Methods and compositions are provided for prophylactic and antibacterial therapy for Helicobacter, particularly for Helicobacter pylori, infection of humans. The immunogenic composition of the invention is composed of a plurality of multimeric complexes, each complex being composed of recombinant, enzymatically inactive Helicobacter pylori urease. Each multimeric complex is composed of six Urease A subunits and six Urease B subunits. Alternatively, the composition is composed of a mixture of multimeric complexes, wherein each multimeric complex in the mixture is composed of six Urease A subunits and six Urease B subunits or four Urease A subunits and four Urease B subunits.

BACKGROUND OF THE INVENTION 
This invention relates to methods and immunogenic compositions (e.g., 
vaccines) for preventing and treating Helicobacter infection. 
Helicobacter is a genus of spiral, gram-negative bacteria which colonize 
the gastrointestinal tracts of mammals. Several species colonize the 
stomach, most notably Helicobacter pylori, Helicobacter heilmanii, 
Helicobacter fells, and Helicobacter mustelae. H. pylori is the species 
most commonly associated with human infection. H. heilmanii is also 
associated with human infection, but at a lower frequency than H. pylori. 
Helicobacter infects over 50% of adult populations in developed countries, 
and nearly 100% in developing countries and some Pacific rim countries, 
making it one of the most prevalent infections of humans worldwide. 
Infection with H. pylori results in chronic stomach inflammation in all 
infected subjects, although the clinical gastroduodenal diseases 
associated with Helicobacter generally appear from several years to 
several decades after the initial infection. H. pylori is the causative 
agent of most peptic ulcers and chronic superficial (type B) gastritis. H. 
pylori infection is also associated with atrophy of the gastric mucosa, 
gastric adenocarcinoma, and non-Hodgkin's lymphoma of the stomach. The 
role of H. pylori in these diseases has been summarized in numerous 
reviews (Blaser, J. Infect. Dis. 161:626-633, 1990; Scolnick et al., 
Infect. Agents Dis. 1:294-309, 1993; Goodwin et al., "Helicobacter 
pylori," Biology and Clinical Practice, CRC Press, Boca Raton, Fla., 465 
pp, 1993; Northfield et al., "Helicobacter pylori," Infection, Kluwer 
Acad. Pub., Dordrecht, 178 pp, 1994). 
Helicobacter colonizes the stomach mucus gel and the underlying gastric 
epithelial cells. Normally, the low pH of the stomach provides a chemical 
barrier to infectious agents. Helicobacter successfully breaches this 
barrier by producing urease, an enzyme present on the bacterial cell 
surface. Urease catalyzes the hydrolysis of urea to produce ammonium 
hydroxide, which provides a buffering cloud of ammonia around the 
bacterium, allowing it to withstand the low pH environment of the stomach 
and establish infection (Ferrero et al., Microb. Ecol. Hlth. Dis. 
4:121-134, 1991). The structural genes encoding H. pylori urease, ureA and 
ureB, have been cloned and sequenced (Clayton et al., Nucl. Acids. Res. 
18:362, 1990; Labigne et al., J. Bacteriol. 173:1920-1931, 1991), and a 
recombinant H. pylori urease has been purified (Hu et al., Infect. Immun. 
60:2657-2666, 1992). 
If untreated, H. pylori infection and the associated gastritis persist 
lifelong, despite the host's systemic and local immune responses to the 
bacterium (Crabtree et al., "Host responses," in Northfield TC et al. 
(Eds.), Helicobacter pylori Infection, Kluwer Acad. Pub., Dordrecht, pp. 
40-52, 1991; Kist "Immunology of Helicobacter pylori," in Helicobacter 
pylori in peptic ulceration and gastritis. Marshall et al., Eds., 
Blackwell Sci. Pub., Oxford, pp. 92-110, 1991; Fox et al., Infect. Immun. 
61:2309-2315, 1993). Conventional treatment of peptic ulcer disease 
associated with H. pylori infection involves the use of one or more 
antibiotics combined with a proton pump inhibitor or an H.sub.2 -receptor 
antagonist. Such treatment regimens are unsuccessful in 10% to 70% of 
patients. Moreover, successful eradication of H. pylori infection with 
antibiotics does not prevent subsequent reinfection. The most effective 
conventional treatment is a triple therapy with bismuth, metronidazole, 
and either amoxicillin or tetracycline. The triple therapy is complicated 
by a complex and prolonged dosing regimen, a high incidence of 
side-effects, poor compliance, and emergence of resistant bacterial 
strains (Hentschel et al., N. Engl. J. Med. 328:308-312, 1993). 
There is a clear need for prophylactic and therapeutic regimens for 
Helicobacter infection which are simple to administer, well-tolerated, and 
result in long-lasting immunity against infection and/or reinfection. 
SUMMARY OF THE INVENTION 
We have shown that vaccine compositions containing multimeric, recombinant 
Helicobacter pylori urease are effective for preventing and treating 
Helicobacter infection. 
Accordingly, in a first aspect, the invention features a vaccine for 
inducing a protective or therapeutically effective mucosal immune response 
to Helicobacter infection in a patient (e.g., a human patient). The 
vaccine of the invention contains a plurality of multimeric complexes 
(e.g., octamers, hexamers, or tetramers) of recombinant, enzymatically 
inactive urease 25 (e.g., H. pylori urease) in a pharmaceutically 
acceptable carrier substance (e.g., sterile water or 2% weight/volume 
sucrose). In a preferred embodiment, the multimeric complexes of 
recombinant, enzymatically inactive urease may be freeze-dried prior to 
administration. 
In a first embodiment, the vaccine contains multimeric complexes containing 
eight Urease A subunits and eight Urease B subunits, multimeric complexes 
comprising six Urease A subunits and six Urease B subunits, or multimeric 
complexes comprising four Urease A subunits and four Urease B subunits. 
In a second embodiment, the vaccine contains multimeric complexes 
containing eight Urease A subunits and eight Urease B subunits (octomer), 
multimeric complexes comprising six Urease A subunits and six Urease B 
subunits (hexamer), and multimeric complexes comprising four Urease A 
subunits and four Urease B subunits (tetramer). 
The vaccine may be administered to a mucosal surface, e.g., a nasal or oral 
surface. The vaccine may be administered with gastric neutralization 
using, e.g., sodium bicarbonate, or without gastric neutralization. In a 
preferred embodiment, oral administration is carried out without gastric 
neutralization. 
In addition to the multimeric complexes of recombinant, enzymatically 
inactive Helicobacter urease, the vaccine may contain a mucosal adjuvant, 
e.g., an adjuvant derived from the heat-labile enterotoxin of 
enterotoxigenic Escherichia coli, or derived from cholera toxin. 
Fragments, mutants, or analogs of adjuvants that maintain adjuvant 
activity may be used in vaccine of the invention. 
In a second aspect, the invention features methods of preventing or 
treating Helicobacter infection in a patient. In these methods, an 
immunogenically effective amount of a composition containing a plurality 
of multimeric complexes of recombinant, enzymatically inactive 
Helicobacter (e.g., Helicobacter pylori) urease is administered to a 
mucosal surface (e.g., an intranasal or oral surface) of the patient. An 
advantage to intranasal delivery is that less antigen and adjuvant are 
required in order to produce a protective or therapeutic immune response. 
In a first embodiment, the composition contains multimeric complexes 
comprising eight Urease A subunits and eight Urease B subunits, multimeric 
complexes comprising six Urease A subunits and six Urease B subunits, or 
multimeric complexes comprising four Urease A subunits and four Urease B 
subunits. 
In a second embodiment, the composition contains multimeric complexes 
comprising eight Urease A subunits and eight Urease B subunits, multimeric 
complexes comprising six Urease A subunits and six Urease B subunits, and 
multimeric complexes comprising four Urease A subunits and four Urease B 
subunits. 
The composition may be administered with gastric neutralization using, 
e.g., sodium bicarbonate, or without gastric neutralization. In a 
preferred embodiment, oral administration is carried out without gastric 
neutralization. 
In a preferred embodiment, the composition is administered in association 
with a mucosal adjuvant, e.g., an adjuvant derived from heat-labile 
enterotoxin of enterotoxigenic Escherichia coli or cholera toxin. 
In a further preferred embodiment, the multimeric complexes of recombinant, 
, enzymatically inactive Helicobacter (e.g., Helicobacter pylori) urease 
is freeze-dried prior to administration. 
By "vaccine" is meant a composition containing at least one antigen which, 
when administered to a patient, elicits or enhances an immune response to 
the antigen that is effective in the prevention of disease, or in the 
treatment of disease associated with a pre-existing infection. 
By "immunogenically effective amount" is meant an amount of a composition 
that is effective in eliciting an immune response (e.g., a humoral or a 
mucosal immune response) when administered to a patient (e.g., human 
patient). 
By "therapeutically effective immune response" is meant an immune response 
which is effective in treating a preexisting disease, particularly a 
preexisting bacterial infection. 
By "Helicobacter" is meant any bacterium of the species Helicobacter, 
particularly a Helicobacter bacterium which infects humans (e.g., H. 
pylori). 
By "urease" is meant an enzyme (e.g., H. pylori urease) which catalyzes the 
conversion of urea into ammonium hydroxide and carbon dioxide. Urease 
activity thereby causes an increase in the pH of the medium or other 
environment in which the enzyme is located. 
By "multimeric complex" is meant a macromolecular complex composed of 
polypeptides (e.g., urease polypeptides). The polypeptides may be 
associated in the complex by a variety of intermolecular interactions, 
such as covalent bonds (e.g., disulfide bonds), hydrogen bonds, and ionic 
bonds. 
By "buffer free" or "free of buffer" is meant that the composition contains 
no compounds which effect a significant increase in pH, and/or is not 
administered to a patient in conjunction with any such pH-raising 
compound. "pH-raising buffer compounds," as meant herein, particularly 
refers to those compounds which effect an increase in stomach acid pH 
following oral administration to a human patient. 
By "mucosal adjuvant" is meant a compound (e.g., an immunomodulator) which 
non-specifically stimulates or enhances a mucosal immune response (e.g., 
production of IgA antibodies). Administration of a mucosal adjuvant in 
conjunction with an immunogenic composition facilitates the induction of a 
mucosal immune response to the immunogenic compound. 
Other features and advantages of the invention will be apparent from the 
following description of the preferred embodiments thereof, and from the 
claims.

CLONING OF THE UREA AND UREB GENES 
The structural genes encoding urease, ureA, and ureB, have been cloned 
(Clayton et al., Nucl. Acids. Res. 18:362, 1990; Labigne et al., J. 
Bacteriol. 173:1920-1931, 1991), and the recombinant urease encoded by 
these genes has been purified (Hu et al., Infect. Immun. 60:2657-2666, 
1992). For use in the present invention, urease was cloned from a clinical 
isolate of H. pylori (CPM630) obtained from a clinical specimen provided 
by Dr. Tabaqchali, St. Bartholomew's Medical College, University of 
London. A genomic DNA library of strain CPM630 was prepared in the lambda 
phage vector EMBL3. Plaques were screened for reactivity with rabbit 
anti-Helicobacter urease polyclonal antibody, and a single reactive plaque 
was isolated. This clone contained a 17 kb SalI fragment that encoded the 
ureA and ureB genes. The 17 kb fragment was subcloned onto pUC18 and 
designated pSCP1. A 2.7 kb TaqI fragment was subcloned (pTCP3) and 
completely sequenced. The 2.7 kb TaqI fragment encoded both ureA and ureB. 
The primers BL1 (CGG GAT CCA CCT TGA TTG CGT TAT GTC T; SEQ ID NO: 4) and 
BL2 (CGG AAT TCA GGA TTT AAG GAA GCG TTG; SEQ ID NO: 5) were used to 
amplify and clone a 2.5 kb fragment from pSCP1. BL1 and BL2 correspond to 
nucleotides 2605-2624 of GenBank accession number M60398 (the BL1 primer) 
and nucleotides 2516-24998 of EMBL accession number X17079 (the BL2 
primer). A restriction enzyme map of the 2.5 kb fragment PCR product is 
shown in FIG. 1. The 2.5 kb fragment contains the entire coding region of 
ureA and ureB, as well as translational start signals from H. pylori 
upstream of ureA. 
Expression of recombinant urease 
The purified 2.5 kb PCR fragment containing the genes encoding ureA and 
ureB was digested with EcoRI and BamHI and inserted into the expression 
vector pET24+(Novagen) to produce the plasmid pORV214 (FIG. 2). 
pET24+contains the colE1 origin of replication, the filamentous phage (f1) 
origin of replication for single strand rescue, and the kanamycin 
resistance gene of Tn903. The fragment was inserted downstream of the T7 
promoter, which provides transcription initiation for the urease genes. 
