Note: Descriptions are shown in the official language in which they were submitted.
wo 95/10300 ~ ~ 7~ PCT~S94/11424
VIBRIO CHOLERAE 01 (CVD1 1 1 ) AND NON-01 (CVD112 AND CVD1 1 2RM)
SEROGROUP VACCINE STRAINS AND METHODS OF MAKING SAME
SAME AND PRODUCTS THEREOF
This application is a continuation-in-part of U.S.
Patent applications 08/133,438 and 08,133/439, both of
which were filed on October 8, 1993, and the contents of
which are incorporated herein by reference. Both U.S.
Patent applications 08/133,438 and 08/133,439 are
continuation-in-parts of U.S. Patent Application
07/931,943, filed August 12th, 1992, which is a
continuation-in-part of applicants' U.S. Patent
application 07t821,872, filed January 16th, 1992, which
is a continuation of applicants' U.S. Patent application
07/533,315, filed June 5th, 1990, each of which are
incorporated herein by reference. U.S. Patent
application 07/533,315 is a continuation-in-part of ap-
plicants' U.S. Patent application 06/581,406 filed
February 17th, 1984, which is a continuation-in-part of
U.S. Patent application 06/472,276, filed March 4th,
1983, each of which are incorporated herein by reference.
U.S. Patent application 07/533,315 is also a
continuation-in-part of applicant's U.S. Patent
Application 07/363,383, filed June 5th, 1989 and allowed
December 12th, 1989, which is a continuation of U.S.
Patent Application 06/867,633, filed May 27th, 1986,
which is a continuation of U.S. Patent Application
06/472,276, filed March 4th, 1983, each of which are
incorporated herein by reference. The research detailed
in this application was supported by the National
Institute of Health and in part by the Medical
Biotechnology Center of the Maryland Biotechnology
Institute.
~CR~ROUND OF THE lNv~NlION
Vibrio cholerae (V. cholerae) is a non-invasive
enteropathogen of the small bowel that does not penetrate
the mucosal surface. Local Siga mediated immunity at the
WO95/10300 PCT~S94/11424 -
Z ~ 7 ~
mucosal surface is therefore implicated as a protective
mechanism. Pathogenic V. cholerae 01 elaborate a protein
enterotoxin (also know as cholera enterotoxin, or
choleragen, or cholera toxin) which is responsible for
induction of copious secretion by the intestine resulting
in watery diarrhea, the clinical consequence of cholera
infection. The genes responsible for cholera enterotoxin
are the ctx genes (also known as the tox genes). Cholera
diarrhea can be extraordinarily severe and result in loss
of so much body water and salts that dehydration,
acidosis, shock, and death ensue without prompt therapy.
There is known in the region of the V. cholerae
chromosome containing the ctx genes that multiples copies
of a 2700 base pair sequence called RSl (for repetitive
sequence) can be found. Mekalanos, Cell 35, 253-263
(1983).
Applicants have also discovered that a second
enterotoxin is produced by V. cholerae which has been
named zonula occludens toxin, reported in Fasano et al,
Vibrio cholerae Produces a Second enterotoxin Which
Affects Intestinal Tiqht Junctions, Proc. Nat. Acad. Sci.
(USA) 88, 5242-5246 (1991).
The cholera vaccines that have been developed can be
broadly divided into two categories; those aiming to
stimulate antitoxic immunity and those intending to
induce antibacterial immunity. Experiments with animal
models support a protective role for either or both
antitoxic and antibacterial immunity. It has been
suggested that when both types of immunity work in
unison, there is a synergistic effect. [Holmgren, J. et
al. J. Infect. Dis. 136 SuPpl., S105-S1122 (1977);
Peterson, J.W. Infect. Immun. 26, 594 (1979); Resnick,
I.G. et al. Infect. Immun. 13, 375 (1980); Svennerholm,
A.-M. et al. Infect. Immun. 13, 735 (1976)]. However, it
appears that protective immunity in humans can be
conferred without such synergistic effect, that is by
either antitoxic immunity or antibacterial immunity
~73~
~ WO95/1~300 PCT~S94/11424
[Eubanks, E.R. et al. Infect. Immun. 15, 533 (1977);
Fujita, K. et al. J. Infect. Dis. 125, 647 (1972);
Holmgren, J., J. Infect. Dis., supra; Lange, S. et al.
Acta Path. Microbiol. Scand Sect. C 86, 145 (1978);
Peterson, J.W., supra (1979); Pierce, N.F. et al. Infect
Immun. 37, 687 (1982); Pierce, N.F. et al. Infect. Immun.
