WO1995004133A1

WO1995004133A1 - Production of gonorrheal pi proteins and vaccines in e. coli and salmonella

Info

Publication number: WO1995004133A1
Application number: PCT/US1994/008586
Authority: WO
Inventors: Christopher Elkins; P. Frederick Sparling
Original assignee: The University Of North Carolina At Chapel Hill
Priority date: 1993-07-30
Filing date: 1994-07-29
Publication date: 1995-02-09
Also published as: CA2167938A1; EP0717774A1; JPH11501203A; AU7408494A

Abstract

The present invention provides a recombinant Salmonella or E. coli cell that expresses a gonococcal porin protein or a fragment thereof. The invention further provides a vaccine comprising the recombinant Salmonella cell and a pharmaceutically acceptable carrier as well as methods for prevention and treatment of gonorrheal infection.

Description

PRODUCTION OF GONORRHEAL PI PROTEINS AND VACCINES IN E. COLI AND

SALMONELLA

BACKGROUND OF THE INVENTION

This application is a continuation-in-part of Serial Number 08/100,588 filed July 30, 1993, which is incorporated herein by reference.

Neisseria αonorrhoeae. the gonococcus, infects only humans and produces the disease gonorrhea. Gonorrhea remains the most frequently reported infectious disease in the United States and is an important cause of morbidity and infertility in both developed and undeveloped countries.

No gonococcal vaccine is currently available. Of the several candidates for a gonococcal vaccine, the gonococcal outer membrane porin protein, referred to in the literature as PI or Por, is a prime candidate (Gotschlich 1984; Blake etal. 1989; Elkins and Sparling 1992). PI is an antigenically stable, surface-exposed protein, and antibodies against PI are potentially protective (Heckels et al. 1990; Joiner et al. 1985; Kohl et al. 1989; Virji et al. 1987; Virji et al. 1986).

PI is typical of most gram negative porins in structure, and exists as a non- covalent trimer complex while exhibiting extensive beta sheet structure in the native state. When denatured by boiling in sodium dodecyl sulfate solution and analyzed by polyacrylamide gel electrophoresis, however, PI dissociates from a trimer configuration into monomers and migrates with an estimated molecular mass of 32- 39kDa (Johnston etal. 1976; McDade and Johnston 1980).

PI is found in two major immunochemical classes termed serogroups PIA and PIB (Tarn etal. 1982; Knapp etal. 1984). PIA and PIB are the products of variants/alleles of the same gene (Carbonetti et al., 1988; Carbonetti and Sparling, 1987). All gonococci contain PI of either serogroup, but not both, and no PI minus mutants are known to exist. PI is stable and does not undergo phase or antigenic variation (Judd 1989).

The DNA and amino acid sequence of PIA and PIB, as well as hybrid PIA/PIB and fragments thereof are known (Carbonetti et al., International Application Nos. PCT/US92/02006, filed March 13, 1992; PCT/US92/02090; PCT/US88/04225; Carbonetti and Sparling 1987; Carbonetti et al. 1988; Carbonetti et al. 1990).

Since the overwhelming majority of gonococcal infections involve the mucus membranes of the genitourinary tract, it is there that an effective vaccine would ideally direct an immune response. Studies have shown that locally produced antibodies of the secretory IgA isotype are among the most important protective factors at mucosal surfaces (McGhee etal. 1992). Specialized lymphoid tissues, termed bronchus- associated and gut-associated lymphoid tissues (BALT and GALT), are sites of induction in the development of mucosal responses (see Holmgren 1991; Holmgren etal. 1992; and McGhee etal. 1992 for reviews).

Several antigen delivery systems have been developed to induce a response at mucosal surfaces. For example, attenuated Salmonella strains can colonize the GALT and stimulate a generalized protective immune response to Salmonella. One system employs live, attenuated Salmonella typhimurium to express and deliver foreign antigens to the GALT. (Curtiss III and Kelly 1987; Dougan et al. 1988; Dougan et al. 1987; Dougan et al. 1989; Morona et al. 1991; Nakamaya et al. 1988; Strugnell et al. 1990; Tarkka et al. 1988; Walker et al. 1992). In addition to the attenuated mouse strains of Salmonella known to be useful for testing expressed genes in animal models, human strains of Salmonella with potential for human clinical trials (S. typhi) are also being tested (Tacket etal. 1992).

Stimulation of GALT by attenuated, recombinant Salmonella requires at least transient growth of the Salmonella host strain in the gut (Dougan etal. 1989), and therefore expression of the foreign protein should not be rapidly lethal to the host. Previous reports (Carbonetti and Sparling 1987; Gotschlich etal. 1987; Carbonetti et al. 1988) have indicated that expression of some gonococcal proteins, including PI, was lethal for E. coli. Since E. coli and S. typhimurium are similar (Neidhardt et al.), gonococcal porin proteins would also be expected to be toxic to Salmonella hosts. The expression of gonococcal porin proteins in Salmonella or of intact gonococcal porin proteins in E. coli has not been reported before the present invention.

Accordingly, there is a need for the construction of recombinant Salmonella or E. coli cells that are able to express gonococcal porin proteins. There particularly is a need to produce vaccine compositions using such recombinant Salmonella or E. coli cells to prevent or treat gonorrheal infection.

SUMMARY OF INVENTION

The invention provides a recombinant Salmonella cell that expresses a gonococcal porin protein or a fragment thereof. The invention further provides a plasmid capable of expressing a gonococcal porin protein in a Salmonella cell comprising DNA encoding such protein and suitable regulatory elements arranged within the plasmid so as to permit expression of the gonococcal porin protein in the Salmonella cell.

The invention also provides a plasmid that expresses an antigen in a Salmonella or E. coli host cell comprising DNA encoding such antigen and suitable regulatory elements, including a gonococcal porin promoter, arranged within the plasmid so as to permit expression of the antigen in the host cell.

The invention also provides a vaccine composition comprising the recombinant Salmonella cell containing a plasmid for expression of a gonococcal porin protein and a pharmaceutically acceptable carrier, as well as methods for treating or preventing gonorrheal infection by administering the vaccine composition.

DESCRIPTION OF THE FIGURES

Figure 1. Reassembly of a complete port gene from gonococcal strain FA19 in E. coli. The 720 bp Smal to Tagl fragment of pUNCH 11 contains the complete port promoter (500 bp of sequence upstream rom the start codon) and coding sequence for 58 amino acids of the protein. The 820 bp Tacfi to Sau3AI of pUNCH 15 contains the remaining coding sequence and 12 bp of downstream port sequence. These two fragments were ligated between Hinctt and BamHI sites of pGEM-2 in the E. coli host M16 to form pUNCH 30. Open white bars indicate adjacent non-coding DNA sequences; small open arrows with the letter p adjacent indicate the promoter region; hatched arrows indicate the position and orientation of por, Amp indicates the position of the beta-lactamase gene in all figures. Restriction sites in parentheses indicate that the site was lost during construction.

Figure 2. Constitutive expression of recombinant Por-1_(A, F_AIS>) and Por-5_(A/_B, _FAW_M) from a native promotor in E. coli. Whole cell lysates of gonococci (2.5 ug of protein) or E. coli (10 ug of protein) were subjected to Western blotting using monoclonal antibodies specific for P1 A (panel A) or PIB (panel B) epitopes. Lanes: 1-3, gonococcal strains FA19 (Por-1_(A)), FA6434 (Por-5_{(A B})), and MS11 (Por-2_(B)), respectively; 4-6, E. coli strain M16 containing plasmids pGEM-2 (no insert), pUNCH 30 (port), pUNCH 535 (por-5), respectively; and 7-9, E. coli strain DH5a MCR containing plasmids pGEM-2 (no insert), pUNCH 30 (port), pUNCH 535 (por-5) respectively. The weak band in panel B, lane 5 represents spillover from lane 6 and was not seen in other similar blots.

