Note: Descriptions are shown in the official language in which they were submitted.
WO 95/04133 21 ~ 7 ~ ~ 8 PCT/US94108586
PRODUC~ION OF GONORRHEAL Pl PROTEINS AND VACCINES IN E. COLI AND
SALMONELLA
.
BACKGROUND OF THE INVENTION
This a~.plicalio" is a continuation-in-part of Serial Number 08/100,588 filed July
30, 1993, which is incorporated herein by reference.
Neisseria gonorrhoeae. the gonococcus, infects only humans and produces
the disease gonorrhea. Gonorrhea remains the most frequently reported infectiousdisease in the United States and is an important cause of morbidity and infel lilily in
15 both developed and undeveloped countries.
No gonococcal vaccine is currently available. Of the several ca~1didales for a
gonococcal vaccine, the gonococcal outer membrane porin protein, referred to in the
literature as Pl or Por, is a prime candidate (Gotschlich 1984; Blak~ et a/. 1989; Elkins
20 and Sparling 1992). Pl is an anliyenically stable, surface-exposed protein, and
antibodies against Pl are potentially protet,li-/e (Heckels et al.1990; Joiner et al.1985;
Kohl et al.1989; Virji et al.1987; Virji et al.1986).
Pl is typical of most gram negative porins in structure, and exists as a non-
25 covalent trimer complex while exhibiting exlensive beta sheet structure in the nativestate. When denatured by boiling in sodium dodecyl sulfate solution and analyzed by
polyacrylamide gel electrophoresis, however, Pl dissoci~tes from a trimer
--1--
WO 95/04133 PCT/US94/08586
2167938 ~
configuration into monomers and migrates with an estimated molecular mass of 32-39kDa (Johnston et a/.1976; McDade and Johnston 1980).
Pl is found in two major immunochemical classes termed serogroups PIA and
PIB (Tam et a/. 1982; Knapp et a/.1984). PIA and PIB are the products of
variants/alleles of the same gene (Ca, ~onelLi et al., 1988; Carbonetti and Sparling,
1987). All gonococci contain Pl of either serogroup, but not both, and no Pl minus
mutants are known to sxist. Pl is stable and does not u "dergo 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., Intemational 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 res~,onse. Studies have shown that locally produced antibodies o
the sec,~tory IgA isotype are among the most important protective factors at mucosal
2 0 surfaces (McGhee et al. 1992). ~peci~ ed Iymphoid tissues, termed bronchus-
associatecl and gut-associated Iymphoid tissues (BALT and GALT), are sites of
induction in the development of mucosal responses (see Holmgren 1991; Holmgren
et al. 1992; and McGhee et al. 1992 for reviews).
Several antigen delivery systems have been developed to induce a response
at mucosal surfaces. For example, attenuated Salmonella strains can colonize theGALT and stimulate a generalized protective immune response to Salmonella. One
system employs live, attenuated Salmonella typhimurium to express and deliver
WO 95/04133 2 ~ 6 7 9 3 ~ PCT/US94/08586
foreign antigens to the GALT. (Curtiss lll 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
5 genes in animal models, human strains of Salmonella with ~.otenlial for human clinical
trials (S. typhi) are also being tested (Tacket et a/.1992).
Stimulation of GALT by attenuated, r~co"lL.. ,ant Salmonella requires at least
transient growth of the Salmon~lla host strain in the gut (Dougan et al.198~), and
10 therefore ex~.r~ssion of the foreign protein should not be rapidly lethal to the host.
Previous reports (Carbonetti and Sparling 1987; Gotschlich et a/. 1987; Carbonetti et
al.1988) have indicated that e~c~.ression of some gonococcal proteins, including Pl,
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.
15 The expression of gonococc~l porin proteins in Salmonella or of intact gonococcal
porin proteins in E. coli has not been r~po, led before the present invention.
Accordingly, there is a need for the construction of recombinant Salmonella or
E. coli cells that are able to express go,1ococcal porin ,c r,te;. ,s. There particularly is a
2 0 need to produce vaccine compositions using such recornbinant Salmonella or E. coli
cells to prevent or treat gonorrheal infection.
SUMMARY OF INVENTION
2 5 The invention provides a recombinant Salmonella cell that ~ .r~sses a
gonococcal porin protein or a fragment thereof.
: ! ',
WO 95/04133 216 7 9 3 8 PCT/US94/08586
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 cle."e"ls arranged within the plasmid so as to permit ex~r~ssion of the
gonococcal porin protein in the Salmonella cell.
The invention also provides a plasmid that expresses an anli~en in a
Salmonella or E. coli host cell co-n~.risi"g DNA encoding such antigen and suitable
regulatory elements, including a gonococcal porin promoter, arranged within the
plasmid so as to permit ex~,r~ssi~l I of the anli~el, in the host cell.
The invention also provides a vaccine composition comprising the recombinant
Salmonella cell containing a plasmid for expression of a gonococc~l porin protein and
a pharmaceutically acceptable carrier, as well as methods for treating or preventing
gonorrheal infection by ad~"i,1isl~ri"9 the vaccine composition.
DESCRIPTION OF THE FIGURES
Figure 1. Re~sembly of a compiete por-1 gene from gonococcal strain FA19 in E.