21, 185 (1978); Pierce, N.F. et al. J. Infect. Dis. 135,
888 (1977); Resnick, I.G. et al., suPra; Svennerholm, A.-
M. et al, supra].
KILLED WHOLE CELL VACCINES
1. Parenteral Whole Cell Vaccines
For almost a century, killed whole V. cholerae have
been employed as parenteral vaccines; these vaccines are
still commercially available. Experience with the paren-
teral whole cell vaccines has been reviewed in Joo, I.
"Cholera Vaccines." In Cholera. (Barua D. and Burrows
W., eds.), Saunders, Philadelphia, pp. 333-355 (1974) and
in Feeley, J.D. et al. In Cholera and Related Diarrheas.
43rd Nobel Symp., Stockholm 1978. (O. Oucherlong, J.
Holmgren, eds.) Karger, Basel, pp. 204-210 (1980). Such
vaccines stimulate high titers of serum vibriocidal
antibodies. They also stimulate increases in intestinal
Siga antibody to V. cholerae somatic O antigen when given
to Pakistanis but not to Swedes [Svennerholm, A.-M. et
al. Infect. Immun. 30., 427 (1980); Svennerholm, A.-M. et
al. Scan. J. Immun. 6, 1345 (1977)]. It has been
suggested that the Pakistani vaccine recipients respond
in this way because they are already immunologically
primed from prior antigenic contact, while persons living
in a non-endemic area (e.g., Sweden) are not. In field
trials parenteral killed whole cell vaccines have been
shown to confer significant protection against the
homologous V. cholerae serotype, but usually for a period
of less than one year [Joo, I. supra; Feeley, J.C. ,
supra; Svennerholm, A.-M. et al. supra, (1980);
Svennerholm, A.-M. et al. supra, (1977); Mosley, W.H. et
WO95/10300 PCT~S94/11424 -
2 1~ 3 ~ ~ ~ 4~ r
al. Bull. Wld. Hlth. Org. 49, 13 (1973); Philippines
Cholera Committee, Bull. Wld. Hlth. Orq. 49, 381 (1973)].
There is some evidence to suggest that parenteral whole
cell Inaba vaccine provides good, short term protection
against Ogawa, as well as against Inaba cholera, while
Ogawa vaccine is effective only against Ogawa.
By use of adjuvants, it has been possible to
maintain a vaccine efficacy of approximately 70% for up
to one-and-one-half years with parenteral vaccine (see,
e.g., Saroso, J.S. et al. Bull. Wld. Hlth. Orq. 56, 619
(1978)). However, the adverse reactions encountered at
the site of inoculation with adjuvanted vaccines (which
include sterile abscesses) are sufficiently frequent and
severe to preclude routine use of such adjuvanted
vaccines.
2. Oral Whole Cell Vaccines
Killed whole vibrios administered orally
stimulate the appearance of local intestinal anti-vibrio
antibody. [Freter, R. J. Infect Dis. 111, 37 (1972);
Freter R. et al. J. Immunol. 91 724 (1963); Ganguly, R.
et al. Bull. Wld. Hlth. Orq. 52, 323 (1975)]. Other
investigators have shown substantial vaccine efficacy,
but a large proportion of the vaccines developed diarrhea
after subsequent challenge with pathogenic vibrios [Cash,
R.A. et al. J. Infect. Dis. 130, 325 (1974)].
TOXOIDS
Immunizing agents intended to prevent cholera by
means of stimulating antitoxic immunity include:
1) Formaldehyde-treated cholera toxoid
2) Glutaraldehyde-treated cholera toxoid;
3) Purified B subunit; and
4) Procholeragenoid (with or without formaldehyde
35treatment).
1. Formaldehyde-Treated Cholera Toxoid
~ WO95/10300 ~ I 7 ~ ~ Y~ ~ PCT~S94/l1424
Treatment of purified cholera toxin in vitro
with formaldehyde eradicates its toxicity, resulting in a
toxoid that exhibits little toxic biological activity but
stimulates antitoxic antibodies following parenteral
immunization of animals. However, when the first toxoid
of this type was administered to either monkeys or man as
a parenteral vaccine, the toxoid reverted to partial
toxicity causing unacceptable local adverse reactions at
the site of inoculation [Northrup, R.S. et al. J. Infect.