Figure 3. Reassembly of the por-2 gene from gonococcal strain MS11 in E. coli. (A): Reassembly of the complete por-2 structural gene. Plasmid pUNCH 23 contains the entire por-2 promoter (157 bp of sequence upstream from the start codon) and DNA sequence encoding the first 105 amino acids of Por-2_(B. S_H). Plasmid puNCH 22 contains the remaining 3' coding sequences of por-2 as well as 1.0 kb of downstream DNA. The checkered pattern in the bars and arrows indicates por-2. The cloning strategy was to double-digest pUNCH 22 and pUNCH 23 with Kpnl and EcoRI, gel purify each relevant fragment and ligate them together. Repeated attempts to reassemble the complete por-2 gene were unsuccessful in that very few transformants were obtained and none expressed serogroup PIB epitopes or contained appropriate sized plasmids. To verify that the proper ligation product was present in the above ligation reaction, 0.1 ul of the ligation mixture was subjected to PCR using primers which anneal upstream and downstream from por-2. The 5' oligonucleotide (5'-GGCGMTTCCGGCCTGCTTA TTTCTTA-3')contains an engineered EcoRI cloning site and anneals to the sequence from 44 to 63 bp of the published por-2 sequence. The 3' oligonucleotide (5'-

GCGAAGCTTATTAGAATTTGTGGCGCAGA-3') contains an engineered HindlllsWe and anneals to the sequence from 1992 to 2011 bp of por-2 sequence. Thus the PCR product contains the -10 promoter region but lacks the -35 promoter region (see Carbonetti (1988) for MS11 DNA sequence showing -10 and -35 promoter region sequences. MS11 chromosomal DNA was similarly amplified as a positive control. The conditions of the amplification were: Denature for 1 min at 94°C; anneal for 30 s at 42°C; extend for 2 min at 72°C for a total of 28 cycles; and extend 5 min at 72°C for the last cycle. (B): Analysis of PCR products by 1% agarose gel electrophoresis. Lanes: 1 , 1 kb ladder molecular weight standard (BRL); 2, PCR amplification of por-2 ligation reaction including T4 DNA ligase; 3, PCR amplification of mock por ligation reaction without T4 DNA ligase; 4, PCR amplification of MS11 chromosome; 5, mock PCR reaction lacking template DNA. The amplified products in lanes 1 and 3 from panel (B) were restricted with EcoRI and Hindi II, ligated to similarly cut and gel- purified pGEM3Zf(-) vector. E. coli strain BL21 DE3 [pLysS] was transformed, and greater than one thousand ampicillin-resistant colonies were obtained. Ampicillin resistant colonies were screened for Por production without IPTG induction by colony lift using Mabs and the majority expressed serogroup PIB epitopes. (C): Expression of recombinant Por-2 by E. coli transformants. Whole cell lysates of gonococci (2.5 ug of protein) or E. coli transformants (10 ug of protein) which bound anti-PIB mAb in the colony lift were electrophoresed on a 0.1% SDS-12% polyacrylamide gel and subjected to Western blotting using mAb specific for PIB. Lanes: 1 and 2, gonococcal strains MS11 (Por-2_(B)) and FA19 (Por-1_(A)), respectively; 3-5, BL21 (DE3) [pLysS] E. coli containing pUNCH 540 (amplified from the por-2 ligation), pUNCH 541 (amplified from the MS11 chromosome), and pGEM3Zf(-) vector control. Cultures were not induced with IPTG. Figure 4. Construction of a plasmid containing a hybrid por gene (por-5 from strain FA6434) behind an intact port promoter. Plasmids pUNCH 30 (Fig. 1) and pUNCH 50 were restricted with Ncol and Sstll. The large fragment of pUNCH 30 was gel- purified and ligated with the small gel-purified fragment of pUNCH 50. The resulting plasmid was termed pUNCH 535. The hatched regions indicate port coding sequence and the checkered region indicates por-2 coding sequence.

Figure 5. Integration of FA19 port into the Salmonella chromosome using chromosomal integration vector pDEL-1. Plasmid pDEL-1 contains 4.1 kb of S. typhimurium aroC flanking DNA (indicated by the stippled boxes) with the majority of aroC deleted (both wild type and deleted aroC sequences are indicated by cross- hatching). Cloning of port into pDEL-1 was accomplished by ligation of the gel- purified Hindi to EcoRI fragment of pUNCH 30 (containing the promoter and complete coding sequence of port) into the gel-purified Sma\ to EcoRI large fragment of vector pDEL-1 , and transformation into both E coli M16 and DH5aMCR. Similar transformants (based on restriction analysis) were obtained from both strains and each expressed epitopes for Por (Figure 6). A representative M16 E. coli transformant was chosen and its plasmid was termed pUNCH 527. Plasmid pUNCH 527 DNA was transformed into S. typhimurium BRD 835 (r^"m⁺) in order to methylate the DNA (not shown in the figure). Methylated plasmid pUNCH 527 DNA was then transformed into S. typhimurium polA strain BRD 207 with selection for ampicillin. Since ColE1 replicons, such as pUNCH 527 and pDEL-1 , cannot replicate in polA mutants, only those bacteria which integrate the plasmid marker into the chromosome can grow in the presence of ampicillin (see bottom of figure 5). One of 4 colonies obtained, termed FX501 , expressed Por-1 _(A( FAI9> weakly, whereas control transformant FX502, obtained by transformation with pDEL-1 , does not express Por- 1(A, FA 9) (Figure 6). Figure 6. Comparison of chromosomal and plasmid expression of Por in S. typhimurium. After electrophoresis on a 0.1% SDS-12% polyacrylamide gel, whole cell lysates of gonococci (2.5 ug of protein), E. co//^'M16 (10 ug of protein) or S. typhimurium (10 ug of protein) were subjected to Western blotting using a mAb specific for a PIA epitope. Lanes: 1 , gonococcal strains FA19 (Por-1 _(A)); 2, MS11 (por-2(_B)); 3-4, E. coli strain M16 carrying pDEL-1 or pUNCH 527; 5-6, S. typhimurium BRD 835 carrying pDEL-1 or pUNCH 527; 7, S. typhimurium FX501 (pUNCH 527 transformant of BRD 207): FX502 (pDEL-1 transformant of BRD 207).

Figure 7. Construction of plasmids suitable for use in asd mutant S. typhimurium vaccine hosts. A 1.75 kb Bgl II fragment containing a Salmonella asαfgene was recovered from pYA292, blunted by filling in the ends with Klenow, and ligated with Sea l-restricted plasmids pUNCH 30 (port), pUNCH 535 (por-5) and pGEM-2 (no insert). Each ligation was transformed into E. cσ//X6212 asd. Transformants were termed pUNCH 537, pUNCH 536, and pUNCH 539, respectively. These plasmids were initially moved into S. typhimurium X3730 (r^"m⁺) and then into mouse vaccine strain X4072. The hatched regions indicate port coding sequence, the checkered region indicates por-2 coding sequence, and the stippled bar indicates asd sequence.

Figure 8. Plasmid expression of por from asdE. coli and S. typhimurium hosts. Whole cell lysates of gonococci (2.4 ug of protein), E. coli (10 ug of protein) or S. typhimurium (10 ug of protein) were subjected to Western blotting using Mabs specific for PIA (A) or PIB (B) epitopes. Lanes: 1-3, gonococcal strains FA19 (Por-1_(A)), FA6434 (Por-5_{(A B)}), and MS11 (Por-2_(B)), respectively; 4-6, E. co//X6212; 7-9, S. typhimurium X3730, 10-12, S. typhimurium X4072. E. coli and S. typhimurium strains contained plasmids pUNCH 539 (control plasmid, no poή, pUNCH 537 (port), and pUNCH 536 (por-5), in their respective lanes. Figure 9. Figure 9 shows the antigenic sequences corresponding to fragments 1 - 6. Each fragment optionally contains an N-terminal cysteine residue. The amino acid numbers correspond to the amino acid residues of Por-1_(A) from N. αonorrhoeae strain FA19 (fragments 1-4) or of Por-2_(B) from N. αonorrhoeae strain MS11 (fragments 5 and 6).

