvant
  • 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-21 in PBS (pH6) ; (b) 100 ⁇ g of A1P0 4 in saline; or (c) PBS (pH7) only. The animals were bled on weeks 0, 4 and 6. Pooled sera was used for all analyses.
  • 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 A1P0 4 in saline; (c) 50 ⁇ g MPLTM in PBS (pH 7); (d) 100 ⁇ g of A1P0 4 and 50 ⁇ g MPLTM in saline; or (e) PBS (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. meningi tidis was also achieved with the addition of StimulonTM QS-21.
  • Franz media [1.3 g/L glutamic acid, 20 mg/L cysteine, 10 g/L ⁇ a 2 HP0 4 -7H 2 0, 90 mg/L KC1, 6 g/L NaCl, 2 g/L yeast dialysate and 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 spotted with an aliquot 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.
  • Figure 3A the antibodies against the meningococcal chimeric class I rpilin protein bound along the length of the pili.
  • normal serum week 0
  • did not show any binding to the pili Figure 3B
  • Sprague-Dawley infant rats (4-5 days old) were 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 injection of normal guinea pig serum (week 0) diluted 1:5 in PBS. Twenty- four hours later, the animals were challenged i.p. with approximately 5 x
  • mice 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 those animals immunized with normal guinea pig serum. This difference was statistically significant, with a p value of ⁇ 0.05.
  • 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, 1 and 2.
  • CT-CRM cross-reactive mutant form of cholera toxin
  • 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.
  • FBS fetal bovine serum
  • Nasal The nasal passages were washed once with 0.5 mL of RPMI 1640 and 20 ⁇ L of FBS 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.
  • the initial step in meningococcal disease in human beings is the colonization of the nasopharynx by the bacteria.
  • pili are believed to play a major role in mediating the inital attachement to the epithelial cells.
  • 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 the efficacy of meningococcal vaccines (35) .
  • a nasal colonization model for N. meningi tidis using adult outbred mice has been developed.
  • mice were immunized with meningococcal chimeric class I rpilin adjuvanted with MPLTM subcutaneously on weeks 0, 4 and 8.
  • the animals received an intraperitonal injection of 2 mg iron dextran (Sigma) and were challenged intranasally with approximately 1 x 10 7 cfu of mid-log phase piliated meningococci in a volume of 10 ⁇ L which also contained 40 ⁇ g of iron dextran.
  • day 1 after challenge half the 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 16.
  • 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 (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 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.
  • Antisera elicted against partially purified meningococcal chimeric 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) .

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US20110182981A1 (en) * 2008-08-25 2011-07-28 Peixuan Zhu Gonococcal vaccines
US9802988B2 (en) * 2009-05-20 2017-10-31 University Of Maryland, Baltimore Engineered type IV pilin of Clostridium difficile
EP2635593B1 (en) * 2010-11-05 2016-09-14 Institut National de la Sante et de la Recherche Medicale (INSERM) Vaccines for preventing meningococcal infections
JP5991740B2 (ja) * 2012-06-21 2016-09-14 キヤノン株式会社 ライセンス管理装置、ライセンス管理方法、およびプログラム
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EP0049945A3 (en) * 1980-09-15 1982-12-01 Bactex Incorporated Determining a hierarchy of piliated organisms and a vaccine prepared from such organisms
US4443431A (en) * 1981-05-27 1984-04-17 The United States Of America As Represented By The Department Of Health And Human Services Neisseria gonorrhoeae vaccine
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CU22302A1 (es) * 1990-09-07 1995-01-31 Cigb Secuencia nucleotidica codificante para una proteina de la membrana externa de neisseria meningitidis y uso de dicha proteina en preparados vacunales
WO1992013871A1 (en) * 1991-01-31 1992-08-20 Washington University Polypeptides and polynucleotides useful for the diagnosis and treatment of pathogenic neisseria
AU3416693A (en) * 1991-12-18 1993-07-19 State Of Oregon Acting By And Through The Oregon State Board Of Higher Education On Behalf Of Oregon State University, The Antigenic preparations that stimulate production of antibodies which bind to the pili of type IV piliated bacteria
DK0761231T3 (da) * 1992-06-25 2000-05-08 Smithkline Beecham Biolog Vaccinepræparat indeholdende adjuvanser
AU5403594A (en) * 1992-10-07 1994-04-26 State Of Oregon, Acting By And Through The Oregon State Board Of Higher Education On Behalf Of The Oregon Health Sciences University Pilin variants and uses thereof
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