2(~ coli. The 720 bp Smal to Taql fragment of pUNCH 11 contains the complete por-1
promoter (500 bp of sequence upstream rom the start codon) and coding sequence
for 58 amino acids of the protein. The 820 bp Taql to Sau3AI of pUNCH 15 contains
th~ remaining coding sequence and 12 bp of downstream por-1 sequence. These
two frayl"enls were ligated between Hincll and BamHI sites of pGEM-2 in the E. coli
25 host M16 to form pUNCH 30. Open white bars indicate ~dj~cent non-coding DNA
sequences; small open arrows with the letter p ~ ^ent indicate the promoter region;
hatched arrows indicate the position and orientation of po~, Amp in~ tes the position
WO 95/04L33 216 7 ~ ~ 8 PCT/US94/08586
of the beta-lactamase gene in all figures. Re:jlri~;tion sites in parentheses indicate
th2t the site was lost during construction.
Figure 2. Constitutive expression of recombinant Por-1 (A, FA1~) and Por-5(AB, FA6434)
5 from a native promotor in E. coli. Whole cell Iysates of gonococci (2.5 ug of protein)
or E. coli (10 ug of protein) were subjected to Western blotting using monoclonal
antibodies specific for P1A (panel A) or PIB (panel B) epitopes. Lanes: 1-3,
gonococc~l strains FA19 (Por-1 (A)), FA6434 (Por-5(A~B)), and MS11 (Por-2(B)),
r~spe~i-/ely; 4-6, E. coli strain M16 containing plasmids pGEM-2 (no insert), pUNCH
30 (por-1), pUNCH 535 (por-5), respectively; and 7-9, E. coli strain DH5a MCR
containing plasmids pGEM-2 (no insert), pUNCH 30 (por-1), pUNCH 535 (por-5)
respectively. The weak band in panel B, lane 5 re,l~resenls spillover from lane 6 and
was not seen in other similar blots.
15 Figure 3. Re~-ssel"bly of ths por-2 gene from gonococcal strain MS11 in E coli.
(A): Re~-ssembly 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) andDNA sequence encoding the first 105 amino acids of Por-2(B Ms1l). Plasmid puNCH
22 contains the remaining 3' coding sequences of por-2 as well as 1.0 kb of
2 0 downstream DNA. The checkered pattern in the bars and arrows indicates por-2.
The cloning ~lrateyy was to double-digest pUNCH 22 and pUNCH 23 with Kpnl and
EcoRI, gel purify each relevant fragment and ligate them together. Repeated
aller"pls to reassemble the complete por-2 gene were unsucces~rul in that very few
l,dr,s~ormants were obtained and none ex~.ressed serogroup PIB epitopes or
2 5 contained appro~,l iala sized plasmids. To verifv that the proper ligation product was
present in the above ligation reaction, 0.1 ul of the lijdlioll mixture was subjected to
PCR using primers which anneal l"~st,aar,~ and dov;"~ am from por-2. The 5'
oligonucleotide (5'-GGCGMTTCCGGCCTGCTTAAAI I I C I IA-3')contains an
wo 95/04133 216 7 ~ 3 8 PCT/US94/08586
engineered EcoRI cloning site and anneals to the sequence from 44 to 63 bp of the
published por-2 sequence. The 3 oligonucleotide (6 -
GCGMGCTTATTAGAA I I I GTGGCGCAGA-3') contains an engineered Hindlll site
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 a,-"~ ic-alion were: Denature for 1 min at 94C; anneal for 30 s
at 42C; extend for 2 min at 72C for a total of 28 cycles; and axtend 5 min at 72C for
the last cycle. (B): Analysis of PCR products by 1% agarose gel electrophoresis.Lanes: 1 1 kb ladder molQcu'ar weight standard (BRL); 2 PCR ampliricalion of por-2
ligation reaction including T4 DNA ligase; 3 PCR ampliricalion of mock por ligation
reaction without T4 DNA ligase; 4 PCR a",plificalion of MS11 chromosome; 5 mock
PCR reaction lacking te"")lale DNA. The amplified products in lanes 1 and 3 frompanel (B) were resl,ic~ed with EcoRI and Hindlll, ligated to similarly cut and gel-
purified pGEM3Zf(-) vector. E, coli strain BL21 DE3 [pLysS~ was l,~ns~or")ed, and
greater than one thousand ampicillin-resistant colonies were obtained. Ampicillin
resistant colonies wers screened for Por production without IPTG induction by colony
lift using Mabs and the majority ex~,ressed serogroup PIB epitopes. (C): Expression
of recombinant Por-2 by E colitransformants. Whole cell Iysates of gonococci (2.5
ug of protein) or E. colitransformants (10 ug of protein) which bound anti-PlB mAb in
the colony lift were electrophoresed on a 0.1% SDS-12% polyac~lamide 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 wcre not
induced with IPTG.
WO 95/04133 216 7 9 3 g PCT/US94/08586
. .
Figure 4. Construction of a plasmid containing a hybrid porgene (por-5from strain
FA6434) behin~ an intact por-1 promoter. Plasmids pUNCH 30 (Fig. 1) and pUNCH
50 were rest,icted 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 por-l coding
sequence and the checkered region inrlic~tes por-2 coding sequence.