Lactose operator (lacO) sequences are present between the T7 promoter and 
the cloning sites to provide inducible expression of the urease genes. A 
T7 transcription terminator sequence is located downstream of the cloning 
sites. The vector also contains the lactose repressor gene (lacI) to 
ensure complete repression of expression. Other sequences present in the 
vector are derived from pBR322, which served as the backbone for vector 
construction. 
The initial ligation mixture, containing the 2.5 kb PCR EcoRI-BamHI 
fragment and the pET24+ vector digested with EcoRI and BamHI, was used to 
transform XL1-Blue (Stratagene, La Jolla, Calif.) prepared by the 
CaCl.sub.2 method. XL1-Blue is an E. coli strain that does not express T7 
RNA polymerase. Kanamycin resistant colonies were directly screened by PCR 
using urease specific primers. Plasmid DNA from several positive colonies 
was extracted using Qiagen minispin columns (Qiagen, Chatsworth, Calif.) 
and checked for the correct restriction digestion pattern. 
Purified pORV214 DNA was used to transform the E. coli strain BL21-DE3 
(Novagen) prepared by the CaCl.sub.2 method. BL21-DE3 is an E. coli B 
strain that is lysogenized with lambda phage DE3, a recombinant phage that 
encodes T7 RNA polymerase under the control of the lavUV5 promoter. BL21 
is deficient in ion and ompT proteases, as well as the hsdS.sub.B 
restriction/modification system and dcm DNA methylation. DNA was prepared 
from kanamycin resistant colonies with Qiagen mini spin columns and 
screened by restriction enzyme analysis using BamHI and EcoRI to confirm 
the presence of the plasmid. Urease expression was assessed by examination 
of BL21-DE3 (pORV214) cell lysates by SDS-PAGE and Western blot. Several 
positive clones had the correct restriction endonuclease digestion pattern 
and expressed urease. 
A single clone containing pORV214 was selected, grown on LB plates 
containing kanamycin (50 .mu.g/ml), harvested, and stored at -80.degree. 
C. in LB containing 50% glycerol. A research cell bank was prepared by 
growing a sample from the glycerol stock on LB plates containing 
kanamycin, selecting an isolated colony, and inoculating a LB broth 
culture containing kanamycin. This culture was grown to OD.sub.600 of 1.0, 
pelleted, and resuspended in an equal volume of LB containing 50% 
glycerol. These research cell bank (RCB) aliquots (100 .mu.l) were then 
stored at -80.degree. C. A master cell bank (MCB) was similarly prepared 
using an isolated colony from the research cell bank, and a manufacturer's 
working cell bank (MWCB) was prepared using an isolated colony from the 
MCB. 
The MCB and MWCB cells were viable, kanamycin resistant, and displayed a 
normal E. coli colony morphology. T7 RNA polymerase expression and lambda 
phage lysogeny was confirmed using appropriate tests, which are well known 
in the art. Urease expression was IPTG-inducible in the MCB and MWCB 
cells, as determined by examination of cultures grown in the presence of 
IPTG, and analysis of lysates from these cell cultures on SDS-PAGE. 
Production of 60 kDa and 29 kDa proteins (UreB and UreA, respectively) by 
the MCB and MWCB cells increased with the incubation time. 
Plasmid DNA was isolated from the MCB and MWCB cells and was tested by 
restriction enzyme analysis, restriction fragment length polymorphism 
(RFLP), and DNA sequence analysis to confirm plasmid structure. The MCB 
harbored a plasmid with the appropriate restriction endonuclease digestion 
pattern with no deletions or rearrangements of the plasmid. Likewise, 
there were no differences between the RFLP fingerprint of pSCP1 and the 
RFLP fingerprints of plasmid DNA from MCB and MWCB, indicating that the 
urease genes had not undergone detectable (&gt;50 bp) deletions or 
rearrangements in the cloning process or in the manufacture of the cell 
banks. 
The coding regions of ureA and ureB, and the sequences of the promoter and 
termination regions of the plasmid isolated from MCB cells was sequenced. 
Sequencing reactions were performed using the Di-Deoxy Cycle Sequencing 
Kit, according to the manufacturer's instructions (Applied Biosystems, 
Inc., Foster City, Calif.) using fluorescent-labeled dideoxynucleotides. 
The sequences of the ureA and ureB genes, with predicted protein 
sequences, as well as the DNA sequences of flanking regions are shown in 
FIG. 3. 
Large-scale production of recombinant urease 
The fermentor(s) to be used was cleaned and sterilized according to 
approved procedures. The culture medium contained 24 g/L yeast extract, 12 
g/L tryptone, 6-15 g/L glycerol, in RO/DI water. Since pORV214 was 
sufficiently stable in the absence of antibiotics, no antibiotics were 
present in the large-scale fermentation cultures. Antifoam was added and 
the unit was sterilized in place. 
For production in the 40 liter fermentor, a vial of the MWCB was thawed and 
used to inoculate a four liter shaker flask containing one liter of LB 
broth (1% tryptone, 0.5% yeast extract, 1% NaCl) without antibiotics. The 
culture was shaken at 37.degree. C. for 16-24 hours. The inoculum is then 
transferred to the 40 liter fermentor (30 liter working volume) containing 
the production media described above. Fermentation was carried out with 
aeration and agitation until the cell density determined by OD.sub.600 was 
approximately 8-10. The inducer isopropyl B-D-thiogalactopyranoside (IPTG) 
was then added to a final concentration of 0.1 mM. Induction was allowed 
to proceed for 16-24 hours. The cells were harvested by centrifugation and 
the wet paste was aliquoted into polypropylene storage containers and 
stored at -80.degree. C. For production at the 400 liter scale (300 liter 
working volume), the inoculum and culture in the 40 liter vessel were 
prepared as described above. When the 40 liter vessel reached an 
OD.sub.600 of approximately 5.0, it was used to inoculate the 400 liter 
fermentor. The culture was further incubated to an OD.sub.600 of 8-10. The 
culture was then induced, harvested, and stored, as described above. This 
procedure required 2-3 generations more than the 40 liter process. 
Purification of recombinant H. pylori urease 
Containers of cell paste produced by the fermentation processes described 
above were removed from storage, thawed at room temperature, and 
re-suspended in 20 mM sodium phosphate/1 mM EDTA buffer, pH 6.8. The 
resuspended cells were disrupted by extrusion through a narrow orifice 
under pressure (Microfluidizer Cell Disruptor). Disrupted cells were 
centrifuged at 4.degree. C. to sediment cell debris and the supernatant 
containing soluble urease was collected. 
A solution of 3M sodium chloride was added to the cell supernatant to a 
final concentration of 0.1M. The supernatant was then applied to a 
DEAE-Sepharose column equilibrated in 20 mM sodium phosphate/1 mM EDTA, 
and the pass-through was collected for further processing. The 
pass-through was diafiltered into 20 mM sodium phosphate/1 mM EDTA, pH 
6.8, with a 100 kDa cutoff membrane to remove low molecular weight 
contaminants and to reduce ionic strength. 
This material was applied to a second DEAE-Sepharose column equilibrated in 
20 mM sodium phosphate/1 mM EDTA. The pass-through was discarded and the 
column rinsed with 20 mM sodium phosphate/1 mM EDTA, pH 6.8. The bound 
material was eluted with approximately three column volumes of 0.1M NaCl/1 
mM 2-mercaptoethanol. Effective elution of the bound urease was controlled 
by the volume and flow rate of the elution buffer. 
The partially purified urease was diafiltered against 25 mM Tris-HCl, pH 
8.6, and applied to a Q-Sepharose column. The column was washed with 
approximately two column volumes of the same buffer and the pass-through 
containing highly purified urease was collected. 
The pass-through from the Q-Sepharose step was then concentrated and 
diafiltered into 2% sucrose in water for injection (WFI), pH 7.5. 
Characterization of the antigenicity and subunit composition of Purified 
recombinant H. pylori urease 
To compare the antigenicity and subunit composition of recombinant H. 
pylori urease to native H. pylori urease, native H. pylori urease was 
purified and used as an antigen to produce polyclonal anti-urease 
antibodies, as well as mono-specific polyclonal anti-UreA, anti-UreB, and 
anti-urease holoenzyme antibodies. 
Native H. pylori urease was purified using a modification of the procedure 
reported by Hu and Mobley (Infect. Immun. 58:992-998, 1990). H. pylori 
strain ATCC 43504 (American Type Culture Collection, Rockville, Md.) was 
grown on Mueller-Hinton agar (Difco Laboratories, Detroit, Mich.) 
containing 5% sheep red blood cells (Crane Labs, Syracuse, N.Y.) and 
antibiotics (5 .mu.g/ml trimethoprim, 10 .mu.g/ml vancomycin and 10 U/ml 
polymyxin B sulfate) (TVP, Sigma Chemical Co., St. Louis, Mo.). Plates 
were incubated 3-4 days at 37.degree. C. in 7% CO.sub.2 and 90% humidity, 
and bacteria were harvested by centrifugation. The bacteria were suspended 
in water or 20 mM phosphate, 1 mM EDTA, 1 mM .beta.-mercaptoethanol (pH 
6.8) containing protease inhibitors, lysed by sonication and clarified by 
centrifugation. The clarified supernatant was mixed with 3M sodium 
chloride to a final sodium chloride concentration of 0.15M and passed 
through DEAE-Sepharose (Fast Flow). Active urease that passed through the 
column was collected, concentrated in a Filtron Macrosep 100 centrifugal 
filtration unit, and then passed through a Superose-12 or Superdex 200 
size exclusion column. Size-exclusion chromatography was performed using 
Pharmacia FPLC prepacked columns. The fractions containing urease activity 
were pooled, concentrated, and further purified by FPLC anion-exchange 
chromatography on a Mono-Q sepharose column prepacked by Pharmacia. The 
bound urease was eluted using a sodium chloride gradient. The fractions 
with urease activity were pooled and concentrated using Macrosep 100 
centrifugal filters from Filtron Inc. For some lots, a final purification 
was achieved by analytical size-exclusion FPLC on Superose-12 columns. 
Polyclonal antiserum to H. pylori urease was produced by immunizing three 
female New Zealand white rabbits with purified native H. pylori urease. 
The animals were pre-bled to confirm non-immune status and then immunized 
subcutaneously with 150 .mu.g urease in complete Freund's adjuvant. Two 
booster doses of 150 .mu.g each were administered subcutaneously 27 and 45 
days later with Freund's incomplete adjuvant. After confirmation of the 
immune response by ELISA and Western blotting against purified urease, the 
animals were exsanguinated. The blood was clotted at 4.degree. C. 
overnight and serum was harvested by centrifugation. Serum IgG was 
purified by ammonium sulfate precipitation (50%) overnight at 4.degree. C. 
The precipitate was resuspended in PBS and dialyzed to remove ammonium 
sulfate. The anti-urease titer of the IgG from each animal was found to be 
1:10.sup.7 and the antibodies from the three animals were pooled. The 
protein concentration was determined to be 17.3 mg/ml. Aliquots of 0.2 ml 
each were prepared and stored at -80.degree. C. 
Mouse polyclonal ascites against H. pylori urease holoenzyme ("M") was 
prepared by injecting five mice subcutaneously with 10 .mu.g native urease 
holoenzyme in complete Freund's adjuvant on Day 0. The mice were boosted 
on Days 10 and 17, bled on day 24 to confirm anti-urease immune response, 
and boosted again on Day 26. On Day 28 the mice were injected 
intraperitoneally with Sarcoma 180 cells. A final intraperitoneal booster 
dose of 10 .mu.g urease was given on Day 31, and ascitic fluids collected 
seven days later. 
The ascitic fluids were incubated for two hours at room temperature and 
then at 4.degree. C. for 16 hours; clots were disrupted by vortexing and 
removed by centrifugation at 10,000 rpm for 10 minutes. After overnight 
incubation at 4.degree. C. in plastic tubes, the fluids formed solid 
clots. These were homogenized, diluted five-fold with PBS, and reclarified 
by centrifugation at 10,000 rpm for 10 minutes. Thirty-six ml of diluted 
ascitic fluids were collected and frozen at -20.degree. C. in 300 .mu.l 
aliquots. Western blot analyses confirmed that the antibody reacts with 
UreA and UreB subunits. An endpoint titer of 1:300,000 was achieved in 
ELISA against urease. 
Mouse polyclonal ascites against H. pylori UreA ("M") was prepared by 
injecting mice subcutaneously with native UreA subunit H. pylori urease 
isolated by electroelution from SDS-PAGE gels. Subsequent steps in the 
preparation of anti-ureA ascites were performed as described above for 
generation of antibodies against the holoenzyme. 
Mouse polyclonal ascites against native H. pylori UreB subunit ("M") was 
prepared by injecting mice subcutaneously with native UreB isolated by 
electroelution from SDS-PAGE gels. Subsequent steps in the preparation of 
anti-UreB ascites were performed as described above for generation of 
antibodies against the holoenzyme. 
MAB71 is an IgA monoclonal antibody against H. felis urease which 
recognizes a protective epitope on the B subunit. Preparation of this 
antibody is described in Czinn et al., J. Vaccine 11(6):637-642, 1993. 