Figure 10. Por-5 surface structure. This figure is based on the model of gonococcal porin proposed by van der Ley (van der Ley et al., 1991). Putative surface-exposed regions are drawn above the upper dotted line, membrane spanning regions between the dotted lines, and periplasmic-exposed regions below the lower dotted line. Each surface exposed loop is numbered in Roman numerals. Bold type indicates the regions where the homologous recombination (double cross-over) occurred during the construction of gonococcal strain FA6434. The structure of Por-5 was deduced from DNA sequencing of pUNCH 50, which contains Por-5. Bactericidal Mabs SM101, 4G5 (PIA Mabs), SM24 and 15A4A (PIB Mabs) bind Por-5.

Figure 11. Immune response to rPor-5. Shown are the ELISA values for individual rabbits (5 per adjuvant group) immunized with rPor-5. The coating antigen was rPor-1 or rPor-2, 100ng/well. The dilution of primary anti-Por-5 was 1:50,000. The secondary antibody was goat anti-rabbit conjugated to horseradish peroxidase and was used at a 1 :10,000 dilution. Pre-immune sera are not shown and their OD at this dilution was less than 0.05.

Figure 12. Dot blot analysis of the immune response to surface exposed epitopes of Por. Gonococcal strains FA19 (Por-1 ), FA6434 (Por-5) or MS11 (Por-2) were immobilized onto nitrocellulose (duplicate columns) and probed with a 1:50 dilution of anti-Por-5 sera for 1 hour. Protein A alkaline phosphatase (1 :2000) was used to detect bound antibody. The numbers to the left and right of the dots are the individual rabbits (no adjuvant, 41-45; BCG, 46-50; CFA, 51-55). The upper 6 dots of each rabbit show the reaction of the pre-immune serum and the lower 6 dots show the post-immune reaction. Mab controls are 4G5 (anti-PIA, N-terminus) and 5.51 (anti- PIB, central region). For demonstration purposes only some of the rabbit data are shown. In the original dot blots, the discrimination between positive and negative reactions was more clear cut than in this reproduction, and these original results are summarized in Table 1.

Figure 13. Western blotting of anti-Por-5 sera. Whole cell lysates (approximately 2 X 10⁷CFU) of gonococcal or E. coli were subjected to Western blotting using 2 selected antisera that bound all three gonococci (expressing Por-1 , Por-2 and Por-5) in dot blots. Lane 1, FA19; lane 2 FA19 Drmp; lane 3, MS11; lane 4, MS11 Drmp; lane 5,

FA6434; lane 6 E. co//MC4100 (parent, wild type for ompA); lane 7, Bre2413 OompA.

Pre, Pre-immune; Post, Post-Immune, the rabbit antisera was diluted 1:1000 and the secondary reagent was protein A alkaline phosphatase. In panel A the rabbit shown was immunized with rPor-5 in the detergent DBM without additional adjuvant. In panel B the rabbit was immunized using KLH as the adjuvant.

Figure 14. por-5 DNA sequence from pUNCH50.

DESCRIPTION OF THE INVENTION

This invention provides a recombinant Salmonella or E. coli cell containing DNA for expression of a gonococcal porin protein (Por). The Por DNA may be integrated into the Salmonella or E. coli cell chromosome or into a suitable cloning/expression vector that is inserted into the Salmonella or E. coli cell. In a preferred embodiment of the invention, the cell contains a vector, preferably a plasmid, comprising any DNA that produces a gonoccocal porin protein. Preferably, the protein is expressed on the surface of the Salmonella or E. coli cell and the cell thereby preferably is capable of stimulating antibody production. Preferably, the protein is expressed at moderate, as compared to high, expression levels. In another preferred embodiment, the porin protein expressed by the cell is used to produce antibodies to protect against gonorrheal infection in a mammal, preferably a human.

The gonococcal porin proteins may be any Por-1, Por-2, or hybrid Por-1/Por-2 proteins. The gonococcal porin proteins are preferably proteins or fragments thereof produced by port, por-2, or por-5 genes, port is herein defined as a por gene assembled from the N. gonorrhoeae strain FA19 (serogroup PIA). por-2 is herein defined as a por gene assembled from the N. gonorrhoeae strain MS11 (serogroup PIB). por-5 is herein defined as a por gene assembled from the N. gonorrhoeae strain FA6434 (see Table 1 and Fig. 10). por-5 expresses a hybrid Por isolated as class 9 (Carbonetti, et al. 1988) containing epitopes from serogroups PIA and PIB. The Por proteins produced by the port, por-2, andpor-5 genes are designated Por- 1(A), Por-2(β), and Por-5_(A^), respectively. The phenotypes of these Por proteins are designated herein by a subscript indicating serogroup and strain, e.g., Por-1_(A,FAi9), Por-2(_B. sιi), and Por-5_(A/B,FA6434), respectively.

L gonorrhoeae porins from serogroups PIA or PIB or chimeric PIA/PIB porin proteins may be expressed by recombinant Salmonella or E. coli cells. Examples of porin proteins and genes are described in International Application No. PCT/US88/04225, filed November 23, 1988, incorporated herein by reference, and hybrid porin proteins are described in PCT/US92/02090 (Fragments of full length porin proteins and genes are suitable)., filed March 13, 1992, also incorporated herein by reference. GOL-1-T hybrid fragments.

FRAGMENTS Fragments containing antigenic sequences of Por-1_(A), Por-2(_B) and Por-5(A/B) may be selected on the basis of generally accepted criteria of potential antigenicity and/or exposure. Such criteria include the hydrophilicity and relative antigenic index, as determined by surface exposure analysis of Por-1 _(A) and Por-2_(B) proteins.

Promising candidates are prepared and tested for antigenicity and immunogenicity.

Fragments 1-6, which are shown in Figure 9, are suitable antigenic sequences. Fragments 1-4 contain amino acid sequences found in Por-1(_A> of gonococcal strain FA19. Fragments 5 and 6 contain amino acid sequences found in Por-2_(B) of gonococcal strain MS11.

PREPARATION OF A RECOMBINANT SALMONELLA OR E. COLI CELL CONTAINING A por GENE

Recombinant Salmonella or E. coli cells that express porin proteins may be produced by known methods. For example, the porin gene may be inserted into a plasmid cloning vector which functions as the unit of replication of the gene. The recombinant plasmid is inserted into a compatible host cell, i.e. Salmonella or E. coli, whereby the gene product is expressed.

The recombinant plasmids described herein enable the stable expression in, without toxicity to, a host cell of the Por proteins. In accordance with the invention, Por proteins may be produced by inserting the cloned sequence of the Por-1(A) or Por- 2_(B) proteins or hybrids thereof into an appropriate cloning/expression vector, such as a plasmid, for insertion into E. coli or Salmonella. Alternatively, the gene may be introduced into the Salmonella or E. coli chromosome using known methods (see Strugnell et al., Gene 88:57-63 (1990), for example).

In order to achieve transcription and translation of the inserted gene inserted into a plasmid or chromosome, the gene must be placed under the control of regulatory elements from, or compatible with, the chosen host cell. Preferably, the regulatory elements include one or more native gonococcal porin protein regulatory elements, such as the porin protein's native promoter.

In another embodiment of the invention, the gonococcal porin promoter may also be used to express in E. coli, Salmonella, or N. gonorrhoeae antigens other than porin proteins not normally expressed in E. coli, Salmonella, or N. gonorrhoeae. For constitutive expression of the desired antigen in E. coli or Salmonella, the antigen DNA is fused to a gonococcal porin promoter and inserted into an appropriate cloning/expression vector, such as a plasmid, for insertion into E. coli or Salmonella. The antigens fused to the gonococcal porin promoters for expression in E. coli, Salmonella, or N. gonorrhoeae may be, for example, gonococci transferrin binding proteins such as B transferrin, lactoferrin, heme binding proteins and other vaccine candidates, as well as meningococci proteins FrpA, FrpB, and FrpC.