Figure 5. Integration of FA19 por-l into the Salmonella chromosome using
chromosoma HllleylaliGl) vector pDEL-1. rlas",icl pDEL-1 contains 4.1 kb of S
tJ/phimurium aroCflanking 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 por-1 into pDEL-1 was accoinplisl1ed by ligation of the gel-
purified Hincll to EcoRI fragment of pUNCH 30 (containing the prol"o~er and
co",~l~lete coding sequence of por-l) into the gel-purified Smal to EcoRI large
fragment of vector pDEL-1, and transformation into both E. coliM16 and DH5aMCR.
Similar transformants (based on re~l.iclion analysis) were obtained from both strains
and each expressed e~ o~Jes for Por (Figure 6). A representative M16 E coli
transforrnant was chosen and its plasmid was termed pUNCH 527. Plasl"icl pUNCH
527 DNA was tra"s~o""ed into S. ~phimurium BRD 835 (r~m') in order to methylate
2 ~ the DNA (not shown in the figure). Methylated plasmid pUNCH 527 DNA was then
l,a"sformed into S. ~yphimurium poM strain BRD 207 with selection for ampicillin.
Since ColE1 replicons, such as pUNCH 527 and pDEL-1 cannot replic~le 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
2 5 obtained, termed FX501 expressed Por-1 (A, FAl9) weakly whereas control
transformant FX502 obtained by transformation with pDEL-1 does not express Por-
1 (A, FAl9) (Figur~ 6).
WO 95/04133 PCT/US94/08586
21~7~38
Figure 6. Comparison of chromosomal and plasmid expression of Por in S.
typhimurium. After el~c~, ,pl-or~sis on a 0.1% SDS-12% polyacrylamide gel, wholecell lysates of gonococci (2.5 ug of protein), E. con M16 (10 ug of prot~in) or S.
~phimurium (10 ug of protein) wers subjected to Western blotting using a mAb
specific for a PIA ~pilope. Lanes: 1, gonococcal strains FA19 (Por-1 (A)); 2, MS11
(por-2(B)); 3-4, E colistrain M16 carrying pDEL-1 or pUNCH 527; 5-6, S. typhimurium
BRD 835 car~ing pDEL-1 or pUNCH 527; 7, S. ~phimurium FX501 (pUNCH 527
transfonnant of BRD 207): FX502 (pDEL-1 transformant of BRD 207).
Figure 7. Construction of plasmids suitable for use in asdmutant S. typhimurium
vaccine hosts. A 1.75 kb Bgl ll fragment containing a Salmonella asdgene was
recovered from pYA292, blunted by filling in the ends with Klenow, and ligated with
Sca l-resl,icted plasmids pUNCH 30 (por-1), pUNCH 535 (por-5) and pGEM-2 (no
insert). Each ligation was transformed into E. coliX6212 asd. Transror",a"ls were
termed pUNCH 537, pUNCH 536, and pUNCH 539, respectively. These plasmids
were initially moved into S. ~phimurium X3730 (r~m~) and then into mouse vaccinestrain X4072. The hatched regions indicate por-1 coding sequence, the checkered
region indicates por-2 coding sequence, and the stippled bar indicates asd sequence.
Figure 8. Plasmid ex~,ression of porfrorn asd E coa and S. ~phimurium hosts.
Whole cell Iysates 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) e,uilopes. Lanes: 1-3, gonococc~l strains FA19 (Por-1(A)),
FA6434 (Por-5(A~B)), and MS11 (Por-2(B)), respectively; 4-6, E coli X6212; 7-9, S.
typhimurium X3730,10-12, S. yphimurium X4072. E coli and S. typhimurium strains
contained plasmids pUNCH 539 (control plasmid, no po~, pUNCH 537 (por-1), and
pUNCH 536 (por-5), in their respective lanes.
WO 95/04L~3 21 6 7 9 3 8 PCT/US94/08586
.',, ~.
Figure 9. Figure 9 shows the allliyellic sequences corresponding to fragments 1-6. Each fragment optionally contains ~n N-terminal cysteine residue. The amino acid
numbers correspond to the amino acid residues of Por-1 (A) from N. gonorrhoeae strain
FA19 ffray",enls 1-4) or of Por-2(B) from N. aonorrhoeae strain MS11 (fragments 5
and 6).
Figure 10. Por-5 surface structure. This figure is based on the model of gonococcal
porin proposecl by van der Ley (van der Ley et al., 1991). Putative surface-ex~.osed
regions are drawn above the upper dotted line, membrane spar,l ,i"y 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 gonococc~l strain FA6434. The structure of Por-5 was ~educed 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, 1 00ng/well. The dilution of primary anti-Por-5 was 1:50,000. The
2 o 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 exrosed epitopes of
Por. Gonococcal strains FA19 (Por-1), FA6434 (Por-5) or MS11 (Por-2) were
immobi';~ed 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
WO 95/04133 PCT/US94/08586
21~7~38 ~
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 ~erum and the lower 6 dots show the
post-immune reaction. Mab CGI llrols are 4G5 (anti-PlA, N-terminus) and 5.51 (anti-
PIB, central region). For demon~l,dlion purposes only some of the rabbit data are
5 shown. In the original dot blots, the dis,cli",ination between positive and negative
reactions was more clear cut than in this r~roduction, and these original results are
summarized in Table 1.