The antibodies described above were used in Western blot experiments to 
characterize the purified recombinant H. pylori urease. 
SDS-PAGE and Western blot analysis of recombinant urease 
Recombinant urease was first analyzed by SDS-PAGE (12.5%) run under 
reducing conditions. Two major protein bands (29 kDa and 60 kDa) and 
several lighter bands (approximately 38 kDa) were evident. To identify the 
proteins in these bands, Western blots were performed using the 
anti-urease and anti-urease subunit antibodies described above. The 60 kDa 
protein reacted with M (anti-urease holoenzyme) and M (anti-UreB), 
but not with M (anti-UreA). The 29 kDa protein reacted with M 
(anti-urease holoenzyme) and M (anti-UreA), but not with M 
(anti-UreB). The lighter 38 kDa band reacted with M anti-urease 
holoenzyme) and M (anti-UreB), indicating that this protein is a 
portion of UreB. 
Two faint high molecular weight (&gt;150 kDa) bands were evident in SDS-PAGE 
gels. Both bands reacted faintly with antibodies to both UreA and UreB, 
indicating that a minor portion of recombinant urease subunits form 
covalent units resistant to sulfhydryl reduction under the conditions 
used. No other protein bands were apparent in Coomassie-stained gels. 
Hence, all proteins detected in the purified product are either UreA, 
UreB, or a derivative of UreA or UreB. 
The wet Coomassie-stained SDS-PAGE gel was scanned using an Ultroscan XL 
laser densitometer and a Gel Scan XL software program (Pharmacia-LKB 
Biotechnology, Piscataway, N.J.). The densitometry data were consistent 
with a 1:1 molar ratio of the UreA:UreB subunits, as expected from the 
structure of native H. pylori urease. UreA and UreB accounted for more 
than 95% of the total protein present in the purified urease preparation. 
The estimated average value for the combined UreA+UreB peaks was 95.2% 
.+-.1.2%. 
Analytical size-exclusion HPLC of purified recombinant urease 
The purity and molecular composition of purified recombinant urease was 
determined by analytical size-exclusion high performance liquid 
chromatography. Chromatography was performed using a Beckman System Gold 
HPLC consisting of Pump 126, diode array dual wavelength detector 168, 
System Gold Software Version V7.11, Progel-TSK G4000SWXL (5 mm.times.30 cm 
i.d) column, and SWXL guard column from SupelCo. Chromatography was 
performed under isocratic conditions using 100 mM phosphate, 100 mM sodium 
chloride buffer, pH 7.0. The column was calibrated using molecular weight 
markers from Pharmacia-LKB. 
A typical HPLC separation profile of representative purified bulk sample is 
shown in FIG. 4. The purified urease product shows a prominent protein 
peak with a retention time between that of thyroglobulin (MW 669 kDa) and 
ferritin (MW 440 kDa). An apparent molecular weight of 550-600 kDa was 
estimated based on a series of runs. This peak was tentatively designated 
as hexameric urease. The area of this hexameric urease was at least 70% of 
the total protein in different lots of product. 
A second prominent protein peak with a lower retention time (higher 
molecular weight) was also detected. The area of this peak ranged from 
5-20%. This peak, with a molecular weight &gt;600 kDa, was designated as 
octomeric urease. The total area of the octomeric plus hexameric urease 
peaks was over 90%. 
These two characteristic peaks were isolated for further characterization. 
The two peaks were purified by HPLC from a preparation of reconstituted, 
freeze-dried urease and stored at 4.degree. C. for over one week. During 
storage for this period of time, octomeric urease increases while the 
hexameric form decreases. The separated peak fractions were analyzed by 
reducing SDS-PAGE and ELISA reactivity with monoclonal and polyclonal 
antibodies to urease. On SDS-PAGE, both peaks showed urease A and B bands 
in comparable ratios. In addition, both showed nearly identical 
immunoreactivity in ELISA with polyclonal anti-urease holoenzyme 
antibodies (M) and a monoclonal anti-UreB antibody (MAB71). 
Enzymatic (urea hydrolytic) activity of recombinant urease 
Three methods were used to investigate the enzymatic activity of 
recombinant urease: urease-specific silver staining following 
electrophoresis, the pH-sensitive phenol red urea broth assay, and direct 
detection of ammonia. Urease-specific silver staining, described by 
deLlano et al. (Anal. Biochem. 177:37-40, 1989), is based on the reaction 
of urease with urea to produce ammonia. The reaction leads to a localized 
increase in pH which facilitates a photographic redox reaction leading to 
disposition of metallic silver. Enzymatic activity of native H. pylori 
urease was detected with only a 1.0 .mu.g sample. In contrast, 20 .mu.g 
purified recombinant urease exhibited no urease activity. 
The pH-sensitive phenol red broth assay, which is well known in the art, is 
based on a change in pH due to ammonia generation as a result of urea 
hydrolysis. Urease activity was demonstrated with as little as 0.2 
.mu.g/ml purified H. pylori urease. In contrast, no urease activity was 
associated with purified recombinant urease, even at concentrations up to 
750 .mu.g/ml. 
Direct estimation of ammonia produced by hydrolysis of urea by urease was 
quantitated using Nesslers' reagent (Koch et al., J. Am. Chem. Society 
46:2066-2069, 1924). Urease activity of native H. pylori urease was 
detectable at a concentration of 1 .mu.g/ml. No activity was detected in 
assays containing up to 500 .mu.g/ml purified recombinant urease. 
Based on the results of the urease-specific silver staining assay, the 
pH-sensitive phenol red broth assay, and direct estimation of ammonia, 
there is no detectable urease activity in the recombinant urease product. 
Protective and therapeutic activity of purified recombinant urease in 
animal infection models 
Since H. pylori does not readily infect laboratory animals, the H. felis 
model in rodents was used to test the efficacy of recombinant H. pylori 
urease in prophylaxis and antibacterial therapy. In this model, 
colonization of the stomach is readily established and is accompanied by 
gastric inflammation. This animal model is a well established system for 
the study of Helicobacter, and has been used extensively in laboratory 
investigations of the pathogenesis and treatment of Helicobacter-induced 
disease (Fox et al., Infect. Immun. 61:2309-2315, 1993; Goodwin & Worsley, 
Helicobacter pylori, Biology and Clinical Practice, CRC Press, Boca Raton, 
Fla., 465 pp, 1993). Antigenic cross-reactivity between H. pylori and H. 
felis ureases allows use of the human vaccine candidate of the invention, 
recombinant H. pylori urease (rUre), to immunize animals infected, or 
subsequently challenged, with H. felis. 
Both germ-free and conventional mice are susceptible to orogastric 
challenge with H. felis, and develop life-long infection of the gastric 
epithelium, characterized by infiltration of inflammatory cells (Fox et 
al., ibid.). Dose response studies indicated that 100% of Swiss-Webster 
specific pathogen-free (SPF) mice become infected after a single oral 
challenge with 10.sup.4 H. felis. Unless otherwise specified, a single 
dose of 10.sup.7 was used to infect mice prior to therapeutic immunization 
or challenge mice after prophylactic immunization. A challenge of 
.about.10.sup.3 times the infectious dose (I.D..sub.90) with this 
Helicobacter spp. represents a severe test of immunity. 
Assays for gastric infection 
Several methods were used to detect Helicobacter in gastric tissue, 
including measurement of gastric urease activity, histologic examination, 
and culture of gastric tissue. Gastric urease activity was measured both 
qualitatively (presence or absence) and quantitatively. In the qualitative 
assay, stomachs were divided longitudinally into two halves, from the 
gastroesophageal sphincter to the pylorus. One longitudinal piece, 
representing approximately 1/4 of the stomach, was placed in 1 mL of urea 
broth (0.1 g yeast extract, 0.091 g monopotassium phosphate, 0.095 g 
disodium phosphate, 20 g urea, and 0.1 g/L phenol red, pH 6.9). A 
distinctive color change (due to hydrolysis of urea by the enzyme, 
production of ammonia, and increased pH) after four hours incubation at 
room temperature indicated a positive result. For quantitative 
determinations, urease activity was determined by measuring absorbance at 
550 nm of clarified urea broth incubated with whole stomach sections for 4 
hours. This assay can detect as few as 1-2.times.10.sup.4 H. felis/0.1 g 
stomach tissue. This assay provides the same sensitivity as commercially 
available urease test kits used for human samples. Commercial kits have 
proven to be 100% specific and 90-92% sensitive compared to 
biopsy/histology (Szeto et al., Postgrad. Med. J. 64:935-936, 1988: 
Borromeo et al. J. Clin. Pathol. 40:462-468, 1987). 
Quantitative gastric urease assays by spectrophotometric measurement of 
A.sub.550 were slightly more sensitive than visual determinations, and 
allowed estimation of the severity of infection. The cut-off value for a 
negative urease assay was defined as 2 standard deviations above the mean 
A.sub.550 for unchallenged/uninfected mice. The cut-off for a positive 
gastric urease assay was defined by 2 standard deviations below the mean 
A.sub.550 of unimmunized/challenged mice. Individual animals with 
low-grade infections had values intermediate between the negative and 
positive cut-offs. Visual grading of the urease response identified 11/12 
(92%) of the positive samples. 
Histologic examination was performed by fixing stomach tissue in 10% 
formalin. The tissue was then embedded in paraffin, sectioned and stained 
with a modified Warthin-Starry silver stain (Steiner's stain; Garvey, et 
al. Histotechnology 8:15-17, 1985) to visualize H. felis, and by 
hematoxylin and eosin (H & E) stain to assess inflammatory responses in 
the tissue. Stained sections were examined by an experienced pathologist 
blinded to the specimen code. 
A semi-quantitative grading system was used to determine the number of 
bacteria and intensity of inflammation. The system used is a modification 
of the widely-accepted Sydney System for histological characterization of 
gastritis in humans (Price, Gastroenterol. Hepatol. 6:209-222, 1991). 
Full-thickness mucosal sections were examined for the intensity of 
inflammation (increase in lymphocytes, plasma cells, neutrophils and 
presence of lymphoid follicles) and depth of infiltration of these cells 
and graded on a scale of 0-4+. Grading the density of H. felis was 
accomplished by counting the number of bacterial cells with typical spiral 
morphology in an entire longitudinal section of gastric antrum (or corpus, 
if specified). Grades were assigned according to a range of bacteria 
observed (0=none; 1+=1-20 bacteria; 2+=21-50 bacteria; 3+=51-100 bacteria; 
and 4+=&gt;100 bacteria). 
Route of immunization 
The effect of the route of administration upon the efficacy of the vaccine 
of the invention was examined in the mouse infection model. Six to eight 
week-old female specific pathogen free (SPF) Swiss-Webster mice were 
immunized with 200 .mu.g recombinant H. pylori urease, either with or 
without 0.24M NaHCO.sub.3. The recombinant urease was co-administered with 
10 .mu.g of cholera toxin (CT) as a mucosal adjuvant in all animals. 
Intragastric (IG) immunization was performed by delivering the antigen in 
0.5 ml through a 20-gauge feeding needle to anesthetized animals. Oral 
immunization was performed by delivering the antigen in a 50 .mu.l volume 
via a pipette tip to the buccal cavity of unanesthetized animals. For 
parenteral immunization, mice received 10 .mu.g recombinant urease 
subcutaneously. Freund's complete adjuvant was used in the first 
subcutaneous immunization and Freund's incomplete adjuvant was used in 
subsequent boosters. For all routes of administration, a total of four 
doses of vaccine were administered at seven day intervals. Mice were 
challenged with 10.sup.7 H. felis two weeks after the final vaccine dose, 
and necropsied two weeks after challenge. Gastric H. felis infection was 
detected by urease activity and histology. Protection in an individual 
mouse was defined by a negative urease assay and by a 0 or 1+ bacterial 
score by histology. 
Oral and intragastric administration of recombinant urease provided 
significant protection to challenge with H. felis (86-100%) (Table 1). 
Oral administration was effective both with and without co-administration 
of NaHCO.sub.3 with the recombinant urease. Intragastric administration 
was more effective when the recombinant urease was co-administered with 
NaHCO.sub.3. Parenteral injection of the vaccine antigen was least 
effective. Immunized mice had significantly lower numbers of bacteria in 
gastric tissue after challenge than unimmunized controls. IgA antibody 
responses in serum and secretions were highest in mice immunized by the 
oral route. IgA antibodies were not elicited by parenteral immunization. 