A number of methods exist for the insertion of DNA into vectors in vitro. DNA ligase is an enzyme which seals single-stranded nicks between adjacent nucleotides in a duplex DNA chain; this enzyme may therefore be used to covalently join the annealed cohesive ends produced by certain restriction enzymes. Alternately, DNA ligase can be used to catalyze the formation of phosphodiester bonds between blunt- ended fragments. Finally, the enzyme terminal deoxynucleotidyl transferase may be employed to form homopolymeric 3' - single-stranded tails at the ends of fragments; by addition of oligo (dA) sequences to the 3' end of one population, and oligo (dT) blocks to 3' ends of a second population, the two types of molecules can anneal to form dimeric circles. Any of these methods or other known methods may be used to ligate the gene segment promoter and other regulatory elements into specific sites in the vector. Thus, the gene encoding the Por proteins is ligated into the chosen vector in a specific relationship to the vector promoter and control elements, so that the sequence is in the correct reading frame. The vector employed will typically have a marker function, such as ampicillin resistance or tetracycline resistance, so that transformed cells can be identified. The vector employed may be any of the known cloning or expression vectors or their derivatives; among the most frequently used plasmid vectors include pBR 322, pAC 105, pVA 5, pACYC 177, PKH 47, pACYC 184, pUB 110, pmB9, pBR325, Col El, pSC101, pBR313, pML21, RSF2124, pCR1, RP4, or, preferably, the pGEM series (Promega Corp.) (see Table I for preferred vectors).

Any recombinant Salmonella or E. coli that expresses a foreign protein, preferably on its surface, may be used to stimulate the production in vivo of antibodies against the porin protein. The E. coli or Salmonella is preferably one that is effective in expressing the foreign porin protein to stimulate an effective immune response against gonorrheal infection.

Preferred bacterial strains and plasmids are shown in Table 1 below.

TABLE 1

Bacterial strains and plasmids.

Bacterial Genotype/Phenotype Source or reference Strains

N. gonorrhoeae

FA19 port, Por-1(_A,FAi9) (Carbonetti and Sparling 1987) MS11 por-2, Por-2_(B,_Msιi) (Carbonetti etal. 1988) FA6434 por-5,

(class 9 hybrid) (Carbonetti etal. 1988) tn

I

S. typhimurium

BRD f C2 metfiι22H1-b H2-e,n,x, nml (Fels2) fla-66 rpsL120 xyl- (Strugnell etal. 1990) 835(LB5010) 404 metE551 hasL6 hsdSA29 hsdSB121 ilv-452 leu-3121 galE856

BRD207 SL5388 fla-66 metA22, trp-2, xyl-401, (rm⁺) leu polA 1 (Strugnell et al. 1990) FX501 BRD207 expressing Por-1_(A, FAi9) from the This specification chromosome

FX502 BRD207 control transformant (vector only) This specification X3730 LT2-Z pStLTI OO^' leu hsdLT galE trpD2 rpsL 120 AasdA 1 Azhf- R. Curtiss III, personal

Bacterial Genotype/Phenotype Source or reference Strains

4::Tn10)metE551 metA22 hsdSA hsdSB ilv communication

X4072 SR-11 pStLTI 00^" gyrA 1816 Acya- 1 Acrp- 1 AasdA 1 Azhf-4::Tn 10) (Na ayama etal. 1988)

E. coli

BL21 (DE3) F^"o pT(r_b ^"m_b ^') λDE2(/acf /acUV5-T7 RNA polymerase) (Studier et al., 1990) [pLysS] pLysS(T7 lysozyme)

DHδaMCR F mcrA mcrB mrr fφβøαf lacZAMIS) endA 1 recA 1 hsdR (r_k ^"-m_k) Bethesda Research Labs hsdM supE44 thi-1X gyrA relA1 A(lacZYA argF)U169 i M 16 (MC4100) F- lacP 0⁺?Y*A⁺ P. Bassford and Elliott Altman*

I

X6212 F mrr$80dlacZAM15) A(lacZYA-argF)W 69 recAl endAI hsdR S. Tinge and hsdM supE44 thi-1 λ^' tyrA relA 1 AasdA4 A(zhf-2::Tn10) R. Curtiss III (unpublished)

Plasmids Genotype Phenotype Source or reference pGEM-2 Ap^R Promega, Madison, Wl, USA pGEM-3Zf(-) Ap^R Promega pUNCH 11 730bp fragment from strain FA19 containing port (Carbonetti and Sparling 1987) promoter and upstream sequences in pGEM-2 pUNCH 15 860bp fragment containing the 3^* portion of the FA19 (Carbonetti and Sparling 1987)

port gene in pBR322 pUNCH 30 port in pGEM-2, expresses Por-1 from its own This specification promoter pUNCH 50 por-5 from FA6434 without native promoter in pGEM-2 (Carbonetti et al. 1988)

(see Figures 10 and 14 for sequence of por-S) pDEL-1 ApΑaroC (S. typhimurium) (Strugnell et al. 1990) pUNCH 527 HincM to EcoRI fragment of pUNCH 30 ligated into This specification pDEL-1 , expresses Por-1 _(A, FAI9). from its own promoter pUNCH 535 NCo\ and SSfll fragment from pUNCH 50 ligated into This specification the same sites of pUNCH 30 creating a hybrid porin gene (por-5) expressed from the port promoter pUNCH 536 pUNCH 535 with asd ligated into the Seal site. This specification pUNCH 537 pUNCH 30 with asd ligated into the Seal site. This specification pUNCH 539 pGEM-3Zf(-) with asd ligated into the Seal site. This specification pUNCH 540 pGEM-3Zf(-) containing the PCR product amplified This specification from the MS11 por-2 ligation reaction (bp -44 to 2011 of por-2) pUNCH 541 pGEM-3Zf(-) containing the PCR product amplified This specification from the MS11 por-2 chromosome (bp -44 to 2011 of por-2)

* M16 is a derivative of strain MC4100 (K12) which tolerates several membrane proteins that are toxic for some other E. coli h (P. Bassford and E. Altman, personal communication).

VACCINES:

Methods are described herein which permit the formulation of vaccine compositions. In preferred embodiments, vaccine compositions comprise Salmonella or E. coli producing Por proteins such as Por-1_(A) or Por-2_(B) or hybrid Por_(A/_B) proteins and fragments thereof.

FORMULATION OF A VACCINE

The Salmonella or E. coli cells containing the por genes, preferably in a plasmid, are useful in the preparation of a vaccine composition for prevention of or treatment of gonorrheal infection. The bacterial host is preferably S. typhimurium.

The vaccine may be combined with any of the commonly used pharmaceutically acceptable carriers, such as water, physiological saline, ethanol, polyols, such as glycerol or propyleneglycol, or vegetable oils, as well as any of the vaccine adjuvants known in the art, such as muramyl peptides, lymphokines, such as interferon, interleukin-1 and interleukin-6, or bacterial adjuvants. As used herein,

"pharmaceutically acceptable carriers" is included to encompass any and all solvents, dispersion media, coatings, antibacterial and antifungal agents; isotonic and absorption delaying agents and the like. The use of such agents for pharmaceutically active substances is known in the art. Except insofar as any conventional medium is incompatible with the active ingredient, its use in the therapeutic composition is contemplated. Supplemental active ingredients may also be incorporated.

A particularly useful embodiment of the present invention is a vaccine in which the active immunogen is a hybrid Por-1_(A Por-2(B) protein, the construction of which is described in Intemational Application No. PCT/US88/04225, filed November 23, 1988, or fragments of the hybrid Por-1(_A Por-2(_B) protein, the construction of which is described in Intemational Application PCT/US92/02090, filed March 13, 1992. Furthermore, the hybrid protein may be any of the hybrid gene structures (classes 1- 9) (Carbonetti et al. 1988). The hybrid Por-1_(A/Por-2(_B) may be contained in plasmids pUNCH50, pUNCH535, and pUNCH536.