Figure 13. Western blotting of anti-Por-5 sera. Whole cell Iysates (approximately 2 X
10 10'CFU) of gonococcal or E coliwere 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 Dm7p; lane 5,
FA6434; lane 6 E. coliMC4100 (parent, wild type for ompA); lane 7, Bre2413 DompA.
Pre, Pre-immune; Post, Post-lmmune. the rabbit antisera was diluted 1:1000 and the
15 secondary reagent was protein A alkaline phosphatase. In panel A the rabbit shown
was immunized with rPor-5 in the dete~gen~ 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 recol"binant Salmonella or E. coli cell containing
DNA for expression of a gonococc~l porin protein (Por). The Por DNA may be
25 i, lleyr~ted 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, co""~risi,lg any DNA that produces a gonoccocal porin protein. Preferably,
--10--
WO 95/04133 2 ~ & ~ ~ 3 8 PCT/USg4/08586
the protein is ex~r~ssed on the surface of the Salmoneila or E. coli cell and the cell
thereby prQrerably is car~hle of stimulating antib~y production. Plefe,dbly, theprotein is expressed at mod6r~le, as compared to high, expression levels. In another
prefer,ed embodimen~, the porin protein expressed by the cell is used to produce5 antibodies to protect against gonorrheal i"fec~ion in a mammal, preferably a human.
.
The gonococc~l porin prGt~i. ,s may be any Por-1, Por-2, or hybrid Por-1/Por-2
proteins. The gonococc~l porin pr~tei"s are preferably proteins or fragments thereof
produced by por-l, por-2, or por-5 genes. por-l is herein defined as a por gene
10 assel"bled from the N. gonorrhoeae strain FA19 (serogroup PIA). por-2is herein
defined as a porgene asser" 'od 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 asclass 9 (Carbonetti, et al.1988) containing epitopes from serogroups PIA and PIB.
15 The Por proteins produced by the por-l, por-2, andpor-5 genes are designated Por-
1 (A). Por-2~s)~ and Por-5(A~B), respectively. The phenotypes of these Por proteins are
designated herein by a sul~scri,ul indicating serogroup and strain, e.g., Por-1 (A, FAl9)~
Por-2(B Ms1l), and Por-5(A~BFA6434). respectively.
N. gonorrhoeae porins from serogroups PIA or PIB or chimeric PIA/PIB porin
protei"s may be expressed by recombinant Salmonella or E. coli cells. Examples of
porin proteins and genes are desc,iL~ed in International Application No.
PCT/US88/04225, filed November 23,1988, incorporated herein by ~eference, and
hybrid porin protei. ,s are descril,ed in PCT/US92/02090 (Fragments of full length
2 5 porin ,c rotei. ,s and genes are suitable)., filed March 13,1992, also incorporated herein
by reference. GOL-1-T hybrid fray",enls.
FRAGMENTS
wo 95/04133 21~ 7 ~ 3 8 PCT/US94/08586
Fragments containing antigenic sequences of Por-1 (A). por-2(B) and Por-5
may be selected on the basis of generally accepted criteria of potential antigenicity
and/or exposure. Such criteria include the hydrophilicity and relative anliye"ic index,
5 as deler",i"ed by surface exposure analysis of Por-1(A) and Por-2(B) protei.,s.
Pro",is~ng candidates are prepared and tested for al,li~el,icily and immunogenicity.
Fragments 1-6, which are shown in Figure 9, are suitable anliyenic sequences.
Frag",en~s 1~ contain amino acid sequences found in Por-l(A) of gonococc~l 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 ~or 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 1~3pli&~lion of the gene. The
recombinant plasmid is inserted into a cG"~pa~ le host cell, i.e. Salmonella or E. coli,
2 0 whereby the gene product is exl ressed.
The recol"~ nant plasmids described herein enable the stable exl,ression in,
without toxicity to, a host cell of the Por proteins. In accordance with the invention,
Por protei. ,s may be produced by inserting the cloned sequence of the Por-1 (A) or Por-
25 2(B) prolei,1s or hybrids thereof into an appropriate cloning/ex,~,ression vector, such asa plasmid, for insertion into E. coli or Salmonella.
--12--
WO 95/04133 2 ~ ~ 7 ~ 3 ~ PCT/US94/08586
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 l.a"scliplion and translation of the inserted gene inserted
into a plasmid or chromosome, the gene must be placed under the control of
reglJ~tory elements from, or co"".alible with, the chosen host cell. Preferably, the
regl ll~tory elements include one or more native gonococc~l 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
conslilutive expression of the desired antigen in E. coli or Salmonella, the antigen
DNA is fused to a gonococcal porin pro",oler and inserted into an appropriate
clo"i, .y/expression vector, such as a plasmid, for insertion into E. coli or Salmonella.