TABLE 1 
__________________________________________________________________________ 
Recombinant H. pylori urease protects mice from challenge 
with H. felis after mucosal but not parental immunization 
% PROTECTED 
(# PROTECTED/TOTAL) 
ROUTE OF UREASE 
VACCINE 
IMMUNIZATION 
ADJUVANT 
BICARBONATE.sup.a 
ASSAY 
HISTOLOGY.sup.b 
__________________________________________________________________________ 
PBS oral CT No 0 (0/8) 
0 (0/7) 
200 .mu.g rUre 
oral CT No 100 (8/8)* 
100 (7/7)* 
200 .mu.g rUre 
oral CT Yes 100 (7/7)* 
86 (6/7)* 
200 .mu.g rUre 
intragastric 
CT No 75 (6/8)* 
38 (3/8) 
200 .mu.g rUre 
intragastric 
CT Yes 100 (7/7)* 
100 (7/7)* 
10 .mu.g rUre 
subcutaneous 
Freund's 
NA 38 (3/8) 
25 (2/8) 
__________________________________________________________________________ 
.sup.a 0.24 M sodium bicarbonate was administered with vaccine and 
adjuvant. 
.sup.b Percent protected (number mice with 0-1+ bacterial score/number 
tested). 
*p &lt; 0.01, Fisher's exact test, compared to mice given CT alone. 
The effect of immunization route upon the anti-urease antibody response was 
examined in mice. Swiss-Webster m ice were immunized four times at ten day 
intervals with either: 1) 200 .mu.g recombinant purified H. pylori urease 
with 10 .mu.g CT, either with or without NaHCO.sub.3, by oral 
administration; 2) 200 .mu.g recombinant purified H. pylori urease and 10 
.mu.g CT with NaHCO.sub.3, by intragastric administration; or 3) 10 .mu.g 
recombinant purified H. pylori urease with Freund's adjuvant by 
subcutaneous administration. One week after the fourth vaccine dose, 
mucosal and serum antibody responses were examined by ELISA using 
microtiter plates coated with 0.5 .mu.g of native H. pylori urease. Serum 
samples were diluted 1:100 and assayed for urease-specific IgA and IgG. 
Fresh fecal pellets, extracted with a protease inhibitor buffer (PBS 
containing 5% non-fat dry milk, 0.2 .mu.g AEBSF, 1 .mu.g aprotinin per ml, 
and 10 .mu.M leupeptin), were examined for fecal anti-urease IgA antibody. 
In some experiments, fecal antibody values were normalized for total IgA 
content determined by ELISA, with urease-specific fecal IgA expressed in 
A.sub.405 units/mg total IgA in each sample. Saliva samples were collected 
after stimulation with pilocarpine under ketamine anesthesia, and tested 
for urease-specific IgA at a dilution of 1:5. 
No significant differences between antibody responses of mice immunized 
orally with or without NaHCO.sub.3 were detected, and the data were pooled 
for analysis. Mice given subcutaneous antigen developed urease-specific 
serum IgG, but serum and fecal IgA responses were elicited only when 
antigen was delivered by mucosal routes (oral or intragastric). 
Mice immunized either orally or intragastrically with urease/CT were 
challenged with 10.sup.7 H. felis. The pre-challenge antibody levels of 
orally or intragastrically immunized mice were correlated with 
histologically-determined bacterial scores after H. felis challenge (FIGS. 
5A, 5B and 5C). Although IgA responses varied considerably between 
individual mice, on the whole, mice with higher serum and fecal IgA levels 
had reduced bacterial scores. These data indicate a role for IgA in 
suppression of, and protection from, infection. In contrast, serum IgG 
antibodies did not correlate with protection. While some protected mice 
had no detectable IgA antibodies, high levels of anti-urease IgA were not 
observed animals that developed 4+ infections after challenge. These 
results not only support a role for mucosal immune responses in 
protection, but also suggest that immune mediators other than fecal and 
serum IgA play a role in eradication of H. felis infection. 
These data show that: i) mucosal immunization is required for protective 
immunity; ii) the oral route of immunization is as effective, or more 
effective, than the intragastric route; iii) neutralization of gastric 
acid with NaHCO.sub.3 is required for effective intragastric (but not 
oral) immunization; and iv) parenteral immunization does not stimulate 
mucosal immunity or provide effective protection against challenge. 
Schedule of immunization 
Alternative immunization schedules were compared in order to determine the 
optimal immunization time-table to elicit a protective mucosal immune 
response. Swiss-Webster mice were immunized with 100 .mu.g recombinant 
urease in two, three, or four oral administrations, on a schedule shown in 
Table 2. The mucosal adjuvant CT (10 .mu.g) was co-administered with the 
recombinant urease. As assessed by qualitative gastric urease assay, mice 
which received four weekly doses of antigen exhibited the highest levels 
of protection. Significant protection was also observed in mice given 
three doses of antigen on days 0, 7, and 21. Secretory IgA antibody 
responses were highest for mice given a total of four doses of recombinant 
urease at one week intervals. On the basis of protection ratio and 
antibody response, the latter schedule was selected for further evaluation 
of therapeutic and prophylactic vaccine activity. 
TABLE 2 
______________________________________ 
Effect of different schedules of oral immunization on the 
prophylactic efficacy of urease vaccine 
VACCINE DOSE/ 
SCHEDULE (DAY 
IMMUNIZATION OF % PROTECTED 
GROUP (.mu.g).sup.a 
IMMUNIZATION) 
(#/TOTAL).sup.b 
______________________________________ 
1 25 0, 7, 14, 21 70 (7/10)* 
2 50 0, 14 20 (2/10) 
3 50 0, 14 30 (3/9) 
4 33.3 0, 7, 21 40 (4/10)* 
5 50, 25, and 25 
0, 7, 21 60 (6/10)* 
6 None 0, 7, 14, 21 0 (0/10) 
______________________________________ 
.sup.a Each group of mice were immunized orally with a total dose of 100 
.mu.g recombinant H. pylori urease. CT (10 .mu.g) was used at each 
immunization, suspended in sterile distilled water, as the mucosal 
adjuvant. Group 6 received CT alone on the same schedule as group 1 and 
acted as the control. Groups 4 and 5 compared the effect of booster doses 
administered after two previous weekly immunizations. 
.sup.b Percent protected (No. protected/No. tested) from a 10.sup.7 
challenge dose of H. felis two weeks postimmunization. Mice were 
sacrificed two weeks after challenge and protection was determined by the 
qualitative gastric urease assay. 
*p &lt; 0.05, Fisher's exact test, compared to mice given CT alone. 
The effect of different immunization schedules upon anti-urease antibody 
production was examined in mice. Antibody responses of mice immunized by 
one of the five different immunization schedules described in Table 2 were 
examined. Significant protection was observed in mice that received 
vaccine in four weekly doses or two weekly doses with a boost on day 21. 
Mice immunized by either of these two immunization schedules also had the 
highest average immune responses. The serum IgG and salivary IgA levels 
were highest in mice vaccinated on a schedule of four weekly doses. 
Dose-protection relationship 
Graded doses of recombinant urease were orally administered to mice to 
determine the minimal and optimal doses required for immunization and 
protection. Recombinant urease doses of 5, 10, 25, 50, and 100 .mu.g were 
administered to groups of eight mice by the oral route with 10 .mu.g CT in 
PBS. The antigen was given on a schedule of four weekly doses. As assessed 
by gastric urease and histologic bacterial score, significant protection 
of mice against challenge with H. felis was observed at all doses, with no 
significant differences between dose groups (Table 3). A dose response 
effect was clearly demonstrated in serum and mucosal antibody responses to 
recombinant urease, with the highest immune response at the 100 .mu.g dose 
level. 
TABLE 3 
______________________________________ 
Recombinant H. pylori urease at doses of 5 .mu.g or more, 
protects mice against challenge with H. felis 
% PROTECTED.sup.A 
ROUTE OF 
(# PROTECTED/TOTAL) 
IMMUNI- UREASE 
VACCINE ADJUVANT ZATION ASSAY HISTOLOGY 
______________________________________ 
none 10 .mu.g CT 
Oral 0 (0/7) 
0 (0/7) 
5 .mu.g rUre 
10 .mu.g CT 
Oral 86 (6/7)* 
71 (5/7)* 
10 .mu.g rUre 
10 .mu.g CT 
Oral 88 (7/8)* 
63 (5/8)* 
25 .mu.g rUre 
10 .mu.g CT 
Oral 100 (9/9)* 
100 (9/9)* 
50 .mu.g rUre 
10 .mu.g CT 
Oral 100 (8/8)* 
88 (7/8)* 
100 .mu.g rUre 
10 .mu.g CT 
Oral 100 (7/7)* 
71 (5/7)* 
______________________________________ 
.sup.a Percent protected (number mice with 0-20 bacteria per 
section/number tested) as determined by examination of silverstained 
stomach sections. 
*Comparison to shamimmunized controls, Fisher's exact test, p .ltoreq. 
0.02. 
The effect of recombinant urease dosage upon the anti-urease antibody 
response was examined in mice. Swiss-Webster mice were immunized orally 
with graded doses (0, 5, 10, 25, 50, or 100 .mu.g) of recombinant urease 
plus 10 .mu.g CT as a mucosal adjuvant. IgA antibody responses in serum, 
feces, and saliva increased with the amount of recombinant urease 
administered. A 100 .mu.g dose of recombinant urease produced the highest 
antibody levels. 
Swiss-Webster mice were orally immunized with graded doses (0, 5, 25, or 
100 .mu.g) of recombinant urease, with 25 .mu.g enterotoxigenic E. coli 
heat-labile toxin (LT) as a mucosal adjuvant. One group of mice received 
25 .mu.g recombinant urease and 10 .mu.g CT as a mucosal adjuvant for 
comparison. The mice were immunized orally 4 times every 7 days. Serum, 
feces, and saliva were collected 10 to 13 days after the last immunization 
and urease-specific antibody levels were determined by ELISA. Mice 
immunized with recombinant urease and LT developed serum and secretory 
antibodies against urease, with a clear dose-response effect. Strong 
salivary IgA antibody responses were observed in these animals. 
Mice immunized with urease and LT as described above were challenged with 
10.sup.7 H. felis (see FIG. 4). The pre-challenge antibody responses of 
the urease/LT immunized mice were correlated with 
histologically-determined bacterial scores (FIGS. 6A, 6B 6C and 6D). Fully 
protected animals (0 bacterial score) and those with low-grade infections 
(1+ bacterial score) had higher levels of antibodies in all compartments 
than animals with more severe infections. A few protected mice had no 
detectable immune response, suggesting that immune mediators other than 
IgA antibodies play a role in eradication of H. felis infection. 
The ability of high doses of recombinant urease to elicit a mucosal immune 
response was examined by administering intragastrically 1 .mu.g, 200 
.mu.g, or 5 mg without adjuvant. One group of mice were intragastrically 
immunized with 200 .mu.g urease plus 10 .mu.g CT as a control. Blood, 
feces, and saliva were collected 5 to 8 days after the last immunization. 
Animals were then challenged with 10.sup.7 H. felis 10 days after the last 
immunization and sacrificed 14 days after challenge. H. felis infection 
was detected by urease activity in stomach tissue. 
High doses of recombinant urease elicited urease-specific serum IgG, but 
elicited comparatively low levels of mucosal antibodies as detected by 
ELISA. Animals which exhibited a urease-specific serum IgG response were 
fully susceptible to H. felis challenge, indicating that serum IgG does 
not play a role in protection. 
In summary, data from the above experiments to investigate different 
administration routes, administration schedules, and mucosal adjuvants 
demonstrate that, when administered with an effective mucosal adjuvant, 
oral or intragastric administration of recombinant urease at relatively 
low doses elicits secretory IgA antibody and serum IgA and IgG responses. 
Secretory IgA antibody provides protection while serum IgG responses do 
not. When protection is measured by histological bacterial counts, animals 
with higher IgA antibody titers were fully protected, or had significantly 
reduced infections, compared to animals with lower IgA antibody titers. 
Animals which did not exhibit detectable IgA antibody levels developed 
severe infections after challenge. Antibody responses were dose-dependent 
and differed by the schedule of administration of antigen. The highest 
levels were achieved at antigen doses of 100-200 .mu.g. Administration of 
four doses of antigen at one week intervals provided the optimal schedule 
for immunization. 
An additional administration route, intranasal, was investigated, as 
follows: 
Intranasal vaccination with recombinant urease 
Swiss Webster mice were immunized either orally or intranasally (IN) with 
recombinant urease, or formalin-fixed urease. The amounts of antigen and 
adjuvant, routes of administration (IN or oral), and immunization 
schedules are shown in FIG. 7. The formalin-fixed urease (Form-ure) was 
prepared according to the following protocol: 
(1) 1 mg vial of recombinant urease is reconstituted with 150 .mu.L of 
RO/DI water; 
(2) 50 .mu.L of formalin (37% formaldehyde) diluted 1:1000 in RO/DI water 
is added to the 1 mg vial (final concentration of urease is 5 mg/ml); 
(3) the vial is then incubated at 35.degree. C. for 48 hours. 
Serum IgG, IgA, fecal IgA and salivary IgA responses in IN+CT group were 
higher than the oral+CT group, despite higher doses of rUrease and 
adjuvants that were used for oral immunization (FIG. 7 and Table 4). 