The vaccine may be administered to a mammal by methods known in the art. Such methods include, for example, oral, intravenous, intraperitoneal, subcutaneous, or intramuscular administration. The preferred method is oral administration.

The vaccine may be administered for prevention prior to gonorrheal infection, or for treatment during gonorrheal infection.

PROBES USING POR POLYPEPTIDES OR ANTIBODIES

Por polypeptide probes:

The Por polypeptide expressed by, and preferably on the surface of, the recombinant Salmonella or E. coli cell of the invention may be used to detect the presence of antibodies specific for porin proteins in a sample. The method comprises preparing a polypeptide containing a segment having an amino acid sequence that is substantially homologous to a porin protein. The polypeptide may be prepared using methods known in the art. Preferably, the polypeptide comprises a segment having an amino acid sequence that is present in the porin protein.

The sample may, for example, be from a patient suspected of being infected with N. gonorrhoeae. Suitable assays are known in the art, such as the standard ELISA protocol described by R.H. Kenneth, "Enzyme-Linked Antibody Assay with Cells Attached to Polyvinyl Chloride Plates" in Kenneth et al, Monoclonal Antibodies. Plenum Press, N.Y., page 376 (1981).

Briefly, plates are coated with antigenic polypeptide at a concentration sufficient to bind detectable amounts of the antibody. After incubating the plates with the polypeptide, the plates are blocked with a suitable blocking agent, such as, for example, 10% normal goat serum. The sample, such as patient sera, is added and titered to determine the endpoint. Positive and negative controls are added simultaneously to quantitate the amount of relevant antibody present in the unknown samples. Following incubation, the samples are probed with goat anti-human Ig conjugated to a suitable enzyme. The presence of anti-polypeptide antibodies in the sample is indicated by the presence of the enzyme.

Por antibody probes:

The porin proteins expressed by, and preferably on the surface of, the recombinant Salmonella or E. coli cells of the invention may be used to produce antibodies for use as probes to detect the presence of porin proteins in a sample. The antibodies may be polyclonal or monoclonal. The sample may, for example, be a bodily fluid from a mammal, including a human, suspected of being infected with N. gonorrhoeae.

Methods are known for detecting polypeptides with antibodies. For example, a polypeptide may be immobilized on a solid support. Immobilization of the polypeptide may occur through an immobilized first antibody specific for the polypeptide. The immobilized first antibody is incubated with a sample suspected of containing the polypeptide. If present, the polypeptide binds to the first antibody.

A second antibody, also specific for the polypeptide, binds to the immobilized polypeptide. The second antibody may be labelled by methods known in the art. Non-immobilized materials are washed away, and the presence of immobilized label indicates the presence of the polypeptide. This and other immunoassays are described by David, et al., in U.S. Patent 4,376,110 assigned to Hybritech, Inc., La Jolla, California.

The probes described above are labelled in accordance with methods known in the art. Methods for labelling antibodies have been described, for example, by Hunter and Greenwood in Nature 144, 945 (1962) and by David et al in Biochemistry 3, 1014-1021 (1974). Additional methods for labelling antibodies have been described in U.S. patents 3,940,475 and 3,645,090. Methods for labelling oligonucleotide probes have been described, for example, by Leary et al, Proc. Natl. Acad. Sci. USA (1983) 80:4045; Renz and Kurz, Nucl. Acids Res. (1984) 12:3435; Richardson and Gumport, Nucl. Acids Res. (1983) 11:6167; Smith et al, Nucl. Acids Res. (1985) 13:2399; and Meinkoth and Wahl, Anal. Biochem. (1984) 138:267.

The label may be radioactive. Some examples of useful radioactive labels include ³²P, ¹²⁵ 1, ¹³¹l, and ³H. Use of radioactive labels have been described in U.K. 2,034,323, U.S. 4,358,535, and U.S. 4,302,204.

Some examples of non-radioactive labels include enzymes, chromophors, atoms and molecules detectable by electron microscopy, and metal ions detectable by their magnetic properties.

Some useful enzymatic labels include enzymes that cause a detectable change in a substrate. Some useful enzymes and their substrates include, for example, horseradish peroxidase (pyrogallol and o-phenylenediamine), beta-galactosidase (fluorescein beta-D-galactopyranoside), and alkaline phosphatase (5-bromo-4-chloro- 3-indolyl phosphate/nitro blue tetrazolium). The use of enzymatic labels have been described in U.K. 2,019,404, EP 63,879, and by Rotman, Proc. Natl. Acad. Sci., 47, 1981-1991 (1961).

Useful chromophores include, for example, fluorescent, chemiluminescent, and bioluminescent molecules, as well as dyes. Some specific chromophores useful in the present invention include, for example, fluorescein, rhodamine, Texas red, phycoerythrin, umbelliferone, luminol.

The labels may be conjugated to the antibody or nucleotide probe by methods that are well known in the art. The labels may be directly attached through a functional group on the probe. The probe either contains or can be caused to contain such a functional group. Some examples of suitable functional groups include, for example, amino, carboxyl, sulfhydryl, maleimide, isocyanate, isothiocyanate.

The label may also be conjugated to the probe by means of a ligand attached to the probe by a method described above and a receptor for that ligand attached to the label. Any of the known ligand-receptor combinations is suitable. The biotin-avidin combination is preferred.

EXAMPLES:

The Examples which follow are set forth to aid in understanding the invention but are not intended to, and should not be construed to, limit its scope in any way. The Examples do not include detailed descriptions of conventional methods employed in the construction of vectors and plasmids, the insertion of genes encoding polypeptides into such vectors and plasmids or the introduction of plasmids into hosts. Such methods are well known to those of ordinary skill in the art and are described in numerous publications including Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press.

The following examples show the stable expression of certain intact por genes (port and por-5) from their own promoters in E. coli and S. typhimurium when present on a plasmid or from the Salmonella chromosome (port only). Moreover, por-2 was expressed in E. coli without toxicicity when cloned without its full promoter region, although higher level expression from an intact promoter was apparently lethal. High level expression of all forms of por genes tested in E. coli were lethal to E. coli (induction of high levels of Por lysed E. coli BL21 (DE3)[pLysS]).

CLONING AND PLASMID EXPRESSION OF oor IN E. COLI

The Por-1 _{A), Por-2_(B) and hybrid Por-1 _(A)(_B) forms of Por were reassembled and expressed in E. coli. The gonococcal port (Por-1(_A)) promotor was used to express the Por-1(_A) and hybrid Por-1_(A)/(_B) Por proteins.

The port gene from gonococcal strain FA19 (Por-1_(A)) (see Table 1 for list of strains and plasmids) was reassembled (pUNCH 30, Fig. 1) and stably expressed from its own promoter in M16 (Fig. 2, lane 5). Further studies indicated that E. coli DH5a MCR (Fig.2, lane 8), DH5a F, and HB101 could be transformed with pUNCH 30 and could express Por-1_{(Ai FA}i9) without apparent toxicity.

Relatively weak but stable expression of Por-2_(B, _Msn) in E. coli was achieved withoutt uussiinngg the entire por-2 promoter region (i.e. lacking the gonococcal -35 but containing the gonococcal -10 region). The promoter sequences (-35 and -10 regions of the gonococcal porin promoter) are shown in the DNA and amino acid sequence of the PIB gene of MS11 in Carbonetti et ai. (1988)).