Dis. 125, 471 (1972)]. An aluminum-adjuvanted
formalinized cholera toxoid has been administered
parenterally to Bangladeshi volunteers, including
lactating mothers, but no field trials with this vaccine
have been undertaken [Merson, M.H. et al. Lancet I, 931
(1980)]. Formalinized cholera toxoid prepared in the
presence of glycine has also been tried by the parenteral
route, but the vaccine showed no evidence of efficacy
[Ohtomo, N. In Proceedinqs of the 12th Joint Conference
on Cholera, U.S.-Japan Cooperative Medical Science
Program, Sapporo (Fukumi H., Zinnaka Y., eds.) pp. 286-
296 (1976); Noriki, H. In Proceedinqs of the 12th Joint
Conference on Cholera, U.S.-Japan Cooperative Medical
Science Program, Sapporo (Fukumi H., Zinnaka Y., eds.)
pp. 302-310 (1976)].
2. Glutaraldehyde-Treated Cholera Toxoid
Methods have been developed for the large-scale
preparation of a glutaraldehyde-treated cholera toxoid
that is essentially free of contaminating somatic antigen
[Rappaport, E.S. et al. Infect. Immun. 14, 687 (1976)].
It was hoped that this antigen could be used to assess in
a "pure" manner the protective role of antitoxic immunity
alone. A large-scale field trial of this toxoid given as
a parenteral vaccine was carried out in Bangladesh in
1974 [Curlin, G. et al. In Proceedinq of the 11th Joint
Conference on Cholera, U.S.-Japan Cooperative Medical
Science Program. pp. 314-329, New Orleans, (1975)]. The
WO95/10300 PCT~S94/11424 -
~ 1 7 3 ~ (o r
toxoid stimulated high titers of circulating antitoxins
in Bangladeshi recipients. Two waves of cholera, El Tor
Inaba followed by El Tor Ogawa, struck the field area
allowing a fair evaluation of vaccine efficacy. A
protective effect could be demonstrated in only one age
group and was restricted to the period of the Inaba
epidemic, so that glutaraldehyde-treated cholera toxoid
given alone as a parenteral vaccine provided little
protection and was substantially inferior to similar
field trials in the same population with parenteral
killed whole cell vaccines.
The use of glutaraldehyde-treated cholera toxoid as
an oral vaccine has been investigated on the assumption
that toxoid given by this route might be more efficient
by stimulating intestinal antitoxin antibodies[Levine,
M.M. et al. Trans. Roy. Soc. TroP. Med. HYq. 73, 3,
(1979)]. Two groups of volunteers were immunized with
three 2.0 mg, or three 8.0 mg doses of toxoid given
directly into the small intestinal lumen (via intestinal
tube) at monthly intervals. The vaccinees and
unimmunized controls then participated in experimental
cholera challenge studies. In neither challenge study
was the attack rate or severity of diarrhea significantly
diminished in the vaccines when compared with controls.
The lack of efficacy of oral glutaraldehyde-treated
cholera toxoid may be due to the fact that the capacity
of B subunits to bind to GMl ganglioside is greatly
diminished as a consequence of toxoiding with
glutaraldehyde.
3. Purified B Subunit
Cholera enterotoxin is composed of two subunits
designated A and B, encoded by the ctxAB operon. The A
subunit induces the enzymatic changes which lead to fluid
secretion, while the non-toxic B subunit is the
immunogenic moiety that binds to the receptor for toxin
(GM1 ganglioside) on intestinal epithelial cells
[Holmgren, J. Nature 292, 413 (1981)]. It has been shown
2~7~
WO9S/10300 PCT~S94/11424
that purified B subunit given either orally or
parenterally to Bangladeshis stimulates the appearance of
Siga antitoxin in intestinal fluid, a result attributable
to immunological priming in a cholera-endemic area
5 [svennerholml A.-M. et al. Lancet I, 305 (1982)].
The major advantages of B subunit oral vaccine to
stimulate antitoxic immunity include its complete safety
(there is not potential for reversion to toxin as exists
with toxoids) and retention of its capacity to adhere to
toxin receptors on enterocytes. Animal studies suggest,
however, that the purified B subunit is less potent than
native holotoxin in stimulating antitoxin antibodies
[Pierce, N.F. supra, ( 1982)].
It will be understood that the purified B subunit
can be used, if at all, in conjunction with e.g. oral
killed vibrios as a combination oral vaccine intended to
stimulate both antibacterial and antitoxic antibodies.
4. Procholeragenoid
Procholeragenoid is the large molecular weight
toxoid (ca. 1,000,000 MW) that results when cholera
enterotoxin is heated at 65C for at least five minutes
[Finkelstein, R.A. et al. J. Immunol. 107, 1043 (1971)].
It is immunogenic while retaining less that 5~ of the
biological toxic activity of the parent toxin. Heating
for longer times (e.g., 25 minutes) produces less
biological toxicity [Germanier, R. et al. Infect. Immun.