Protection was measured by urease test, as well as by determining bacterial 
score on stomach tissues of sacrificed animals. One hundred percent 
protection was found in the IN group, as compared to 80% in the oral group 
when protection was assayed by urease test. Seven of eight mice from 
orally immunized animals were positive for bacteria on their stomach 
tissues, whereas only 1/10 of IN group was positive for bacteria (see 
groups 3 and 6 in Table 4 and FIGS. 8A, 8B, 8C, and 8D). 
In a second experiment, mice were immunized either intragastrically (IG) or 
intranasally with rUrease co-administered with LT as mucosal adjuvant (see 
FIG. 9 for details of experiment and immunization schedule). In this 
experiment, serum IgG, IgA and saliva IgA responses in the IN+LT group 
were higher than the IG+LT group, despite higher doses of antigen and 
adjuvant that were used for IG immunizations (FIGS. 10A 10B, 10C 10D and 
10E, and Table 5, groups 4 and 8). Mice in the IN+LT group were fully 
protected against H. felis challenge (10/10 compared with 3/9 untreated 
group) as assessed by urease test (see groups 4 and 5 of Table 5). 
TABLE 4 
__________________________________________________________________________ 
Urease 4 h 
Mouse # 
treatment 
Route/adj. 
serum G 
serum A 
fecal A 
salivary A 
urease 
Bact. 
Path. 
__________________________________________________________________________ 
1A1 10 .mu.g/HCHO 
IN/none 
3.398 
3.318 
3.377 
3.339 
+ 
1A2 3.119 
3.218 
0.786 
3.249 
+ 
1A3 2.967 
2.535 
0.593 
3.153 
1A4 3.064 
2.390 
1.065 
3.311 
+ 
1A5 3.009 
2.647 
0.617 
3.244 
+ 
1B1 2.994 
2.068 
0.497 
3.329 
- 
1B2 3.099 
2.611 
-0.010 
3.235 
+ 
1B3 3.306 
1.823 
0.323 
3.295 
+ 
1B4 3.424 
2.931 
0.522 
3.372 
+ 
1B5 3.175 
0.832 
0.803 
2.439 
+ 
2A1 10 .mu.g 
IN/none 
3.021 
2.562 
2.090 
3.204 
+ 
2A2 2.971 
2.806 
1.475 
3.304 
+ 
2A3 2.894 
1.943 
0.584 
3.288 
+ 
2A4 2.918 
2.804 
2.291 
3.363 
+ 
2A5 2.926 
3.264 
0.118 
3.418 
+ 
2B1 3.220 
3.505 
1.634 
3.415 
+ 
2B2 3.383 
2.708 
0.410 
3.336 
+ 
2B3 3.092 
1.814 
0.411 
3.253 
+ 
2B4 3.033 
3.090 
2.672 
3.266 
+ 
2B5 3.055 
2.076 
1.193 
3.263 
+ 
3A1 10 .mu.g 
IN/CT 
2.969 
0.405 
0.192 
1.670 
- 0 2 
3A2 2.871 
0.097 
0.154 
0.526 
- 0 2 
3A3 2.984 
0.677 
0.081 
0.473 
3A4 3.092 
0.563 
1.824 
3.398 
- 
3A5 3.335 
0.256 
1.052 
0.587 
- 1 2 
3B1 3.013 
0.148 
0.142 
0.586 
- 0 2 
3B2 3.068 
0.388 
0.867 
2.410 
- 0 2 
3B3 3.008 
0.255 
0.468 
0.778 
- 0 2 
3B4 2.908 
0.985 
1.086 
1.172 
- 0 2 
3B5 2.879 
0.186 
1.904 
1.317 
- 0 2 
4A1 100 .mu.g/HCHO 
oral/none 
-0.006 
0.024 
0.021 
0.009 
+ 
4A2 0.050 
0.097 
0.079 
0.041 
+ 
4A3 0.001 
0.097 
0.056 
-0.019 
+ 
4A4 3.195 
0.598 
0.190 
2.097 
+ 0 3 
4A5 1.271 
0.027 
0.034 
0.010 
+ 
4B1 0.096 
0.045 
0.165 
0.309 
+ 
4B2 0.021 
0.033 
0.043 
0.059 
+ 
4B3 1.109 
0.055 
0.080 
0.019 
+ 
4B4 0.006 
0.022 
0.083 
0.017 
+ 
4B5 0.013 
0.027 
0.039 
0.010 
+ 
5A1 100 .mu.g/-- 
oral/none 
0.032 
0.042 
0.151 
0.030 
+ 
5A2 0.756 
0.077 
2.957 
0.156 
+ 
5A3 2.362 
0.075 
.0.104 
0.262 
+ 
5A4 0.012 
0.034 
0.146 
0.065 
+ 
5A5 0.011 
0.035 
0.163 
0.013 
+ 
5B1 0.012 
0.042 
0.089 
0.017 
+ 
5B2 0.017 
0.017 
0.076 
0.049 
+ 
5B3 1.583 
0.058 
0.058 
0.182 
+/- 
5B4 0.602 
0.030 
0.093 
0.093 
+ 
5B5 1.672 
0.059 
0.060 
0.093 
+ 
6A1 25 .mu.g/-- 
oral/CT 
0.698 
0.267 
0.076 
0.387 
- 1 2 
6A2 2.139 
0.065 
0.089 
0.206 
- 1 2 
6A3 0.006 
0.013 
0.098 
0.011 
- 3 2 
6A4 2.957 
0.354 
0.185 
2.999 
- 1 2 
6A5 0.011 
0.019 
0.042 
0.026 
- 0 2 
6B1 0.000 
0.022 
0.041 
0.005 
- 1 2 
6B2 0.009 
0.002 
0.067 
0.069 
- 2 2 
6B3 0.320 
0.022 
0.041 
0.048 
+ 4 2 
6B4 0.010 
0.009 
-0.021 
0.023 
+ 4 2 
6B5 2.633 
0.090 
0.047 
0.647 
- 1 2 
7A1 none 0.011 
0.018 
0.074 
0.019 
+ 4 1 
7A2 0.007 
0.025 
0.042 
-0.004 
+ 4 2 
7A3 0.000 
0.019 
0.082 
0.018 
+ 4 2 
7A4 0.004 
0.028 
0.024 
-0.002 
+ 4 2 
7A5 0.011 
0.005 
0.041 
0.029 
X 
7B1 0.019 
0.004 
0.089 
-0.005 
+ 4 1 
7B2 0.006 
-0.003 
-0.009 
0.008 
+ 4 2 
7B3 0.006 
0.024 
0.066 
0.167 
+ 4 2 
7B4 0.015 
0.018 
0.063 
0.018 
+ 4 1 
7B5 0.007 
0.023 
0.111 
0.011 
+ 
plate 1 controls 
+ 2.265 
3.280 
1.651 
3.315 
- -0.010 
0.040 
0.065 
0.056 
plate 2 controls 
+ 1.969 
3.289 
1.700 
3.298 
- -0.001 
0.034 
-0.004 
0.019 
__________________________________________________________________________ 
TABLE 5 
______________________________________ 
serum serum salivary 
2 week sac 
Mouse # 
Treatment G A A A (550) 
______________________________________ 
1AN 25 .mu.g 5X IN 
3.530 0.350 0.340 0.802 
1AL 200 .mu.g 2X IG 
3.263 0.154 1.544 0.768 
1AR 3.231 0.630 0.204 0.805 
1ALL 3.233 0.318 0.251 0.806 
1BN 3.344 0.401 0.112 0.726 
1BL 3.322 0.724 2.015 0.168 
1BR 3.424 0.225 0.426 0.748 
1BLL 3.285 0.591 0.719 0.807 
1BRR 3.262 0.141 0.202 0.675 
2AN 25 .mu.g 5X IN 
3.164 0.054 0.014 0.814 
2AL 200 .mu.g 1X IG 
3.232 0.231 0.747 0.823 
2AR 3.236 0.981 0.412 0.695 
2ALL 3.303 0.122 0.832 0.813 
2BN 3.357 0.690 0.993 0.747 
2BL 3.264 1.002 0.840 0.704 
2BR 3.284 1.377 0.934 0.161 
2BLL 3.241 0.458 0.191 0.748 
2BRR 3.280 0.129 0.134 0.720 
3AN 25 .mu.g 2X IN 
0.165 0.031 0.001 0.791 
3AL 200 .mu.g 2X IG 
3.364 0.332 0.356 0.824 
3AR 1.731 0.031 -0.003 0.810 
3ALL 3.321 0.450 0.541 0.798 
3ARR 3.358 0.158 0.001 0.675 
3BN 3.199 0.276 0.212 0.820 
3BL 3.174 2.463 2.838 0.839 
3BR 3.274 0.607 1.517 0.481 
3BLL 3.284 1.279 2.593 0.829 
3BRR 3.254 0.427 1.684 0.852 
4AN 25 .mu.g 4X IN 
3.375 0.118 0.212 0.111 
4AL with 2 .mu.g LT 
3.482 0.346 0.546 0.107 
4AR 3.330 3.237 3.302 0.109 
4ALL 3.343 3.067 3.229 0.103 
4ARR 3.220 1.625 2.156 0.106 
4BN 3.287 1.263 2.943 0.224 
4BL 3.305 0.496 2.029 0.139 
4BR 3.283 0.809 2.307 0.114 
4BLL 3.313 1.019 3.346 0.106 
4BRR 3.474 0.674 1.683 0.119 
5AN none 0.075 0.023 0.001 0.26 
5AL 0.005 0.016 -0.002 0.881 
5AR 0.011 0.008 -0.001 0.839 
5ARR 0.005 0.007 0.000 0.834 
5BN 0.033 0.016 0.001 0.134 
5BL 0.013 0.009 0.004 0.814 
5BR 0.025 0.014 0.007 0.833 
5BLL 0.028 0.019 0.008 0.155 
5BRR 0.095 0.012 0.003 0.818 
6AN 25 .mu.g 5X IN 
3.353 0.680 0.498 
6AL 200 .mu.g 2X IG 
3.470 0.248 0.100 
6AR 3.387 1.236 0.741 
6ALL 3.362 0.409 0.322 
6ARR 3.244 1.036 1.056 
6BN 3.211 2.749 2.014 
6BL 3.286 0.360 0.328 
6BR 8.269 0.749 1.882 
6BLL 3.347 0.275 0.030 
6BRR 3.321 0.311 0.989 
7AN 25 .mu.g 4X IN 
3.415 0.756 2.584 
7AL with 2 .mu.g LT 
3.428 3.080 3.287 
7AR 3.249 2054 3.188 
7ALL 3.269 1.010 3.240 
7ARR 3.255 0.713 3.243 
7BN 3.310 3.081 3.274 
7BL 3.439 2.298 3.222 
7BR 3.359 1.026 1.757 
7BLL 3.370 1.441 3.025 
7BRR 3.330 0.689 1.169 
8AN 200 .mu.g 4X IG 
0.281 0.031 0.000 
8AL with 10 .mu.g LT 
3.234 0.925 1.872 
8AR 3.285 0.827 2.751 
8ALL 3.313 0.547 0.395 
8ARR 3.363 0.382 1.144 
8BN 3.407 1.189 1.880 
8BR 3.315 1.608 2.536 
8BLL 3.271 0.438 0.599 
9AN none 0.011 0.003 0.000 
9AL 0.017 0.005 0.001 
9AR 0.007 0.002 0.001 
9ALL 0.011 0.013 0.000 
9ARR 0.006 0.019 -0.002 
9BN 0.017 0.010 -0.001 
9BL 0.020 0.008 -0.002 
9BR 0.031 0.012 -0.002 
9BLL 0.005 0.016 0.005 
95BRR 0.019 0.011 0.000 
______________________________________ 
Further support for the efficacy of intranasal immunization is shown in 
FIG. 11. Briefly, this experiment showed that rUrease (10 .mu.g) 
administered by the intranasal route with CT (5 .mu.g) is at least as 
effective as rUrease (25 .mu.g) given by the oral route with CT (10 .mu.g) 
in preventing infection with H. felis. 
Selection of a mucosal adjuvant 
E. coli heat-labile toxin (LT), a multisubunit toxin with A and B 
components, is closely related, both biochemically and immunologically, to 
CT. Because the toxicity of LT is lower than CT, LT is likely to be more 
acceptable as a mucosal adjuvant for use in humans (Walker and Clements, 
Vaccine Res. 2:1-10, 1993). 
Mice were orally immunized with 5 .mu.g, 25 .mu.g, or 100 .mu.g recombinant 
urease and 25 .mu.g recombinant LT (Swiss Serum Vaccine Institute, Berne, 
Switzerland) for a total of four weekly doses. Controls received LT only, 
or 25 .mu.g urease vaccine with CT. Mice were challenged two weeks after 
the fourth dose and necropsied two weeks after challenge. 