The antigens fused to the gonococcal porin promoters for ex~ ression in E. colilSalmonella, or N. gonorrhoeae may be, for example, gGllOCOCCi tra"~er,in bindingprot~i. ,s such as B transferrin, lactoferrin, heme binding prGtei. ,s 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 ~dj~,ent nucleotides
in a duplex DNA chain; this enzyme may therefore be used to covalently join the
annealed cohesive ends produced by certain re~l,i~ion enzymes. Alternately, DN
ligase can be used to catalyze the formation of phosphodiester bonds between blunt-
ended fray"~enls. Finally, the enzyme terminal deoxynucleotidyl transferase may be
employed to form homopolymeric 3' - single-stranded tails at the ends of fray",e"~s;
2 ~ ~ 7 9 3 8 PCT/US94/08586
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 mol~cules can anneal to
form dimeric circles. Any of these methods or other known methods may be used toligate the gene segment promoter and other regul~tory elements into specific sit~s in
5 the vector. Thus, the gene encoding the Por proteins is ligated into the chosen vector
in a specific relalionship to the vector promoter and control ele",enls, so that the
sequence is in the correct reading frame. The vector employed will typically hava a
marker function, such as ampicillin lesi~ldr,cs or tetracycline r~si;~ldnce, so that
transformed cells can be identified. The vector employed may be any of the known10 cloning or e~.~.r~ssion 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 ex~,resses a foreign protein,
preferdbly 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 e~,~,ressi"g ths foreign porin protein to stimulate an effective immune response
2 0 against gononl ,eal i"~c;tion.
P,efer,ed bacterial strains and plasmids are shown in Table 1 below.
--14--
WO 95/04133 216 7 9 3 8 PCTIUS94/08586
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m ' ~ 'a
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o ~ ~ ~ E ~;
~i S yL ~ æ æ ~ ~ E æ
~ o ~ ~ r ' 5~
C~ ~ Q ~ m ~, ~ Q
~ ~s ~ _ N
s 5
@ " r ,~
- m m t~7 2 ~ ) m ~
_15_
WO 95/04133 ` ~ PCT/US94/08586
2~&7938 -- ~
E ~
D C S
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8 ~ c~s O 2 ~ ~g c c Q
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,0 ~ S ~ ~
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c~ ~
~ ~ ~ oE ~ ~ ~ E ~ ~ ~Q ~Q ~
cn L~ L ~ Q 0
o
o
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--16--
WO 95/04133 216 7 9 3 8 i PCT/US94/08586
~Q
o
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a:~ O E
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c ~ ~ c c c c c c --
Q o C Q U~ Q Q Q Q Q C~
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c~ ~5 Q . ~ ~-- o
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e e o e" ~ ' ' ~ ~ Q
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i~ ~ x C~ 3 -- --~ ~ ~ ~ o
a~ c C~ . -- C~ ; C ~;
~ ~ a) 'Q ~ n . ~ r C~ o Q 5 _
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I~In ~ ~ a~ o _ ~
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-- I I I I I I I
Z Z U~ Z Z Z Z Z Z Z '
Q Q Q Q Q Q Q Q Q Q ~ O
~ ~8
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--17--
WO 95/041~33 . PCT/US94/08586
21g7~38 ~
VACCINES:
Methods are described herein which permit the formulation of vaccine
S compositions. In prefer,ed embodiments, vaccine compositions cGr"~,rise Salmonella
or E. coli producing Por proteins such as Por-1 (A) or Por-2(~3) or hybrid Por(A~B) proteins
and fragments thereof.
10 FORMULATION OF A VACCINE
The Salmonella or E. coli cells containing the porgenes, pr~rerably 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 co"lb .,ed with any of the collllllol,ly used
pharm~celJtic~lly 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, Iymphokines, such as
2 0 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, coatinQs, antibacterial and antifungal agents; isotonic an
absorption delaying agents and the like. The use of such agents for pharmaceutically
active sl~st~snces is known in the art. Except insofar as any conve"lional medium is
25 incompatible with the active ingredient, its use in the therapeutic co"".osition is
conler"plated. Supplemental active ingredients may also be incor~Jorated.
A particularly useful el"l)odi."ent of the present invention is a vaccine in which
the active immunogen is a hybrid Por-l (A~/Por-2(B) protein, the construction of which is
--18--
wo 95/04133 21 6 7 ~ 3 ~ PCT/US94/08586
desc,ibed in Inter"dliG"al Applicalion No. PCT/US88/04225, filed November 23,1988,
or fragments of th~ hybrid Por-1 (A~/Por-2(B) protein, the construction of which is
described in International ApF',c~tion 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 intramuscu~r admini~l,alio". 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 poly~ e~.lide ex~,ressed 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
sl~hst~ntially homologous to a porin protein. The polypeptide may be prepared using
methods known in the art. r,eferdbly, the polypeptide co"".rises 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 pro~ocol described by R.H. Kenneth, "Enzyme-Linked Antibody Assay with
--19--
Wo 95/04133 ~ ~ 7 9 3 8 PCT/US94/08586
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
5 sufficient to bind detect~hle amounts of the antibody. After incuh~ting the plates with
the polypeptide, the plates are blocked with a slJit~ 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 qua"lilate the amount of relevant antibody present in the unknown
10 samples. Following incubation, the sa"~les are probed with goat anti-human lgconjugated to a suit~ enzyme. The presence of anti-polypeptide antibodies in thesample is indicated by the presence of the enzyme.