13, 1692 (1976)], and subsequent treatment with
formaldehyde completely abolishes residual biological
toxicity. The resultant formaldehyde-treated
procholeragenoid is at least as potent as the parent
toxin in stimulating serum antitoxin following
immunization of rabbits. Swiss volunteers developed
brisk serum antitoxin responses following parenteral
immunization with 10, 30, or 100 mcg doses of
formaldehyde-treated procholeragenoid [Germanier, R. et
al. J. Infect. Dis. 135. 512 (1977)]. No notable adverse
reactions were observed.
WO9S/10300 PCT~S9~/11424 -
2t73~ 8
As an oral antigen procholeragenoid is more
immunogenic when given in the form without formaldehyde-
treatment. In dogs, untreated procholeragenoid is
tolerated as well as an oral vaccine; oral doses (with
NaHCO3 carrier) up to 500 mcg do not case diarrhea. Five
500 mcg doses spaced over 42 days stimulate significant
protection in dogs against oral challenge with pathogenic
V. cholerae. Doses of 50 mcg and 200 mcg with NaHCO3
have been given to groups of six and four adult
volunteers, respectively, without eliciting adverse
reactions.
It will be understood that pro-choleragenoid can be
used in conjunction with e.g., live vaccines, killed
vibrios or other relevant antigens capable of stimulating
antibacterial immunity so that the antitoxic immunity
induced by procholeragenoid is enhanced.
COMBINATION VACCINES
The major attraction of non-living, oral cholera
vaccine is its safety. An oral vaccine consisting of a
combination of antigens, intending to stimulate both
antibacterial and antitoxic immunity, would be most
likely to succeed for the following reasons: Toxoid
vaccines that stimulate purely antitoxic immunity have
not been shown to be efficacious in protecting man
against cholera, although they may protect animal models.
In addition, oral or parenteral killed whole cell
vaccines that stimulate no antitoxic immunity provide
significant protection against cholera in man, albeit for
a short period of time. Furthermore, combinations of
antigens (such as crude cholera toxin, or toxin plus
lipopolysaccharide) that stimulate both antitoxic and
antibacterial immunity, give synergistic protection.
Two studies so far have been carried out in many
with combination vaccines. In the first, nine volunteers
who ingested glutaraldehyde-treated cholera toxoid (2 mg
weekly for four weeks) plus killed El Tor Inaba vibrios
2~7~ WO9S/10300 PCT~S94/11424
q
(101 vibrios twice weekly for four weeks) were
challenged, along with six unimmunized controls, after
one month with 106 pathogenic El Tor Inaba vibrios.
Diarrhea occurred in only two of nine vaccines, versus
four of six controls (vaccine efficacy 67%) and illness
was clearly attenuated in the two ill vaccinees. More
pertinent, perhaps, is the observation that V. cholerae
could be directly cultured from stools of only two of
nine vaccinees, versus six of six controls. This
demonstrates that immunologic mechanisms impeded the
proliferation of vibrios.
In another study, three doses of B subunit/killed
whole cell combination vaccine was given to adult
volunteers who participated in a vaccine efficacy
challenge. The combination vaccine was given on days 0,
14, and 28. Each of the three doses of vaccine contained
0.5 mg of purified B subunit and 2 x 1011 killed V.
cholerae ( 5 x 101 classical Inaba, 5 x 101 classical
Ogawa, and 1 x 1011 El Tor Inaba).
A group of eleven volunteers immunized with this
combination vaccine were challenged one month after their
last dose with 106 pathogenic V. cholerae El Tor Inaba,
along with seven control volunteers. Diarrhea occurred
in seven of seven controls, but in only four of eleven
vaccinees (p=0.01). The illness in the four vaccinees
was definitely milder.
Thus, results of studies with oral toxoid/killed
whole cell vaccine combinations demonstrate a measurable
degree of efficacy. The protective vaccine efficacy,
however, is only moderate (55-65%) and multiple doses are
required to induce the protection.
ATTENUATED V. CNOLERAE VACCINES
Both classical and El Tor clinical cholera
infections stimulate a high degree of protective immunity
for at least three years in North American volunteers
[Cash, R.A. et al., supra (1974); Levine, M.M. et al. ,
WO95/l0300 PCT~S94/l1424 -
2 ~ 7 ~ o
supra (1979); Levine, M.M. et al. "VoIunteers studies in
development of vaccines against cholera and
enterotoxigenic Escherichia coli: a review," in Acute
Enteric Infections in Children: New ProsPects for
Treatment and Prevention. (T. Holm, J. Holmgren, M.
Merson, and R. Mollby, eds.) Elsevier, Amsterdam, pp.