Several attempts to reassemble the por-2 gene from strain MS11 (Por-2_(B)) with its promoter intact were previously unsuccessful (Carbonetti et al. 1988) despite using E. coli M16 and DH5a MCR and S. typhimurium X3730 as recipients in the transformation. In order to test whether the attempted por-2 ligations contained an assembled por-2 gene, a por-2 ligation reaction containing the 4.7-kb EcoRI to Kpnl fragment of pUNCH 22 and the 469 bp EcoRI to Kpnl fragment of pUNCH 23 was subjected to polymerase chain reaction (PCR) using primers which were predicted to yield a 1 kb product from the assembled gene (Fig. 3A). Agarose gel analysis of the reaction reproducibly yielded a 1 kb band as the major product of PCR from the ligation as well as the control PCR amplification from the MS11 chromosome (Fig. 3B, lanes 2 and 4, respectively). The PCR fragments amplified from the ligation reaction (Fig. 3B, lane 2) and from the MS11 chromosome (Fig. 3B, lane 4) were cloned and both clones (pUNCH 540 and 541, respectively) expressed full length Por-2_(B,_Msιi) in E. coli (Fig. 3C, lanes 3 and 4, respectively.) Since pUNCH 540 and 541 both lack the gonococcal -35 but contain the gonococcal -10 promoter sequences, expression in E. coli either did not require the -35 sequence or there was transcription originating from the upstream T7 promoter in the vector in the absence of induction. These data suggest that the failure to obtain Por-2(_B,_Msn)-expressing transformants with constructs containing the entire promoter (-35 and -10) region of por-2 was due to a problem other than proper reassembly of the gene. Moreover, the results demonstrate that Por-2_(B, _Msn) was stably, although weakly, expressed in E. coli.

or-5^ CLASS 9 HYBRID

A gonococcus expressing an intertypic hybrid porin (termed the class 9 hybrid) which contains epitopes for both Por-1_(A) and Por-1_(B) monoclonal antibodies has been constructed (Carbonetti etal. 1988). The class 9 hybrid porin contains the N and C terminal domains of Por-1_{(Aι FA19)} and a central domain of Por-2_(B, _Msn) (Carbonetti et al. 1988). Cloning of the gene (por-5) for the hybrid porin (Por-5_(A/_B. _FA6 34) from the gonococcal strain FA6434 without its own promoter in pGEM-2 resulted in plasmid pUNCH 50 (Table 1). Since the majority of the class 9 protein was Por-1_(A, _FAι₉), except for a central Por-2(_B,_Msn) surface-exposed region, it was possible to constitutively express por-5 in E. coli without toxicity. Cloning por-5 behind an intact port promoter was successful and resulted in pUNCH 535 (Fig. 4), which expressed epitopes for Por-1_(A,_FA19) and Por-2_{(Bt M}sιi) (Fig.2).

Thus, reassembly and expression in full length of Por-1_{(A> FA19}), Por-2(_B,_Msn) as well as the Por-5_(AB, _{FA6 3} ) hybrid was achieved, although expression of Por-2_(B, _MSn₎ required alteration of the promoter region to avoid toxicity.

CLONING AND EXPRESSION OF PorIN SALMONELLA

a). Chromosomal expression:

Integration of the cloned gonococcal port gene into the chromosome of S. typhimurium strain BRD 207 resulted in strain FX501 (Fig. 5). Southern blotting analysis of FX501 and FX502 (control Salmonella strain with the vector pDEL-1 integrated into the chromosome) using probes for port and aroC indicated that plasmids pUNCH 527 and pDEL-1 both integrated upstream from aroC by a single crossover event. Expression of Por-1 _(A, _FA19> was modest when a single copy of port was present in the Salmonella chromosome compared to expression from multicopy expression plasmids (Fig. 6).

b). Plasmid expression:

Por was introduced into a plasmid expression system in Salmonella using a stable expression system developed by Curtiss and colleages (Galan et al. 1990, Nakayama et al. 1988). The system relies on the complementation of a defective aspartate B-semialdehyde dehydrogenase (asd) chromosomal gene with a plasmid encoded asd gene (Nakayama etal. 1988; Galan etal. 1990). Asd is an enzyme involved in the synthesis of diaminopimelic acid (DAP), an essential component of the cell wall of bacteria that is absent from mammalian cells. Bacteria mutagenized in the chromosomal locus asd that lose their plasmid are unable to synthesize DAP and cannot grow in vivo unless a wild-type asd gene is provided in trans (Galan et al. 1990, Nakayama et al. 1988). Thus asα plasmids remain stable in vitro and in vivo in the absence of antibiotic pressure in asαf hosts (Table I). A BglH fragment containing the asd gene from pYA292 was made blunt by a fill in reaction with Klenow fragment and cloned into the Seal sites of pUNCH30 and pUNCH535 to form pUNCH537 and pUNCH536, respectively. Details of the subcloning of the port gene and por-5 gene are shown in Fig. 7, and the expression of Por in asd hosts is shown in Fig. 8.

Substantial amounts of both Por-1_{(A> FA}ι₉) and Por-5_(As, _FA6434) are made in this system, and the amount of Por is estimated to be one-fifth the amount typically made by the gonococcus. Since it is believed that the gonococcus contains 100,000 to 300,000 copies of Por per cell (Joiner et al. 1985), the recombinant Salmonella of the present invention makes 20,000 to 60,000 copies of Por per cell. Por protein appeared to be stable in Salmonella without detectable proteolysis (Figure 8). Dot blot analysis of whole cells using anti-Por monoclonal antibodies indicated that Por was surface-exposed on all tested Salmonella and E. coli containing Por plasmids except S. typhimurium strain X4072. Lack of detection of Por protein in this strain despite its presence may be due to the shielding of Por by the smooth LPS present in this strain, but absent in others. TOXICITY TESTING

Toxicicity of Por-1_(A, _FAι₉₎ and Por-5(/v_B, _FA6₄3 ) was tested in S. typhimurium strain X4072 (containing pUNCH 537, 536 and 539) by performing growth curves in Luria- Bertani broth. There were no apparent differences in growth rates between strains expressing Por compared to the control containing the vector alone.

pUNCH 30 was sequenced to determine if a mutation accounted for the unexpected lack of toxicity to E. coli and Salmonella hosts. No difference between the originally reported (Carbonetti and Sparling 1987, corrected by Elkins et al. 1992) port sequence and the present invention's port sequence was found.

INDUCTION OF ANTIBODIES TO BOTH SEROGROUPS OF GONOCOCCI BY IMMUNIZATION WITH A GONOCOCCAL HYBRID PORIN

The following example shows the ability of various adjuvants to induce an immune response to both PIA and PIB sequences upon immunization of rabbits with rPor-5.

Experimental procedures

Recombinant Por-5 was purified under relatively non-denaturing conditions. New Zealand white rabbits were immunized four times every two weeks with 100ug of purified Por-5 Mixed with the following adjuvants: none, Complete

Freunds/lncomplete Freunds, Keyhole limpet hemocyanin, Ribi, Bacilie Calmet- Guerin, or Aluminum Hydroxide. Sera were obtained prior to immunization (pre- immune) and one week after the fourth immunization (post-immune). Immunological assays were performed as described in Harlow and Lane (Harlow and Lane, 1988). Results

ELISA titers indicated that all rabbits had detectable anti-Por activity at a 1 :5000 dilution (Fig. 11) using rPor-1 or rPor-2 as the coating antigen. Por-1 domains from the Por-5 hybrid were consistently more immunogenic than the Por-2 (central) domain in the Por-5 hybrid. In order to assess the degree of binding to surface exposed epitopes on whole gonococci, dot blots were performed using immobilized gonococci. As show in Figure 12 all post immune sera bound strain FA19 (Por-1) gonococci, but most failed to bind MS11 (Por-2). However, when the immune response to MS11 by dot blot was compared between each of the adjuvant groups, the group which received no adjuvant responded better than the other 5 adjuvant groups. Four of five rabbits which received no adjuvant bound whole cells of MS11 well.