Por antibody probes:
The porin pl~tei. Is expressed by, and pre~erably 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
b~ polyclonal or monoclonal. The sample may, for example, be a bodily fluid from a
20 mammal, including a human, suspected of being infected with M gonorrhoeae.
Methods are known for detecting poly5.e,ulides with antiho~ies. For example, a
polypeptide may be immobili~ecl on a solid support. Immol.~ lion of the polypeptide
may occur through an immobilized first antibody specitic for the polypeptide. The
25 immobilized first antibody is inclJk~ted with a sample suspe~;ted of containing the
polypeptide. If present, the polypeptide binds to the first antibody.
A second antibody, also specitic for the polypeptide, binds to the immobilized
polypeptide. The second antibody may be labelled by methods known in the art.
--20--
wo 95/04133 X 1 6 7 9 3 8 PCT/USg4/08586
Non-immobi~i~ed materials are washed away, and the presence of immobilized labelindicates the p,esence of the polypeptide. This and other immunoassays are
desclibed by David, et al., in U.S. Patent 4,376,110 assigned to Hybrilecl1, Inc., La
Jolla, California.
The probes clesc,ibed above are labelled in accordance with methods known in
the art. Methods for labelling al~lil)odies have been descril)e-l, for example, by Hunter
and Greenwood in Nature 144, 945 (1962) and by David et al in Bioche",isl,y 13,
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 Me.nkQth and Wahl, Anal. Biochem. (1984) 138:267.
The label may be rA~ c~ctive. Some examples of useful radioactive labels include32p, 125 j, 1311, and 3H. Use of rA~'iozctive labels have been desc,ibed in U.K.2,034,323, U.S. 4,358,535, and U.S. 4,302,204.
Some examples of non-rA~icActive labels include enzymes, chromophors, atoms
and ml~lecl~'~s ~lelecl~hle by electron rl~iCrOSCOpy, and metal ions detect~hle by their
magnetic properties.
Some useful enzymatic labels include enzymes that cause a detectAhlQ change in
a sul)~l,ate. Some useful enzymes and their sul,sl,ates include, for example,
I,or:~erddish ~.eroxidase (pyrogallol and o-phenylenediamine), beta-galactosidase
ffluorescein beta-D-~Al~ctopyranoside), and alkaline phosphatase (5-bromo-4-chloro-
3-indolyl phosphate/nitro blue tetrazolium). The use of enzymatic labels have been
--21--
WO 95/04133 2 ~ 6 7 9 ~ 8 PCT/US94/08586
clescril,ed 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
5 bioluminescent molecules, as well as dyes. Some specific chromophores useful in
the present invention include, for example, fluorescei", rhodamine, Texas red,
phycoerythrin, umbelliferone, luminol.
The labels may be conjugated to the antibody or nucleotide probs by methods that10 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 afunctional group. Some examples of slJit~hle functional groups include, for example,
amino, carboxyl, sulfhydryl, maleimide, isocyanate, isothiocyanate.
15 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 leceplor for that ligand attached to the
label. Any of the known ligand-receptor co",~ . ,ations 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.
25 The Examples do not include detailed descriptions of conventional methods employed
in the construction of vectors and plasmids, the insertion of genes encoding
pol~,ueplicles 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)
wo 95/04L~3 2 1 ~ 7 g 3r8 PCT/US94/08586
MC'QCU'~r Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory
Press.
The f~ w.;"g examples show the stable expression of certain intact porgenes
5 (por-l and por-5J from their own promoters in E. coli and S. typhimurium when
present on a plasmid or from the Salmonella chromosome (por-1 only). Moreover,
por-2 was ex,,.ressed in E. coli without toxic;cily when cloned without its full promoter
region, although higher level expression from an intact promoter was apparently
lethal. High level ~xl,r~ssion of all forrns of porgenes tested in E. coli were lethal to
10 E. coli (induGtion of high levels of Por Iysed E. coli BL21 (DE3)[pLysS]).
CLONING AND PLASMID EXPRESSION OF PorlN 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 por-l (Por-1 (A)) promotor was used to express
the Por-1 (A) and hybrid Por-1 (A~(B) Por protoi. ,s.
Por-1"~,
Ths por-1 gene from gonococc~l strain FA19 (Por-1(A)) (see Table 1 for list of
strains and plasmids) was reasse",bled (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 exlJress Por-1 (A, FA19) without apparent toxicity.
Por-2,~
Relatively weak but stable e,~Jressioo of Por-2(B MSll) in E. coli was achieved
without using the entire por-2 pro",oter region (i.e. Iacking the gonococcal -35 but
Wo 95/04133 2 ~ ~ 7 9 3 8 PCT/US94/08586
containing the gonococc~l -10 region). The promoter sequences (-35 and -10 region~
of the gonococcal porin promoter) are shown in the DNA and amino acid sequence of
the PIB gene of MS11 in Carbonetti et al. (1988)).