443-459 (1981); and Levine, M.M. et al. J. Infect. Dis.
143, 818 (1981)]. Based on these observations in
volunteers, perhaps the most promising approach toward
immunologic control of cholera may be with attenuated
non-toxigenic V. cholerae strains employed as oral
vaccines.
1. Naturally-Occurring V. cholerae O1 Strains
Non-toxigenic V. cholerae O1 serogroup strains
isolated from environmental sources in India and Brazil
have been evaluated in volunteers as potential vaccine
candidates with disappointing results. They either
failed to colonize the intestine of man, or did so
minimally; vibriocidal antibody responses were meager,
and they failed to provide protection in experimental
challenge studies [Cash, R.A. et al. Infect. Immun. 10,
762 (1974); Levine M.M. et al. J. Infect. Dis. 145, 296
(1982)]. Many of these strains appear to lack the toxin
gene, as measured by hybridization with a radioactive DNA
probe [Kaper, J.B. et al. Infect. Immun. 32, 661 (1981)].
2. Mutagenized Attenuated Strains
Classical Inaba 569B has been mutagenized with
nitrosoguanosine (NTG) and hypotoxinogenic mutant
isolated [Finkelstien, R.A. et al. J. Infect. Dis. 129,
117 (1974); Holmes, R.K. et al. J. Clin. Invest. 55, 551
(1975). This mutant strain, M13, was fed to volunteers.
Diarrhea did not occur but the strain colonized poorly.
Challenge studies demonstrated that some protective
efficacy was conferred by immunization with multiple
doses [Woodward, E. et al. Develop. Biol. Stand. 33, 108,
(1976)].
W095/10300 21 7 3 ~ ~ ~ PCT/US94/11424
~i r
El Tor Ogawa 3083 has also been mutagenized [Honda,
T. et al. Proc. Nat. Acad. Sci. 76, 2052 (1979)]. Brute
force selection and analysis of thousands of colonies
yielded one isolate that continued to produce the
5 immunogenic B subunit while failing to produce detectable
A subunit or holotoxin. The one isolate, Texas Star-SR,
fulfilled these criteria. Texas Star-SR produces normal
or increased amount of B subunit but is negative in
assays for holotoxin activity or A subunit activity.
Texas Star-SR has been extensively evaluated in
volunteers (see, e.g., Levine M.M. et al. Acute Enteric,
supra (1981)). Groups of five volunteers received two
109 organism doses one week apart and eighteen more
volunteers ingested two 2 x 101 organism doses one week
15 apart. Some degree of diarrhea was seen in sixteen of
the sixty-eight vaccinees (24%). In only one individual
did the total stool volume exceed 1.0 liter (1464 ml).
Typically, the vaccine-induced diarrhea consisted of two
or three small, loose stools totalling less than 400 ml
20 in volume. Vaccine organisms were recovered from co-
procultures of approximately one-half of the vaccine
recipients. Where jejunal fluid was cultured (recipients
of doses of 108 or more vaccine organisms), cultures were
positive in thirty-five of forty-six vaccines (76%).
25 Hundreds of Texas Star clones recovered from co-
procultures and jejunal fluid cultures were examined for
cholera holotoxin by the sensitive Y-1 adrenal cell
assay; none were positive.
Significant rises in serum antitoxin were detected
30 in only 29% of the vaccinees; however, 93% manifested
significant rises in serum vibriocidal antibody and the
titers were substantially close to those encountered
following infection with pathogenic V. cholerae. In
experimental challenge studies in volunteers, Texas Star-
35 SR was found to confer significant protection against
challenge with both EL Tor Ogawa And El Tor Inaba
vibrios. One or two doses of Texas Star-SR attenuated
WO95/10300 PCT~S94/11424 -
~1 7~6~ ~7
oral vaccine confers good protection against El Tor
cholera.
It is clear that the use of attenuated strains has
intrinsic advantages since such strains mimic infection-
derived immunity to cholera. However, the Texas Star-SR
strains suffers from certain drawbacks. To begin with,
mutagenesis (e.g., with nitrosoguanosine) induces
multiple mutations, not all of which are necessarily
recognized. Furthermore, the precise genetic lesion that
is presumed to be responsible for the attenuation of
Texas Star-SR is not known. In addition, as with any
pathogen mutated with nitrosoguanosine, Texas Star-SR may
revert to virulence.
3. Naturally-Occurring V. cholerae non-Ol Strains
V. cholerae of the Ol serotype is generally
responsible for epidemic cholera. The non-O1 serogroup
has been associated mainly with sporadic cases of
gastroenteritis and extraintestinal infections, but has
not previously had epidemic potential. Recently,
however, several strains of V. cholerae isolated from a
typical cholera-like outbreaks have been obtained.