Gastric urease assays indicated significant protection or suppression of 
the infection in immunized animals at all vaccine doses (Table 6). 
Histological assessment confirmed significant reductions in bacterial 
scores in mice which received .ltoreq.5 .mu.g urease and LT. Protection 
was directly correlated with dose as determined by both gastric urease 
assay and histological examination, with the highest protection conferred 
by 100 .mu.g of recombinant urease co-administered with LT. Antibody 
determinations confirmed a dose-response relationship, with highest 
mucosal immune responses at the 100 .mu.g dose. When recombinant urease 
was administered at equivalent doses (25 .mu.g), LT was superior to CT as 
a mucosal adjuvant. These data indicate that while CT enhances the mucosal 
immune response to orally administered recombinant urease, LT is a better 
mucosal adjuvant and is thus the preferred over CT for co-administration 
with recombinant urease. 
TABLE 6 
______________________________________ 
E. coli heat-labile enterotoxin (LT) as a mucosal adjuvant 
for immunization with recombinant H. pylori urease 
% PROTECTED 
(# PROTECTED/TOTAL).sup.a 
VACCINE ADJUVANT UREASE ASSAY HISTOLOGY 
______________________________________ 
None 25 .mu.g LT 
0 (0/9) 0 (0/9) 
25 .mu.g rUre 
10 .mu.g CT 
70 (7/10)* 60 (6/10)* 
5 .mu.g rUre 
25 .mu.g LT 
787 (7/9)* 56 (5/9)* 
25 .mu.g rUre 
25 .mu.g LT 
90 (9/10)* 80 (8/10)* 
100 .mu.g rUre 
25 .mu.g LT 
100 (8/8)* 100 (8/8)* 
______________________________________ 
.sup.a Percent protected (number of mice with negative urease assay or 
bacterial score 0-1+/number tested) determined by bacterial scores of 
silverstained stomach sections. 
*Comparison to shamimmunized controls, Fisher's exact test, p &lt; 0.03. 
To determine the adjuvant activity of lower doses of LT, mice were orally 
immunized with 25 .mu.g of recombinant urease administered with 1, 5, 10, 
or 25 .mu.g of LT. Similar protection ratios and antibody responses were 
observed at all LT doses. 
Several studies were performed to assess adjuvants other than CT and LT, 
and to determine whether the requirement for a mucosal adjuvant could be 
eliminated by administration of antigen alone at a high dose. Cholera 
toxin B subunit (CTB) was compared with CT as a mucosal adjuvant for 
urease immunization. No protective effect was observed when 200 .mu.g 
urease was co-administered intragastrically with 100 .mu.g CTB 
(Calbiochem, La Jolla, Calif.) in 0.24M sodium bicarbonate, whereas the 
same amount of urease with 10 .mu.g CT gave 100% protection as assessed by 
gastric urease activity. 
An orally-active semi-synthetic analogue of muramyl dipeptide, GMDP, 
(N-acetylgluosaminyl-(b1-4)-N-acetyl-muramyl-L-alanyl-D-isoglutamine), was 
co-administered with 25 .mu.g urease at doses of 2, 20, and 200 .mu.g. 
GMDP co-administered with recombinant urease failed to protect mice 
against H. felis challenge. 
Large doses (200 .mu.g, 1 mg, or 5 mg) of recombinant urease with or 
without CT were intragastrically administered to mice once a week for four 
weeks. The intragastric route was required because volumes for high 
antigen doses exceeded those that could be given orally in a reproducible 
fashion. NaHCO.sub.3 was co-administered to neutralize gastric acid. A 
total of four doses of antigen were administered at ten day intervals. 
Blood, feces, and saliva were collected five to eight days after the last 
immunization. Animals were challenged with 1.times.10.sup.7 H. felis, and 
infection was determined by urease activity in stomach tissue. 
In the absence of CT, high antigen doses did not confer protection against 
H. felis challenge, whereas controls given 200 .mu.g of recombinant urease 
with CT were significantly protected (Table 7). Urease-specific serum IgG 
was induced at the high recombinant urease doses without adjuvant, but 
serum, fecal, and salivary IgA responses were absent or minimal. 
Histological examination of coded specimens from animals given 5 mg doses 
of urease revealed no differences in bacterial scores or leukocytic 
infiltrates, compared with sham-immunized animals. 
TABLE 7 
__________________________________________________________________________ 
A mucosal adjuvant facilitates protection by recombinant 
urease 
% protected 
Mean antibody levels (.+-.SD) 
(# protected/ 
before challenge.sup.a 
Vaccine 
Adjuvant 
tested) 
Serum IgG 
Serum IgG 
Fecal IgA 
Salivary IgA 
__________________________________________________________________________ 
none PBS 0 (0/5) 
0.01 0.03 0.01 0.02 
(.+-.0.00) 
(.+-.0.07) 
(.+-.0.02) 
(.+-.0.03) 
200 .mu.g rUre 
10 .mu.g CT 
88 (7/8)* 
&gt;2.97 &gt;1.02 0.16 0.59 
(.+-.1.84) 
(.+-.1.41) 
(.+-.0.14) 
(.+-.0.73) 
200 .mu.g rUre 
none 0 (0/9) 
&gt;2.06 0.07 0.02 0.06 
(.+-.1.98) 
(.+-.0.11) 
(.+-.0.02) 
(.+-.0.13) 
1 mg rUre 
none 0 (0/9) 
&gt;3.28 0.12 0.02 0.18 
(.+-.1.48) 
(.+-.0.17) 
(.+-.0.03) 
(.+-.0.40) 
5 mg rUre 
none 0 (0/9) 
&gt;2.68 &gt;0.22 0.02 0.08 
(.+-.1.98) 
(.+-.0.17) 
(.+-.0.02) 
(.+-.0.11) 
__________________________________________________________________________ 
.sup.a Mean (.+-.SD) ureasespecific serum IgG or IgA expressed as 
A.sub.405 units of serum (diluted 1:100), fecal extract (diluted 1:20), o 
saliva (diluted 1:5). Means are shown as &gt; when individual values of 4.0 
(the maximum A.sub.405 reading) were included in calculating the mean 
values. 
*Comparison to shamimmunized controls, Fisher's exact test, p = 0.005. 
Therapeutic immunization of mice with H. felis gastritis 
The ability of recombinant urease vaccine to cure infection in mice was 
evaluated by intragastrically infecting Balb/c mice with 10.sup.7 H. felis 
(FIG. 12). Four weeks after infection, groups of infected mice were given 
four weekly oral doses of 100 .mu.g recombinant urease with 10 .mu.g LT. 
Controls received LT only. 
Ten of the mice in each group were necropsied four weeks after the final 
immunization to examine the degree of Helicobacter infection by 
quantitative gastric urease. Four weeks after immunization, nine of ten 
Balb/c mice (90%) were free from infection as measured by quantitative 
urease assay, whereas all controls were infected. 
At four weeks, twelve animals receiving LT and 40 animals receiving 
urease+LT were reinfected with H. felis. Ten weeks after the challenge, 
the twelve animals receiving LT and 40 animals receiving Urease+LT were 
reinfected with H. felis. Ten weeks after this challenge, animals were 
sacrificed to determine the extent of infection by quantitative urease 
assay. Of the nine animals which were given urease+LT, but not subjected 
to reinfection, 5 were protected (57%), as determined by gastric urease 
activity. All twelve LT treated animals which were rechallenged were 
infected. Thirty seven of the 40 mice (93%) which were given urease+LT, 
and then re-challenged with H. felis, were protected as determined by 
reduced gastric urease activity. This experiment shows that urease 
vaccination not only eradicates an existing Helicobacter infection, but 
also protects the host against reinfection. 
Five of fourteen (36%) immunized Swiss-Webster mice were cured or had 
reduced infections, whereas all control animals were infected, although 
the differences in infection ratios between groups was not significant 
(p=0.26, Fisher's exact test, two-tailed). Infection in Swiss-Webster mice 
was more severe than in Balb/c mice, as measured by higher mean gastric 
urease activity in unimmunized animals (p&lt;0.0001, one-way ANOVA), possibly 
explaining the lower cure rates in Swiss-Webster versus Balb/c mice. 
Differences in susceptibility of mouse strains to H. felis has been noted 
(Sakagami et al., Am. J. Gastroenterol 89:1345, 1994). 
By histologic assessment, all unimmunized Balb/c mice had 4+ infections 
(&gt;100 bacteria/section), whereas reduced bacterial scores were seen in 43% 
of immunized mice at four weeks. At ten weeks, four of six had reduced 
urease activity, although only one of six had a reduced bacterial score. 
The role of antibodies in Helicobacter therapy 
The role of anti-urease antibodies in Helicobacter therapy, i.e., the 
clearance of H. felis from infected mice, was examined by first infecting 
Balb/c mice with 10.sup.7 H. felis. Four weeks after infection, the mice 
were orally immunized with 200 .mu.g recombinant urease plus 10 .mu.g CT. 
Control mice were given 10 .mu.g CT only. Antigen was administered 4 times 
at one week intervals. Animals were sacrificed 4 and 10 weeks after the 
last immunization, and serum and fecal samples were collected for ELISA. 
Mice infected with H. felis produced serum anti-urease IgG antibodies, but 
no secretory anti-urease IgA response was detected. However, infected 
animals immunized with urease/CT exhibited high secretory anti-urease IgA 
antibody responses (FIG. 13). There was no significant difference in 
urease specific mucosal IgA levels between immunized mice that remained 
infected and those with reduced bacterial scores. 
These data indicate that H. felis infection does not elicit a secretory 
anti-urease response. Thus, suppression of the IgA antibody response may 
play a role in H. felis's ability to evade clearance by the immune system. 
In contrast, immunization of H. felis-infected mice with urease and a 
mucosal adjuvant resulted in strong mucosal anti-urease responses, which 
correlated with clearance of the infection H. felis. 
Correlation of protection to Helicobacter infection with gastric immune 
responses 
Several of the experiments with the mouse infection model showed that some 
animals rendered resistant to infection by recombinant urease vaccine 
lacked detectable antibody responses or had low antibody levels in serum, 
saliva, or feces. Therefore, the immune response was measured in the 
gastric mucosa itself to determine if such measurements could be more 
precisely correlated with protection. 
Immune responses in gastric mucosa were assessed by detecting IgA 
antibodies and IgA+ antibody secreting cells in intestinal and gastric 
murine tissue by immunohistochemistry. Portions of the stomach comprising 
pylorus-proximal duodenum, antrum, corpus, and cardia were mounted in OCT 
compound, flash-frozen, and cryosectioned. Sections (7 .mu.m thick) were 
fixed in cold acetone, and IgA+ cells were identified by staining with 
biotinylated monoclonal anti-IgA, followed by avidin conjugated to 
biotinylated glucose oxidase (ABC-GO, Vector Laboratories), and 
counterstained with methyl green. Urease-specific antibody secreting cells 
(ASC) were identified by sequentially incubating sections with recombinant 
urease, rabbit anti-urease, biotinylated donkey anti-rabbit Ig (Amersham, 
Arlington Heights, Ill.), ABC-GO, TNBT, and methyl green. Control sections 
were incubated without urease or urease plus rabbit anti-urease to 
determine reactivity with the donkey secondary reagent and background 
endogenous glucose oxidase activity. Cryosections of cell pellets from an 
IgA hybridoma against H. felis ureB (MAB71, S. Czinn, Case-Western Reserve 
University) and an irrelevant IgA monoclonal (HNK20) against F 
glycoprotein of respiratory syncytial virus served as positive and 
negative controls, respectively. 
Swiss-Webster mice were immunized orally with four weekly doses of 100 
.mu.g recombinant urease plus adjuvant (CT). Control mice received 
adjuvant only. Groups of three mice each were necropsied at 3, 7, 14, or 
21 days after the last immunization. Peyer's patches were removed from the 
intestines and lamina propria lymphocytes (LPL) were isolated separation 
on a 40-70% Percoll gradient. IgA+ B cells were detected by ELISPOT assays 
in 96-well filter plates coated with 1 .mu.g/well recombinant urease and 
blocked with bovine serum albumin. Ten-fold serial dilutions of LPL were 
added to the wells, starting at 1.times.10.sup.6 cells. IgA+ ASC were 
detected with a biotinylated anti-mouse IgA reagent followed by 
streptavidin-alkaline phosphatase, and positive cells were counted by 
microscopy. 
Anti-urease IgA+ ASC were found by ELISPOT in intestinal lamina propria as 
early as three days after the last immunization, peaked at seven days, and 
diminished thereafter (Table 8). Urease-specific ASC represented 
.about.10% of the total IgA+ cells observed on immunohistochemistry. 
Two-color immunofluorescence microscopy confirmed that urease-specific ASC 
were also IgA+. These observations confirm that an intense anti-urease IgA 
response occurs at the level of the intestinal mucosa after oral antigenic 
stimulation. The chronology and kinetics of this response are similar to 
those described for other oral vaccines (Czerkinsky et al., Infect. Immun. 