Several allelllp~sto reassemblethepor-2genefrom 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 rec;~.ie, lls in the
transformation. In order to test whether the alle",5)led por-2 ligaliol ,s contained an
assel "bled 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 andboth clones (pUNCH 540 and 541, respectively) ex,. ressed full length Por-2(B MS11) 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, expression2 o in E. coli either did not require the -35 sequence or there was transcription originating
from the upstream 17 promoter in the vector in the absenca of induction. These data
suggest that the failure to obtain Por-2(B Ms1~-eh~ressiog transformants with
constructs containing the entire promoter (-35 and -10) region of por-2 was due to a
problem other than proper reasse,nbly of the gene. Moreover, the results
demo":jl,ale that Por-2(B, MS11) was stably, although weakly, expressed in E. coli.
Por-5,~, ~l '\S!; 9 HYBRID
A gonococcus expressi"g 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
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WO 95/04133 2 1 6 7 9 3 8 PCT/US94/08586
constructed (Carbonetti et al 1988). The class 9 hybrid porin contains the N and C
terminal domains of Por-l(A~ FAl9) and a central domain of Por-2(B~ MS11) (Carbonetti et al.
1988). Cloning of the gene (por-5) for the hybrid porin (Por-5(A~B FA6434) 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, FA19).
except for a central Por-2(B MS11) surface-exposed region it was possi'~le to
constitutively ex~.ress por-5 in E. coli without toxicity. Cloning por-5 behind an intact
por-l promoter was s~ccess~ul and resulted in pUNCH 535 (Fig. 4) which expressedepitopes for Por-1(A FA19) and Por-2(B Ms11) (Fig. 2).
Thus reassembly and expression in full length of Por-1 (A, FA19), Por-2(B~ MS11) as
well as the Por-5(A~B FA6434) hybrid was achieved, although ex,uressio" of Por-2required alteration of the promoter region to avoid toxicity.
CLONING AND EXPRESSION OF DorlN SALMONELLA
a). Chromosomal expression:
Integration of the cloned gonococcal por-l gene into the chromosome of S.
2 0 typhimurium strain BRD 207 resulter~ in strain FX501 (Fig. 5). Southern Llolli. ~y
analysis of FX501 and FX502 (control Salmonella strain with the vector pDEL-1
i"leg,~ted into the chromosome) using probes for por-l 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 por-l
2 5 was present in the Salmonella chromosome compared to expression from multicopy
expression plasmids (Fig. 6).
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wo 95/04133 ~ ~ 6 7 g 3~ PCTIUS94/08586
b). Plasmid expression:
Por was introduced into a plasmid ex~.ressio" system in Salmonella using a
stable expression system developed by Curtiss and coll~ages (Galan et al. 1990
s Nakayama et al. 1988). The system relies on the complementation of a defectiveaspartate B-semialdehyde dehydrogenase (as~ chromosomal gene with a plasmid
e"cocled asd gene (Nakayama et al. 1988; Galan et al. 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
10 chromosomal locus asdthat lose their plasmid are unable to synthesize DAP andcannot grow in vivo unless a wild-type asdgene is provided in trans (Galan et al.
1990 Nakayama et al. 1988). Thus asd plasmids remain stable in vitro and in vtvo in
the absence of antibiotic pressure in asdhosts (Table 1). A Bglll fragment containing
the asd gene from pYA292 was made blunt by a fill in reaction with Klenow fragment
and cloned into the Scal sites of pUNCH30 and pUNCH535 to form pUNCH537 and
pUNCH536 respectively. Details of the subcloning of the por-1 gen~ 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, FA19) and Por-5(A~B, FA6434) are made in this
2 0 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 reco"~bi-,ant 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
25 blot analysis of whole cells using anti-Por monoclonal antibodies indicated that Por
was suRace-ex~osed on all tested Salmonella and E. coli containing Por plasrnidsexcept 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.
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WO 95/04133 2 1 6 7~ 3 8 ~- PCT/US94/08586
TOXICITY I t~ G
Toxicicity of Por-1 (A, FAl9) and Por-5(A~B, FA6434) was tested in S. typhimurium strain
5 X4072 (containing pUNCH 537, ~36 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)
por-l sequence and the present invention's por-1 sequence was found.
INDUCTION OF ANTIBODIES TO BOTH SEROGROUPS OF GONOCOCCI BY
IMMUNIZATION WITH A GONOCOCCAL HYBRID PORIN
The following ex~mple shows the ability of various adjuvants to induce an
immune res~.onse to both PIA and PIB sequences upon immunization of rabbits withrPor-5.
Experimental proc~ lures
Recombinant Por-5 was purified under relatively non-denaturing conditions.
New Zealand white rabbits were immunized four times every two weeks with 1 00ug of
purified Por-5 Mixed with the following adjuvants: none, Complete
Freunds/lncG"",lele Freunds, Keyhole limpet hemocyanin, Ribi, Bacille 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 desc, ibed in Harlow and Lane (Harlow and Lane, 1988).
. !