[Ramamurthy et al., The Lancet, vol. 341, 703-704 (1993),
the entire contents of which are incorporated herein by
reference.] Serological characterization of a large
number of strains isolated in this recent outbreak showed
that they failed to agglutinate with Ol antiserum nor
with any of tested monoclonal antibodies raised against
factor A, B or C of O1 serogroup V. cholerae. As a
result, these vibrios have been identified as non-Ol.
Furthermore, with the exception of one non-O1 strain
tested as above, all other non-O1 strains tested in a
particular new outbreak could not be typed in the panel
of 138 antigens developed for V. cholerae non-O1
serogroup at the Japanese National Institutes of Health,
indicating that the strain associated with this new
outbreak belong to a previously unrecognized, or recently
WO95/10300 2 ~ 7 3 ~ ~ ~ PCT~S94111424
13 :
emergent, non-Ol serotype capable of causing epidemic
cholera.
Upon DNA hybridization analysis, all of the strains
from this outbreak hybridized with both ctx- and zot-
specific probes, but none hybridized with a DNA probespecific for the heat-stable enterotoxin of V. cholerae
non-Ol (NAG-ST). Additionally, production of cholera
toxin was apparently detected by an enzyme-linked
immunosorbent assay. The amount of enterotoxin produced
by these newly isolate strains has been reported to be
similar to that produced in clinical strains of V.
cholerae Ol. Most of these strains have also been
reported to be resistant to, for example, streptomycin
and furazolidone, but sensitive to other commonly used
antibiotics including tetracycline. Resistance to
ampicillin, or its derivatives, was not reported.
In another recent outbreak, although the overall
number of cholera cases did not increase, a large
majority of the V. cholerae isolates from cholergic
diarrhoeal patients screened were non-Ol, and as in the
previous isolates, could not be typed by standard typing
tests.
In another reported case of a non-Ol associated,
potentially epidemic cholera outbreak has been reported
which primarily has been affecting adults [Albert et al.,
The Lancet, vol. 341, 704 (1993), the entire contents of
which are incorporated herein by reference]. Of the
rectal swabs obtained in this report, about 67 percent
yielded V. cholerae non-Ol upon standard testing, and
none were reported to test as V. cholerae 01.
The V. cholerae responsible for this outbreak has
been reported to resemble V. cholerae 01 both
biochemically and in colony morphology, but was reported
to not agglutinate V. cholerae antisera. The V. cholerae
strains tested were non-reactive with a monoclonal
antibody specific for the A factor of V. cholerae Ol.
WO95/10300 PCT~S94/11424 -
13 6 ~
In this second recently reported outbreak, all non-
Ol strains tested were reported as positive for the
production of cholera toxin in the sensitive Y-l adrenal
cell assay, and were reported to be neutralized by rabbit
polyclonal antiserum to cholera toxin. Polymerase chain
reaction analysis using Ol cholera toxin-specific primers
was also reported to amplify cholera toxin sequences.
Selected isolates were also analyzed by the rabbit
intestinal illeal loop assay and were reported to perfuse
watery diarrhea in the reverse illeal loop tie similar to
that due to V. cholerae Ol.
The V. cholerae strain of this second outbreak were
reported to be sensitive to certain antibiotics,
including tetracycline, but were resistant to other
vibriostatic compounds. Such a result contrasts to the
susceptibility of currently prevalent strains in the
area, the majority of which isolates are resistant to
tetracycline.
Due to these outbreaks of V. cholerae non-Ol
outbreaks, it would be highly desirable to produce
vaccines specific for these organisms. In addition, it
would also be highly desirable to produce combination
vaccines that are effective against both Ol and non-Ol
organisms, particularly for use in those region where
virulent strains of both V. cholerae serogroups may be
encountered.
SUMMARY OF THE lNV ~.~r. ION
Applicants of the present invention have made, by
novel methods, and isolated mutants of a virulent strain
of Vibrio cholerae of the Ol and non-Ol serotype, said
mutants being suitable for use as a vaccine for
protection against the symptoms of cholera upon challenge
by virulent V. cholerae strains. The starting strain for
the making the one mutant of the present invention is V.
cholerae strain 1837, which is a non-Ol strain of the
WO95/10300 217 3 ~ ~ ~ PCT~S94/11424
i~
0139 serogroup. The mutants contain deletions in the V.
cholerae enterotoxin core region, which deletions were
made using restriction endonucleases of a newly
identified, characterized and cloned core region of the
ctx locus of the starting strain, although other suitable
methods of deletion known in the art could also be used.