59:996-1001, 1991; McGhee & Kiyono, 1993, ibid.) 
TABLE 8 
______________________________________ 
Kinetics of induction of anti-urease IgA secreting cells 
after oral immunization with recombinant urease 
NUMBER OF ASC/10.sup.6 LAMINA 
PROPRIA LYMPHOCYTES 
IMMUNIZATION 
DAY 3.sup.a 
DAY 7 DAY 14 DAY 21 
______________________________________ 
recombinant 17.sup.c 7400 10 2 
urease + CT.sup.b 
PBS + CT 1 200 0 0 
______________________________________ 
.sup.a Day after the last oral immunization. 
.sup.b Cholera toxin. 
.sup.c Average value from duplicate wells containing intestinal 
lymphocytes from three separate mice. 
To determine whether IgA+ cells are recruited into the gastric mucosa, 
stomachs of orally immunized mice were examined by immunohistochemistry, 
as described above. IgA+ cells were virtually absent in gastric mucosa of 
immunized and control mice, indicating that the stomach is immunologically 
"silent" until stimulated by Helicobacter challenge. 
The role of the stomach as an immunological effector organ in challenged, 
immunized mice was examined. Mice were given four weekly oral doses of 200 
.mu.g recombinant urease with 10 .mu.g CT. Control mice received CT only. 
One week after immunization, the mice were challenged. Mice were 
necropsied prior to challenge and at 7, 14, 28, 70, and 133 days after 
challenge. 
Prior to challenge, no IgA+ ASC were found. At all time intervals after 
challenge, IgA+ ASC were present in large numbers in the gastric mucosae 
of immunized mice, with a peak at seven days (FIG. 14). The number of IgA+ 
ASC greatly exceeded that in unimmunized (CT only) mice, especially 7-28 
days after challenge. The anatomical localization of IgA+ ASC also 
differed, with immunized mice having cells throughout the mucosa, in the 
lamina propria, and around the crypts, but rarely under the surface 
epithelium. Urease-specific and IgA+ASC revealed that the majority of 
urease-specific cells in gastric mucosa were IgA+. 
These observations indicate that the gastric mucosa of hosts primed by 
prior immunization becomes immunologically activated only after antigenic 
stimulation by H. felis. The resulting tissue response is characterized by 
rapid, intense, and long-lasting recruitment of IgA+ B cells, many of 
which are urease specific. This response is quantitatively greater than in 
immunologically naive mice after challenge. Moreover, the localization of 
IgA+ cells in immunized mice differs from that seen in immunologically 
naive mice that are challenged and become persistently infected with H. 
felis. The enhanced IgA+ ASC response in gastric mucosa suggests a basis 
for the protection conferred by immunization against H. felis challenge. 
The data are concordant with studies of cholera vaccine, in which 
immunological memory responses triggered within hours after bacterial 
challenge were sufficient to provide protection (Lycke & Holmgren, Scand. 
J. Immunol. 25:407-412, 1987). 
Correlation of the gastric immune response and bacterial load 
The relationship between the gastric tissue immune response and bacterial 
infection was defined at the structural level. Swiss-Webster mice were 
immunized with 4 weekly doses of 200 .mu.g recombinant urease with 10 
.mu.g CT. One week after the last immunization, the mice were challenged 
with 10.sup.7 H. felis. Animals were sacrificed 0, 1, 7, 14, 28, 70, and 
133 days after challenge, and H. felis colonization was assessed by light 
and electron microscopy. 
Within 24 hours after challenge, both immunized and unimmunized mice had 
substantial numbers of H. felis within the lumen of gastric pits (FIG. 
15). Within seven days after challenge, the bacteria were cleared from the 
immunized mice, but were still present in high numbers in the gastric pits 
and the lumens of unimmunized mice. Bacteria were also associated with the 
apical membrane of mucus-secreting cells of the unimmunized mice. The 
clearance of bacteria from the gastric mucosa of immunized mice 
corresponded to the appearance of IgA+ ASC and anti-urease ASC in the 
gastric tissue. 
These results suggest the following sequence of events: 1) challenge of 
immunized mice results in transient colonization of gastric epithelium 
with H. felis; 2) unimmunized animals remain infected, while animals 
immunized with recombinant urease clear the bacteria from the stomach 
during the first week after challenge; and 3) clearance of infection is 
associated with the recruitment of IgA+ urease-specific ASC to gastric 
mucosa. A similar mechanism may be responsible for the clearance of 
bacteria from the gastric mucosa of chronically infected animals that are 
subjected to therapeutic immunization. 
Antigenic conservation of urease among strains of H. pylori 
The ability of various antisera to bind multiple clinical isolates of H. 
pylori was tested. The antisera included M, a hyperimmune rabbit serum 
prepared against purified H. pylori urease, and sera and secretions 
(gastric wick samples and saliva) from mice immunized with recombinant 
urease. Antibody preparations were tested by immunoblotting for 
recognition of the homologous H. pylori strain (Hp630), ATCC 43504 type 
strain, and five clinical isolates from ulcer patients at St. 
Bartholomew's Hospital, London, collected within the last five years. 
All antisera recognized the UreA and UreB subunits of all H. pylori 
strains, as well as purified native and recombinant H. pylori ureases, 
native and recombinant H. felis urease, and native H. mustelae urease. In 
addition, urease-specific IgA antibodies in gastric secretions and saliva 
of immunized mice reacted with both UreA and UreB of all H. pylori 
strains, as well as heterologous ureases. Immunologic recognition was 
greater for UreB than for UreA. Sham-immunized mice showed no reactivity 
with any urease subunits. These results demonstrate that H. pylori strains 
express ureases that are highly conserved at the antigenic level. Thus, 
antigenic variation among H. pylori strains is not a significant factor 
for development of a recombinant urease vaccine. 
Identification of human patients for administration of recombinant H. 
pylori urease vaccine 
The recombinant H. pylori urease vaccine of the invention may be 
administered to uninfected individuals as a prophylactic therapy or to 
individuals infected with Helicobacter as an antibacterial therapy. 
Individuals selected for prophylactic administration of recombinant urease 
include any individual at risk of Helicobacter infection as based upon 
age, geographical location, or the presence of a condition which renders 
the individual susceptible to Helicobacter infection. Individuals at 
particularly high risk of infection, or who would be most severely 
affected by infection, include individuals in developing countries, 
infants and children in developing and in developed countries, individuals 
with naturally or artificially low gastric acid pH, submarine crews, and 
military personnel. 
Individuals who may receive the recombinant H. pylori urease vaccine as a 
therapeutic include those individuals with symptoms of gastritis or other 
gastrointestinal disorders which may be associated with H. pylori 
infection. The clinical symptoms associated with gastritis, an 
inflammation of the stomach mucosa, include a broad range of 
poorly-defined, and generally inadequately treated, symptoms such as 
indigestion, "heart burn," dyspepsia, and excessive eructation. A general 
discussion of gastritis appears in Sleisenger and Fordtran, In 
Gastrointestinal Disease, 4th Ed., Saunders Publishing Co., Philadelphia, 
Pa., pp. 772-902, 1989. 
Individuals who have a gastrointestinal disorder may also be treated by 
administration of the vaccine of the invention. Gastrointestinal disorders 
includes any disease or other disorder of the gastrointestinal tract of a 
human or other mammal. Gastrointestinal disorders include, for example, 
disorders not manifested by the presence of ulcerations in the gastric 
mucosa (non-ulcerative gastrointestinal disorder), including chronic or 
atrophic gastritis, gastroenteritis, non-ulcer dyspepsia, esophageal 
reflux disease, gastric motility disorders, and peptic ulcer disease 
(e.g., gastric and duodenal ulcers). Peptic ulcers involve ulceration and 
formation of lesions of the mucous membrane of the esophagus, stomach, or 
duodenum, and is generally characterized by loss of tissue due to the 
action of digestive acids, pepsin, or other factors. Alternatively, it may 
be desirable to administer the vaccine to asymptomatic individuals, 
particularly where the individual may have been exposed to H. pylori or 
has a condition rendering the individual susceptible to infection. 
Infection with Helicobacter can be readily diagnosed by a variety of 
methods well known in the art, including, e.g., by serology, .sup.13 C 
breath test, and/or gastroscopic examination. 
Preparation of purified recombinant urease for administration to patients 
The process for formulation of recombinant urease involves combination of 
the urease with a stabilizer, (e.g., a carbohydrate mannitol) and freeze 
drying (i.e., lyophilizing) the product. This process prevents degradation 
by aggregation and fragmentation. In addition, the product is stable for 
months following lyophilization. The mechanism for instability involves 
formation of disulfide bonds between protein subunits and is effectively 
inhibited by lyophilization. 
Recombinant urease is freeze-dried following the final purification step. 
The purified protein product (approximately 4 mg/ml) is dialyzed against 
2% sucrose, and this solution is transferred to lyophilization vials. The 
vialed solution is either: (1) frozen in liquid nitrogen, and then placed 
into the lyophilizer, or (2) cooled to 4.degree. C., and then placed in 
the lyophilizer, where it is frozen to -40.degree. C., or lower. 
Lyophilization is carried out using standard methods. The freeze-dried 
product may be reconstituted in water. 
Mode of administration to human patients 
Recombinant H. pylori urease is administered to a mucosal surface of the 
individual in order to stimulate a mucosal immune response effective to 
provide protection to subsequent exposure to Helicobacter and/or 
facilitate clearance of a pre-existing Helicobacter infection. Preferably, 
recombinant urease is administered so as to elicit a mucosal immune 
response associated with production of anti-urease IgA antibodies and/or 
infiltration of lymphocytes into the gastric mucosa. The recombinant 
urease may be administered to any mucosal surface of the patient. 
Preferable mucosal surfaces are intranasal or oral. In the case of oral 
administration, it is preferable that the administration involves 
ingestion of the vaccine, but the vaccine may also be administered as a 
mouth wash, so that an immune response is stimulated in the mucosal 
surface of the oral cavity, without actual ingestion of the vaccine. 
Alternatively, a systemic mucosal immune response may be achieved by 
administration of the vaccine to a mucosal surface of the eye in the form 
of, e.g., an eye drop or an intraocular implant. 
Dosages of recombinant H. pylori urease administered to the individual as 
either a prophylactic therapy or an antibacterial therapy can be 
determined by one skilled in the art. Generally, dosages will contain 
between about 10 .mu.g to 1,000 mg, preferably between about 10 mg and 500 
mg, more preferably between about 30 mg and 120 mg, more preferably 
between about 40 mg and 70 mg, most preferably about 60 mg recombinant H. 
pylori urease. 
At least one dose of the recombinant H. pylori urease will be administered 
to the patient, preferably at least two doses, more preferably four doses, 
with up to six or more total doses administered. It may be desirable to 
administer booster doses of the recombinant urease at one or two week 
intervals after the last immunization, generally one booster dose 
containing less than, or the same amount of, recombinant H. pylori urease 
as the initial dose administered. Most preferably, the vaccine regimen 
will be administered in four doses at one week intervals. 
Recombinant H. pylori urease may be co-administered with a mucosal 
adjuvant. The mucosal adjuvant may be any mucosal adjuvant known in the 
art which is appropriate for human use. For example, the mucosal adjuvant 
may be cholera toxin (CT), enterotoxigenic E. Coli heat-labile toxin (LT), 
or a derivative, subunit, or fragment of CT or LT which retains 
adjuvanticity. Preferably, the mucosal adjuvant is LT or a derivative of 
LT. The mucosal adjuvant is co-administered with recombinant H. pylori 
urease in an amount effective to elicit or enhance a mucosal immune 
response, particularly a humoral and/or a mucosal immune response. The 
ratio of adjuvant to recombinant urease may be determined by standard 
methods by one skilled in the art. Preferably, the adjuvant is present at 
a ratio of 1 part adjuvant to 10 parts recombinant urease. 
A buffer may be administered prior to administration of recombinant H. 
pylori urease in order to neutralize or increase the pH of the gastric 
acid. Any buffer that is effective in raising the pH of gastric acid and 
is appropriate for human use may be used. For example, buffers such as 
sodium bicarbonate, potassium bicarbonate, and sodium phosphate may be 
used. Preferably, oral administration of the vaccine is buffer-free, i.e., 
no amount of a pH-raising buffer compound effective to significantly 
affect gastric acid pH is administered to the patient either prior to, or 
concomitant with, administration of recombinant urease. 
The vaccine formulation containing recombinant urease may contain a variety 
of other components, including stabilizers, flavor enhancers (e.g., 
sugar), or, where the vaccine is administered as an antibacterial 
therapeutic, other compounds effective in facilitating clearance and/or 
eradication of the infecting bacteria. 