WO 95/04L~3 2 1 6 7 9 3 8 PCT/US94/08586
n e S"ltS
ELISA titers inf~ c~te-l that all rabbits had detectable anti-Por activity at a
1:5000 dilution (Fig. 1 1 ) using rPor-1 or rPor-2 as the coating antigen. Por-1 domains
from the Por-5 hybrid were consistel,lly more immunogenic than the Por-2 (central)
domain in the Por-5 hybrid. In order to ~csess 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 co"~i"" that the immune response against whole gonococci in the
dot blot was specific to Por, modified Western blots were performed on sera fromselected 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
2 0 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 (Plll) (Gotschlich et al., 1987) and
OmpA-like proteins are common in Gram negative bacteria. The regions of homologybetween 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 andcan 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 radioimmunoprecipit~tion.
wO 95/04133 21~ 7 9 3 8 PCT/US94/08S86
This suggests that non-conserved regions of OmpA are more immunogenic than the
conserved regions.
TABLE ll
5 Summary of Anti-Por-5 Rabbit Sera Binding to Gonococci by Dot Blot
Adjuvant # Rabbit a,lti-~ra binding gonococcal strain
FA19 (Por-1) FA6434(Por-5) MS11 (Por-2)
NONE 5/5 515 415
BCG 5/5 515 215
CFA 5/5 5/5 1/5
ALUM 5/5 5/5 0/5
KLH 5/5 515 1/5
RIBI 5/5 515 0/5
~otes to Table ll:
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
10 scored as positive.
Of the Por-1 and Por-2 sequences found in the Por-5 hybrid prote;., of this
example, Por-1 (N- and C- terminal sequences) were more immunogenic than Por-2
15 (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-e~ osed sequences as compared to all
other adjuvant groups.
--29--
WO 9!;/04133 PCT/US94/08586
2~67938 ~
SuPPle "~.,lzl EnaLle l,~.,t
The invention as claimed is enabled in accordance with the specification and
5 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. coli strains carrying the listed plasmids have been deposited
10 with the Agricultural Research Culture Collection (NRRL), Peoria, lL or the American
Type Culture Collection, (ATCC), Rockville, MD and have been assigned the
~ccession numbers indicated.
E. coli Strain Plasmid Accession No.
BL21 (DE3) pUNC7 NRRLB-18263
BL21 (DE3) pUNCH25 ATCC 67775
N. gonorrhoeae Strain
FA6248 ATCC 53808
FA19 ATCC 55073
Each deposited strain is only i"tended as a single illustration of one aspect ofthe 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 RIJ~rest Treaty on the
Intemational Recognition of the Deposit of Microo-yai ,isms for the Purposes of Patent
Procedure and the regulations thereunder (Budapest Treat,v). This assures
maintenance of a viable culture for 30 years from date of deposil. The organisms will
be made available by ATCC under the terms of the Budapest Treaty, and subject to
--30--
21~793~
WO 95/04133 PCT/US94/08586
. . . . .
an agreement between Appllcants and ATCC, which assures unrestrlcted avallablllty
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.
.
--31--
WO 95/04133 , . ' PCT/US94108586
2 ~ 6 7 9 3 8 ~t~t~ENCES
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 suscel,libility to human
serum and antibiotics. Molec. Microbiol. 4:1009-10018.
Carbonetti, N.H., Simnad, V.l., 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). Molecu~r cloning and characterization of
the structural gene for protein 1, the major outer membrane protein of Neisseriagonorrhoeae. Proc Natl Acad Sci U S A. 84(24): 9084-9088.
Curtiss lll, R., Goldschmidt, R.M., Fletchall, N.B., and Kelly, S.M. (1988). Avirulent
Salmonella typhimurium cya~crp- oral vaccine strains ex~,r~ssing a sl,eplococcalcolonization and virulence antigen. Vaccine. 6: 155-160.
Curtiss lll, R., Kelly, S.M., Gulig, P.A., Gentry-Weeks, C.R., and Galan, J.E. (1988).
Avirulent Salmonellae expressing virulence a"liyens from other pathogens for use as
orally adl"i"islered vaccines. Virulence mechanisms of bacterial pathogens.
2 5 Washi~ ~ylol1, Amer Soc for Microbiol. 311 -328.
Curtiss, R.l., and Kelly, S.M. (1987). Salmonella typhimurium deletion mutants lacking
adenylate cyclase and cyclic AMP receplor protein are avirulent and immunogenic.Infect !mmun. 55: 3055-3043.
--32--
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Dougan, G., Chatfield, S., Picard, D., Bester, J., O'Callaghan, D., and Maskell, D.
(1988). Construction and characteri~alio" of vaccine strains of Salmonella harboring
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Dougan, G., Hormaeche, C.E., and Maskell, D.J. (1987). Live oral Salmonella
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the immune system. Parasite Immunology. 9: 151-160.
Dougan, G., Smith, L., and Heffron, F. (1989). Live bacterial vaccines and theiras~ ons as carriers for foreign anlige, ls. 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 Dise~ses New York,
Raven Press.113-139.
Galan, J.E., Nakayama, K., and lll, R.C. (1990). Cloning and characterization of the
asd gene of Salmonella typhimurium: use in stable maintenance of recombinant
2 0 plasmids in Salmonella vaccine strains. Gene. 94: 29-35.
Gotschlich, E.C. (1984). Development of a gonorrhoea vaccine: prospects, strategies
and t~GtiCS. Bull World Health Organ. 62(5): 671-80.
25 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):
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