In order to induce immunogenicity, sequences
encoding the cholera toxin B subunit were re-introduced
in the vaccine strain. Also introduced into the mutant
strains were sequences which encode resistance to heavy
metals, for example mercury, and which allow for the
identification of the presence of the vaccine strain
without the use of potentially therapeutically useful
antibiotic markers.
The V. cholerae non-Ol vaccine strains of the
present invention have thus been specifically altered
through the use of recombinant DNA techniques to render
the strains avirulent without affecting other components
necessary for immunity.
One avirulent non-Ol V. cholerae of the invention is
V. cholerae CVD112 (cep~, zot~, ace~, orfU~, ctxA-, ctxB,
mer, hylA , attRSl , RSl-). Another avirulent V. cholerae
non-Ol of the invention is V. cholerae CVD112RM (cep~,
zot~, ace~, orfU~, ctxA-, ctxB, mer, hylA , recA~ attRSl ,
RSl-).
A characterization of a virulent V. cholerae non-Ol
ctx locus was undertaken, leading to the new finding that
the strain isolated contained two core regions and four
repetitive sequences (RSl sequences). A plasmid is
constructed in which there is a deletion in all, or
substantially all, of the cholera toxin core region, but
retaining extensive lengths of flanking DNA repetitive
sequence of the V. cholerae chromosome at the ctx locus
(i.e., the ctx genetic element). Conjugal gene transfer
of this plasmid into a virulent V. cholerae non-Ol,
followed by homologous recombination and a second
recombination event yielded a V. cholerae non-Ol with
WO9S/10300 ~ 7~ PCI`/US94/11424--
r
only a single core sequence and a single RS1 repetitive
element. The remaining core sequence and RS1 element
were subsequently removed using newly cloned, and then
deleted, 0139 chromosomal DNA. Sequences encoding the
cholera toxin B subunit and sequences encoding a
resistance to heavy metals were subsequently re-
introduced into the mutant V. cholerae chromosome by
homologous recombination.
The non-01, nontoxigenic deletion mutants of the
invention are capable of colonizing the small intestine
and stimulating local, protective immunity directed
against the bacterial cell. After the transient
colonization episode, the vaccine is protective against
subsequent infection with virulent toxigenic V. cholerae
non-01 strains.
The invention also provides for methods of making
avirulent V. cholerae non-01 strains, and vaccines
derived from these strains, including combination
vaccines.
The genes for V. cholerae 01 serogroup cholera toxin
have been cloned [Pearson, G.D.N. et al. Prod. Nat. Acad.
Sci. 79, 2976 (1982); Kaper, J.B. et al. Amer. Soc.
Micribiol. Abstr. Annu. Meeting, Atlanta Georgia, 36
(1982); Kaper, J.B. et al. SYmposium on Enteric
Infections in Man and in Animals: Standardization of
Immunoloqical Procedures, Dublin, Ireland, Abstract No.
2.5 (1982)]. Toxin structural gene deletion mutants of
V. cholerae have been isolated, but only by infection
with mutagenic vibriophages capable of integration at
random sites along to chromosome [Mekalanos, J.J. et al.
Proc. Nat. Acad. Sci. 79, 151, (1982)]. Recombination in
Vibrio cholerae has been reported, but it has not been
used to isolate deletions in the ctx genes for
vaccination purposes [Parker, C. et al. J. Bact. 112, 707
(1972); Johnson, S.R. et al. Molec. Gen. Genet. 170, 93
(1979); Sublett, R.D. et al. Infect. Immun. 32 1132
2173~
~ WO95/10300 PCT~S94/11424
17 r
(1981) and Thomson, J.A. et al. J. Bact. 148, 374
(1981)]-
Avirulent Vibrio cholerae strain of the 0139serotype (V. cholerae 1837), mutated to have a region of
chromosomal DNA deleted to confer avirulence and to
retain capacity to colonize the intestine of a host
animal, while still conferring immunogenicity is
described herein. The DNA fragment deleted includes all,
or substantially all of the cholera toxin core region.
One isolated deletion mutant encompasses all elements
associated with the ctx locus, and therefore has no core
or repetitive sequences (RS1) elements. Sequences
encoding the cholera toxin B subunit and sequences
encoding a resistance to heavy metals were re-introduced
into this deletion mutant. An additional mutant in the
recA gene is also described. Inactivation of the recA
gene product in this strain removes a potential mechanism
for homologous recombination in this vaccine strain.