For prophylactic therapy, the vaccine containing recombinant H. pylori 
urease may be administered at any time prior to contact with, or 
establishment of, Helicobacter infection. Because the vaccine can also act 
as an antibacterial therapy, there is no contraindication for 
administration of the vaccine if there is marginal evidence or suspicion 
of a pre-existing Helicobacter infection (e.g., an asymptomatic 
infection). 
For use of the vaccine as an antibacterial therapy, recombinant H. pylori 
urease may be administered at any time before, during, or after the onset 
of symptoms associated with Helicobacter infection or with gastritis, 
peptic ulcers or other gastrointestinal disorder. Although it is not a 
prerequisite to the initiation of therapy, it may be preferable to confirm 
diagnosis of Helicobacter infection by .sup.13 C breath test, serology, 
gastroscopy, biopsy, or another Helicobacter detection method known in the 
art. 
The progress of immunized patients may be followed by general medical 
evaluation, screening for H. pylori infection by serology, .sup.13 C 
breath test, and/or gastroscopic examination. 
Example of human administration of recombinant H. pylori urease 
A vaccine composed of 60 mg of recombinant H. pylori urease in a total 
volume of 15 ml of water containing 2% w/v sucrose, pH 7.5 is orally 
administered to the patient. Administration of the vaccine is repeated at 
weekly intervals for a total of 4 doses. Symptoms are recorded daily by 
the patient. To determine adverse effects, physician interviews are 
performed weekly during the period of vaccine administration, as well as 1 
week and 1 month after the last immunization. Anti-urease antibodies are 
measured in serum and saliva, and antibody-secreting cells are monitored 
in peripheral blood collected 7 days after the last immunization. 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 3 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 2735 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
TAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCATCCACCTTGATTGCGT60 
TATGTCTTCAAGGAAAAACACTTTAAGAATAGGAGAATGAGATGAAACTCACCCCAAAAG120 
AGTTAGATAAGTTGATGCTCCACTACGCTGGAGAATTGGCTAAAAAACGCAAAGAAAAAG180 
GCATTAAGCTTAACTATGTAGAAGCAGTAGCTTTGATTAGTGCCCATATTATGGAAGAAG240 
CGAGAGCTGGTAAAAAGACTGCGGCTGAATTGATGCAAGAAGGGCGCACTCTTTTAAAAC300 
CAGATGATGTGATGGATGGCGTGGCAAGCATGATCCATGAAGTGGGTATTGAAGCGATGT360 
TTCCTGATGGGACTAAACTCGTAACCGTGCATACCCCTATTGAGGCCAATGGTAAATTAG420 
TTCCTGGTGAGTTGTTCTTAAAAAATGAAGACATCACTATCAACGAAGGCAAAAAAGCCG480 
TTAGCGTGAAAGTTAAAAATGTTGGCGACAGACCGGTTCAAATCGGCTCACACTTCCATT540 
TCTTTGAAGTGAATAGATGCCTAGACTTTGACAGAGAAAAAACTTTCGGTAAACGCTTAG600 
ACATTGCGAGCGGGACAGCGGTAAGATTTGAGCCTGGCGAAGAAAAATCCGTAGAATTGA660 
TTGACATTGGCGGTAACAGAAGAATCTTTGGATTTAACGCATTGGTTGATAGACAAGCAG720 
ACAACGAAAGCAAAAAAATTGCTTTACACAGAGCTAAAGAGCGTGGTTTTCATGGCGCTA780 
AAAGCGATGACAACTATGTAAAAACAATTAAGGAGTAAGAAATGAAAAAGATTAGCAGAA840 
AAGAATATGTTTCTATGTATGGTCCTACTACAGGCGATAAAGTGAGATTGGGCGATACAG900 
ACTTGATCGCTGAAGTAGAACATGACTACACCATTTATGGCGAAGAGCTTAAATTCGGTG960 
GCGGTAAAACCCTAAGAGAAGGCATGAGCCAATCTAACAACCCTAGCAAAGAAGAGTTGG1020 
ATTTAATTATCACTAACGCTTTAATCGTGGATTACACCGGTATTTATAAAGCGGATATTG1080 
GTATTAAAGATGGCAAAATCGCTGGCATTGGTAAAGGCGGTAACAAAGACATGCAAGATG1140 
GCGTTAAAAACAATCTTAGCGTAGGTCCTGCTACTGAAGCCTTAGCCGGTGAAGGTTTGA1200 
TCGTAACGGCTGGTGGTATTGACACACACATCCACTTCATTTCACCCCAACAAATCCCTA1260 
CAGCTTTTGCAAGCGGTGTAACAACCATGATTGGTGGTGGAACCGGTCCTGCTGATGGCA1320 
CTAATGCGACTACTATCACTCCAGGCAGAAGAAATTTAAAATGGATGCTCAGAGCGGCTG1380 
AAGAATATTCTATGAATTTAGGTTTCTTGGCTAAAGGTAACGCTTCTAACGATGCGAGCT1440 
TAGCCGATCAAATTGAAGCCGGTGCGATTGGCTTTAAAATTCACGAAGACTGGGGCACCA1500 
CTCCTTCTGCAATCAATCATGCGTTAGATGTTGCGGACAAATACGATGTGCAAGTCGCTA1560 
TCCACACAGACACTTTGAATGAAGCCGGTTGTGTAGAAGACACTATGGCTGCTATTGCTG1620 
GACGCACTATGCACACTTTCCACACTGAAGGCGCTGGCGGCGGACACGCTCCTGATATTA1680 
TTAAAGTAGCCGGTGAACACAACATTCTTCCCGCTTCCACTAACCCCACCATCCCTTTCA1740 
CCGTGAATACAGAAGCAGAGCACATGGACATGCTTATGGTGTGCCACCACTTGGATAAAA1800 
GCATTAAAGAAGATGTTCAGTTCGCTGATTCAAGGATCCGCCCTCAAACCATTGCGGCTG1860 
AAGACACTTTGCATGACATGGGGATTTTCTCAATCACCAGTTCTGACTCTCAAGCGATGG1920 
GCCGTGTGGGTGAAGTTATCACTAGAACTTGGCAAACAGCTGACAAAAACAAGAAAGAAT1980 
TTGGCCGCTTGAAAGAAGAAAAAGGCGATAACGACAACTTCAGGATCAAACGCTACTTGT2040 
CTAAATACACCATTAACCCAGCGATCGCTCATGGGATTAGCGAGTATGTAGGTTCAGTAG2100 
AAGTGGGCAAAGTGGCTGACTTGGTATTGTGGAGTCCAGCATTCTTTGGCGTGAAACCCA2160 
ACATGATCATCAAAGGCGGATTCATTGCGTTAAGCCAAATGGGCGATGCGAACGCTTCTA2220 
TCCCTACCCCACAACCGGTTTATTACAGAGAAATGTTCGCTCATCATGGTAAAGCTAAAT2280 
ACGATGCAAACATCACTTTTGTGTCTCAAGCGGCTTATGACAAAGGCATTAAAGAAGAAT2340 
TAGGACTTGAAAGACAAGTGTTGCCGGTAAAAAATTGCAGAAATATCACTAAAAAAGACA2400 
TGCAATTCAACGACACTACTGCTCACATTGAAGTCAATCCTGAAACTTACCATGTGTTCG2460 
TGGATGGCAAAGAAGTAACTTCTAAACCAGCCAATAAAGTGAGCTTGGCGCAACTCTTTA2520 
GCATTTTCTAGGATTTTTTAGGAGCAACGCTTCCTTAAATCCTGAATTCGAGCTCCGTCG2580 
ACAAGCTTGCGGCCGCACTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAACA2640 
AAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCC2700 
TTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTG2735 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 238 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
MetLysLeuThrProLysGluLeuAspLysLeuMetLeuHisTyrAla 
151015 
GlyGluLeuAlaLysLysArgLysGluLysGlyIleLysLeuAsnTyr 
202530 
ValGluAlaValAlaLeuIleSerAlaHisIleMetGluGluAlaArg 
354045 
AlaGlyLysLysThrAlaAlaGluLeuMetGlnGluGlyArgThrLeu 
505560 
LeuLysProAspAspValMetAspGlyValAlaSerMetIleHisGlu 
65707580 
ValGlyIleGluAlaMetPheProAspGlyThrLysLeuValThrVal 
859095 
HisThrProIleGluAlaAsnGlyLysLeuValProGlyGluLeuPhe 
100105110 
LeuLysAsnGluAspIleThrIleAsnGluGlyLysLysAlaValSer 
115120125 
ValLysValLysAsnValGlyAspArgProValGlnIleGlySerHis 
130135140 
PheHisPhePheGluValAsnArgCysLeuAspPheAspArgGluLys 
145150155160 
ThrPheGlyLysArgLeuAspIleAlaSerGlyThrAlaValArgPhe 
165170175 
GluProGlyGluGluLysSerValGluLeuIleAspIleGlyGlyAsn 
180185190 
ArgArgIlePheGlyPheAsnAlaLeuValAspArgGlnAlaAspAsn 
195200205 
GluSerLysLysIleAlaLeuHisArgAlaLysGluArgGlyPheHis 
210215220 
GlyAlaLysSerAspAspAsnTyrValLysThrIleLysGlu 
225230235 
(2) INFORMATION FOR SEQ ID NO:3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 566 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
MetLysLysIleSerArgLysGluTyrValSerMetTyrGlyProThr 
151015 
ThrGlyAspLysValArgLeuGlyAspThrAspLeuIleAlaGluVal 
202530 
GluHisAspTyrThrIleTyrGlyGluGluLeuLysPheGlyGlyGly 
354045 
LysThrLeuArgGluGlyMetSerGlnSerAsnAsnProSerLysGlu 
505560 
GluLeuAspLeuIleIleThrAsnAlaLeuIleValAspTyrThrGly 
65707580 
IleTyrLysAlaAspIleGlyIleLysAspGlyLysIleAlaGlyIle 
859095 
GlyLysGlyGlyAsnLysAspMetGlnAspGlyValLysAsnAsnLeu 
100105110 
SerValGlyProAlaThrGluAlaLeuAlaGlyGluGlyLeuIleVal 
115120125 
ThrAlaGlyGlyIleAspThrHisIleHisPheIleSerProGlnGln 
130135140 
IleProThrAlaPheAlaSerGlyValThrThrMetIleGlyGlyGly 
145150155160 
ThrGlyProAlaAspGlyThrAsnAlaThrThrIleThrProGlyArg 
165170175 
ArgAsnLeuLysTrpMetLeuArgAlaAlaGluGluTyrSerMetAsn 
180185190 
LeuGlyPheLeuAlaLysGlyAsnAlaSerAsnAspAlaSerLeuAla 
195200205 
AspGlnIleGluAlaGlyAlaIleGlyPheLysIleHisGluAspTrp 
210215220 
GlyThrThrProSerAlaIleAsnHisAlaLeuAspValAlaAspLys 
225230235240 
TyrAspValGlnValAlaIleHisThrAspThrLeuAsnGluAlaGly 
245250255 
CysValGluAspThrMetAlaAlaIleAlaGlyArgThrMetHisThr 
260265270 
PheHisThrGluGlyAlaGlyGlyGlyHisAlaProAspIleIleLys 
275280285 
ValAlaGlyGluHisAsnIleLeuProAlaSerThrAsnProThrIle 
290295300 
ProPheThrValAsnThrGluAlaGluHisMetAspMetLeuMetVal 
305310315320 
CysHisHisLeuAspLysSerIleLysGluAspValGlnPheAlaAsp 
325330335 
SerArgIleArgProGlnThrIleAlaAlaGluAspThrLeuHisAsp 
340345350 
MetGlyIlePheSerIleThrSerSerAspSerGlnAlaMetGlyArg 
355360365 
ValGlyGluValIleThrArgThrTrpGlnThrAlaAspLysAsnLys 
370375380 
LysGluPheGlyArgLeuLysGluGluLysGlyAspAsnAspAsnPhe 
385390395400 
ArgIleLysArgTyrLeuSerLysTyrThrIleAsnProAlaIleAla 
405410415 
HisGlyIleSerGluTyrValGlySerValGluValGlyLysValAla 
420425430 
AspLeuValLeuTrpSerProAlaPhePheGlyValLysProAsnMet 
435440445 
IleIleLysGlyGlyPheIleAlaLeuSerGlnMetGlyAspAlaAsn 
450455460 
AlaSerIleProThrProGlnProValTyrTyrArgGluMetPheAla 
465470475480 
HisHisGlyLysAlaLysTyrAspAlaAsnIleThrPheValSerGln 
485490495 
AlaAlaTyrAspLysGlyIleLysGluGluLeuGlyLeuGluArgGln 
500505510 
ValLeuProValLysAsnCysArgAsnIleThrLysLysAspMetGln 
515520525 
PheAsnAspThrThrAlaHisIleGluValAsnProGluThrTyrHis 
530535540 
ValPheValAspGlyLysGluValThrSerLysProAlaAsnLysVal 
545550555560 
SerLeuAlaGlnLeuPhe 
565 
__________________________________________________________________________