In order to confirm that the immune response against whole gonococci in the dot blot was specific to Por, modified Western blots were performed on sera from selected rabbits which received no adjuvant (Fig. 13). All post-immune sera recognized Por-1 , Por-2 and Por-5, but some pre and/or post-immune sera recognized gonococcal antigens other than Por. For example, reactivity against E. coli OmpA was seen in several rabbit sera by western blot (Fig 13). OmpA has been shown to be partly homologous to gonococcal Rmp (Pill) (Gotschlich et al., 1987) and OmpA-like proteins are common in Gram negative bacteria. The regions of homology between the two proteins lies in their C-terminal domains which are not believed to be surface-exposed in E. coli and, by analogy, in gonococci. Rmp is immunogenic and can induce antibodies which block bactericidal killing of gonococci by anti-por or other antibodies (Rice et al., 1986; Virji and Heckels, 1988). Although there often were strong reactions with E. coli OmpA in both pre and post immune sera, there was little or no reaction against Rmp by Western blot (Fig. 13) or by radioimmunoprecipitation. This suggests that non-conserved regions of OmpA are more immunogenic than the conserved regions.

TABLE II Summary of Anti-Por-5 Rabbit Sera Binding to Gonococci by Dot Blot

Adjuvant # Rabbit antisera binding gonococcal strain

FA19 (Por-1) FA6434 (Por-5) MS11 (Por-2)

NONE 5/5 5/5 4/5

BCG 5/5 5/5 2/5

CFA 5/5 5/5 1/5

ALUM 5/5 5/5 0/5

KLH 5/5 5/5 1/5

RIBI 5/5 5/5 0/5

Notes to Table II:

Each adjuvant group contained five rabbits. Binding in post immune serum was compared to pre-immune and only those reactions which were judged strong were scored as positive.

Of the Por-1 and Por-2 sequences found in the Por-5 hybrid protein of this example, Por-1 (N- and C- terminal sequences) were more immunogenic than Por-2 (central region sequences) regardless of the adjuvant used or method of evaluation (ELISA or dot blot). However, the group that received Por-5 without adjuvant responded relatively well to Por-2 surface-exposed sequences as compared to all other adjuvant groups.

Supplemental Enablement

The invention as claimed is enabled in accordance with the specification and readily available references and starting materials. Nevertheless, the following cell lines are available in the American Type Culture Collection, Rockville, Maryland in order to facilitate the making and using of the invention:

The following E. coH strains carrying the listed plasmids have been deposited with the Agricultural Research Culture Collection (NRRL), Peoria, IL or the American Type Culture Collection, (ATCC), Rockville, MD and have been assigned the accession numbers indicated.

E. coli Strain Plasmid Accession No.

BL21 (DE3) pUNC7 NRRL B-18263

BL21 (DE3) pUNCH25 ATCC 67775

N. αonorrhoeae Strain

FA6248 ATCC 53808

FA19 ATCC 55073

Each deposited strain is only intended as a single illustration of one aspect of the invention, and any cell lines which are functionally equivalent are within the scope of the invention.

These deposits were made under the provisions of the Budapest Treaty on the

Intemational Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure and the regulations thereunder (Budapest Treaty). This assures maintenance of a viable culture for 30 years from date of deposit. The organisms will be made available by ATCC under the terms of the Budapest Treaty, and subject to an agreement between Applicants and ATCC, which assures unrestricted availability upon issuance of the pertinent U.S. patent. Availability of the deposited strains is not ^• to be construed as a license to practice the invention in contravention of the rights granted under the authority of any government in accordance with its patent laws.

REFERENCES

Blake, M.S., Wetzler, L.M., Gotschlich, E.C., and Rice, P.A. (1989). Developing a gonococcal protein I vaccine. Adv Exp Med Biol. 251(315): 315-27.

Carbonetti, N.C., V.S. Simnad, C. Elkins.a nd P.F. Sparling. 1990 Construction of isogenic gonococci with variable porin structures: effect on susceptibility to human serum and antibiotics. Molec. Microbiol. 4:1009-10018.

Carbonetti, N.H., Simnad, V.I., Seifert, H.S., So, M., and Sparling, P.F. (1988). Genetics of protein I of Neisseria gonorrhoeae: construction of hybrid porins. Proc Natl Acad Sci U S A. 85(18): 6841-6845.

Carbonetti, N.H., and Sparling, P.F. (1987). Molecular cloning and characterization of the structural gene for protein I, the major outer membrane protein of Neisseria gonorrhoeae. Proc Natl Acad Sci U S A. 84(24): 9084-9088.

Curtiss III, R., Goldschmidt, R.M., Fletchall, N.B., and Kelly, S.M. (1988). Avirulent Salmonella typhimurium cya^'crp^* oral vaccine strains expressing a streptococcal colonization and virulence antigen. Vaccine. 6: 155-160.

Curtiss III, R., Kelly, S.M., Gulig, P.A., Gentry-Weeks, C.R., and Galan, J.E. (1988). Avirulent Salmonellae expressing virulence antigens from other pathogens for use as orally administered vaccines. Virulence mechanisms of bacterial pathogens. Washington, Amer Soc for Microbiol. 311 -328.

Curtiss, R.I., and Kelly, S.M. (1987). Salmonella typhimurium deletion mutants lacking adenylate cyclase and cyclic AMP receptor protein are avirulent and immunogenic. Infect Immun. 55: 3055-3043. Dougan, G., Chatfield, S., Picard, D., Bester, J., O'Callaghan, D., and Maskell, D. (1988). Construction and characterization of vaccine strains of Salmonella harboring mutations in two different aro genes. I Infect Dis. 158: 1329-1335.

Dougan, G., Hormaeche, C.E., and Maskell, D.J. (1987). Live oral Salmonella vaccines: potential use of attenuated strains as carriers of heterologous antigens to the immune system. Parasite Immunology. 9: 151-160.

Dougan, G., Smith, L., and Heffron, F. (1989). Live bacterial vaccines and their applications as carriers for foreign antigens. Vaccine Biotechnology. San Diego, Academic Press. 1st, ed. 271-300.

Elkins, C, and Sparling, P.F. (1992). Immunobiology of Neisseria gonorrhoeae. Advances in Host Defense Mechanisms: Sexually Transmitted Diseases. New York, Raven Press. 113-139.

Galan, J.E., Nakayama, K., and III, R.C. (1990). Cloning and characterization of the asd gene of Salmonella typhimurium: use in stable maintenance of recombinant plasmids in Salmonella vaccine strains. Gene. 94: 29-35.

Gotschlich, E.C. (1984). Development of a gonorrhoea vaccine: prospects, strategies and tactics. Bull World Health Organ. 62(5): 671-80.

Gotschlich, E.C, Seiff, M.E., Blake, M.S., and Koomey, M. (1987). Porin protein of Neisseria gonorrhoeae: cloning and gene structure. Proc Natl Acad Sci U S A. 84(22): 8135-8139. Gulig, P.A., and III, R.C. (1987). Plasmid-associated virulence of Salmonella typhimurium. Infect and Immun. 55: 2891-2901.

Harlow, E., and Lane, D. (1988) Antibodies: A laboratory manual. Cold Spring Harbor: Cold Spring Harbor Laboratory.

Heckels, J.E., Virji, M., and Tinsley, CR. (1990). Vaccination against gonorrhoea: the potential protective effect of immunization with a synthetic peptide containing a conserved epitope of gonococcal outer membrane protein IB. Vaccine. 8(3): 225-30.

Hoiseth, S.K., and Stocker, B.A.D. (1981). Aromatic-dependent Salmonella typhimurium are non-virulent and effective as live vaccine. Nature. 291 : 238-239.

Holmgren, J. (1991). Mucosal immunity and vaccination. Fems Microbiol Immunol. 4(1): 1-9.

Holmgren, J., Czerkinsky, C, Lycke, N., and Svennerholm, A.M. (1992). Mucosal immunity: implications for vaccine development. Immunobiology. 184(2-3): 157-79.

Johnston, K.H., Holmes, K.K., and Gotschlich, E.C. (1976). The serological classification of Neisseria gonorrhoeae. I. Isolation of the outer membrane complex responsible for the serotypic specificity. J Exp Med. 143: 741-758.

Joiner, K.A., Warren, K.A., Tam, M., and Frank, M.M. (1985). Monoclonal antibodies directed against gonococcal protein I vary in bactericidal activity. J Immunol. 134(5): 3411-3419. Judd, R.C. (1989). Protein I: structure, function, and genetics. Clin Microbiol Rev. 2: 41-48.

Killar, L.M., and Eisenstein, T.K. (1985). Immunity to Salmonella typhimurium infection in C3H/HeJ and C3H/HeNCrlBR mice: Studies with an aromatic-dependent live S. typhimurium strain as a vaccine. Infect Immun. 47: 605-612.

Knapp, J.S., Tam, M.R., Nowinski, R.C, Holmes, K.K., and Sandstrom, E.G. (1984). Serological classification of Neisseria gonorrhoeae with use of monoclonal antibodies to gonococcal outer membrane protein I. J Infect Dis. 150(1): 44-8.

Kohl, P.K., Olsen, D.A., and Buchanan, T.M. (1989). Monoclonal antibodies to protein I for serotyping of Neisseria gonorrhoeae: correlation of serotype with bactericidal activity. Zentralbl Bakterioi Mikrobiol Hyg A. 270(4): 517-26.

McDade, R.L., Jr., and Johnston, K.H. (1980). Characterization of serologically dominant outer membrane proteins of Neisseria gonorrhoeae. J Bacteriol. 141 : 1183- 1191.

McGhee, J.R., Mestecky, J., Dertzbaugh, M.T., Eldridge, J.H., Hirasawa, M., and Kiyono, H. (1992). The mucosal immune system: from fundamental concepts to vaccine development. Vaccine. 10(2): 75-88.

Nakayama, K., Kelly, S.M., and III, R.C (1988). Construction of an asd⁺ expression- cloning vector: stable maintenance and high level expression of cloned genes in a Salmonella vaccine strain. Biotechnology. 6: 693-697.

Neidhardt, F.C, Editor-in-Chief, Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology, Vol. 1 , American Society for Microbiology, Washington, D.C

O'Callaghan, D., Maskell, D., Liew, F.Y., Easmon, C.S.F., and Dougan, G. (1988). Characterization of aromatic- and purine-dependent Salmonella typhimurium: Attenuation, persistence and ability to induce protective immunity in Balb/C mice. Infect Immun. 56: 419-423.

Plummer, F.A., Simonsen, J.N., Chubb, H., Slaney, L, Kimata, J., Bosire, M., Ndinya, A.J., and Ngugi, E.N. (1989). Epidemiologic evidence for the development of serovar- specific immunity after gonococcal infection. J Clin Invest. 83(5): 1472-1476.

Rice, P.A., Vayo, H.E. Tam, M.R. and Blake M.S. (1986) Immunoglobulin G antibodies directed against protein III block killing of serum- resistant Neisseria gonorrhoeae by immune serum. J. Exp Med 164: 1735-48.

Strugnell, R.A., Maskell, D., Fairweather, N., Picard, D., Cockayne, A., Penn, C, and Dougan, G. (1990). Stable expression of foreign antigens from the chromosome of Salmonella typhimurium vaccine strains. Gene. 88: 57-63.

Tackett, CO., Hone, D.M., Curtiss III, R., Kelly, S.M., Losonsky, G., Guers, L., Harris, A.M., Edelman, R., and Levine, M.M. (1992). Comparison of the safety and immunogenicity of aroC- and cya- crp- Salmonella typhi strains in adult volunteers. Infect and Immun. 60: 536-541.

Tam, M.R., Buchanan, T.M., Sandstrom, E.G., Holmes, K.K., Knapp, J.S., Siadak, A.W., and Nowinski, R.C. (1982). Serological classification of Neisseria gonorrhoeae with monoclonal antibodies, infect Immun. 36: 1042-1053. Tarkka, E., Muotiala, A., Karvonen, M., Saukkonen-Laitinen, K., and Sarvas, M. (1988). Antibody production to a meningococcal outer membrane protein cloned into live Salmonella typhimurium aroA vaccine strain. Microb Path. 6: 327-335.

Virji, M., Fletcher, J.N., Zak, K., and Heckels, J.E. (1987). The potential protective effect of monoclonal antibodies to gonococcal outer membrane protein IA. J Gen Microbiol. 133: 2639-2646.

Virji, M. and Heckels, J.E., (1988) Non-bactericidal antibodies against Neisseria gonorrhoeae: evaluation of their blocking effect on bactericidal antibodies directed against outer membrane antigens. J. Gen Microbiol 134: 2703-2711.

Virji, M., Zak, K., and Heckels, J.E. (1986). Monoclonal antibodies to gonococcal outer membrane protein IB: use in investigation of the potential protective effect of antibodies directed against conserved and type-specific epitopes. J Gen Microbiol. 132: 1621-1629.

Claims

1. A recombinant Salmonella cell that expresses a gonococcal porin protein or a fragment thereof.

2. The recombinant Salmonella cell of claim 1 , wherein the cell contains a vector that expresses a gonococcal porin protein.

3. The cell of claim 2, wherein the vector is a plasmid.

4. A recombinant Salmonella cell of claim 1 , wherein the porin protein is full- length.

5. A Salmonella cell of claim 4, wherein the gonococcal porin protein is Por-1_(A).

6. A Salmonella cell of claim 4, wherein the gonococcal porin protein is Por-1_(B).

7. A Salmonella cell of claim 4, wherein the gonococcal porin protein is a Por- 5(Λ/β) hybrid.

8. A Salmonella cell of claim 7, wherein the Por-5_{(A B)} hybrid is in plasmid PUNCH50.

9. A recombinant Salmonella cell of claim 1 , wherein the porin protein is a fragment.

10. A recombinant Salmonella cell of claim 9, wherein the fragment is a fragment of Por-1_(A).

11. A recombinant Salmonella cell of claim 10, wherein the Por-1 _(A) fragment comprises any DNA sequence selected from the Por-1 _(A) sequences of Figure 9.

12. A recombinant Salmonella cell of claim 9, wherein the fragment is a fragment of Por-1(_B).

13. A recombinant Salmonella cell of claim 10, wherein the Por-2_(B) fragment comprises any DNA sequence selected from the Por-2_(B) sequences of Figure 9.

14. A Salmonella cell of claim 9, wherein the fragment is a fragment of a Por-5_(A/B) hybrid.

15. A Salmonella cell of claim 1 , wherein the Salmonella cell is Salmonella typhimurium.

16. A plasmid capable of expressing a gonococcal porin protein in a Salmonella cell comprising DNA encoding such protein and suitable regulatory elements arranged within the plasmid so as to permit expression of the protein in the

Salmonella cell.

17. A method for expressing a gonococcal porin protein in a Salmonella cell comprising inserting the plasmid of claim 16 into the Salmonella cell under suitable conditions for expressing the protein, whereby the protein is expressed.

18. A method of claim 17, wherein the plasmid is pUNCH50.

19. A plasmid that expresses an antigen in a Salmonella, E. coli, or N. gonorrhoeae host cell comprising DNA encoding such antigen and suitable regulatory elements, including a gonococcal porin promoter, arranged within the plasmid so as to permit expression of the antigen in the host cell.

20. A recombinant Salmonella or E. coli cell containing the plasmid of claim 19.

21. A vaccine composition comprising the Salmonella cell of claim 1 and a pharmaceutically acceptable carrier.

22. A vaccine composition according to claim 21 wherein the composition also comprises a pharmaceutically acceptable adjuvant.

23. The recombinant Salmonella cell of claim 1 and a pharmaceutically acceptable adjuvant.

24. A method for preventing gonorrheal infection by administering the vaccine composition of claim 21 or 22.

25. A method for treating gonorrheal infection by administering the vaccine composition of claim 21 or 22.