A first avirulent non-O1 V. cholerae deletion mutant
of the cholera toxin core and RS1 sequences of non-O1
Vibrio cholerae is described, as is a method of making
this V. cholerae comprising the steps of
(a) constructing a first plasmid comprising DNA of
the Vibrio cholerae cholera toxin core region and
flanking sequences of sufficient length to promote
detectable in vivo recombination, ligated to a gene
encoding a first selectable marker of foreign origin
which confers resistance to a selective agent, wherein
said first plasmid is incapable of replicating
extrachromosomally in V. cholerae;
(b) mating a virulent strain of Vibrio cholerae of
a non-O1 serogroup with a first microorganism carrying
the first plasmid;
(c) selecting for and isolating Vibrio cholerae
expressing the first selectable marker;
(d) growing the V. cholerae isolated in step (c) in
the absence of said selective agent;
WO95/10300 PCT~S94/11424 -
2173~
(e) screening the V. cholerae of step (d) for the
loss of expression of said first selectable marker;
(f) constructing a second plasmid comprising Vibrio
cholerae non-O1 chromosomal sequences which flank the
cholera toxin locus, deleted of DNA of the cholera toxin
core and RS1 sequences, ligated to a second selectable
marker of foreign origin which confers resistance to a
second selective agent;
(g) mating the selected product of step (e) with a
second microorganism carrying said second plasmid; and
(h) selecting for Vibrio cholerae which express the
second selectable marker;
(g) growing the selected product of step (h) in the
absence of the second selective agent;
(h) screening the V. cholerae of step (g) for the
loss of said second selectable marker; and
(i) isolating the screened product of.step (h).
The product of the first method of the invention is
a non-Ol V. chol erae deleted of all cholera toxin core
sequences and the RS1 elements associated with the ctx
locus.
A second avirulent, non-O1 V. chol erae according to
the invention, which has a deletion of the cholera toxin
core and RS1 sequences but expresses re-inserted
sequences of the cholera toxin B subunit, or a part
thereof sufficient to confer immunogenicity, and
sequences encoding a product which confers resistance to
heavy metals is also described, as is a method of making
this second non-O1 vaccine strain. This second method of
the invention comprises the steps of
(a) providing a first plasmid comprising DNA of the
Vibrio cholerae cholera toxin core region and flanking
sequences of sufficient length to promote detectable in
vivo recombination, ligated to a gene encoding a first
selectable marker of foreign origin which confers
resistance to a selective agent, wherein said first
wO gS/10300 2 1 7 3 ~ ~ ~ PCT~S94111424
~ r
plasmid is incapable of replicating extrachromosomally in
V. cholerae;
(b) mating a virulent strain of Vibrio cholerae of
a non-01 serogroup with a first microorganism carrying
the first plasmid;
(c) selecting for and isolating Vibrio cholerae
expressing the first selectable marker;
(d) growing the V. cholerae isolated in step (c) in
the absence of said selective agent;
(e) screening the V. cholerae of step (d) for the
loss of expression of said first selectable marker;
(f) providing a second plasmid comprising Vibrio
cholerae non-O1 chromosomal sequences which flank the
cholera toxin locus, deleted of DNA of the cholera toxin
core and RS1 sequences, ligated to a second selectable
marker of foreign origin which confers resistance to a
second selective agent;
(g) mating the screened product of step (e) with a
second microorganism carrying said second plasmid; and
(h) selecting for Vibrio cholerae which express the
second selectable marker;
(g) growing the selected product of step (h) in the
absence of the second selective agent;
(h) screening the V. cholerae of step (g) for the
loss of said second selectable marker;
(i) isolating the screened product of step (h);
(j) providing a third plasmid comprising V.
cholerae chromosomal sequences of sufficient length to
promote detectable in vivo recombination flanking
sequences of the cholera toxin B subunit sufficient to
confer immunogenicity, and sequences encoding a product
which confers resistance to heavy metals, and ligated to
a third selectable marker of foreign origin, wherein said
third plasmid is incapable of replicating
extrachromosomally in V. cholerae;
(k) mating the screened product of step (h) with a
third microorganism carrying said third plasmid;
WO95/10300 , PCT~S94111424 -
~36~
(1) selecting for Vibrio cholerae which express the
third selectable marker; and
(m) isolating the selected product of step (1).
The non-O1 V. cholerae of the second method of the
invention is deleted of all cholera toxin core and RS1
sequences at the chromosomal ctx locus, but expresses
sequences of the cholera toxin B subunit sufficient to
confer immunogenicity and sequences which confer
resistance to heavy metals.
A third avirulent, non-O1 V. cholerae of the
invention has a deletion of the cholera toxin core and
RS1 sequences of non-Ol Vibrio cholerae but expresses re-
inserted sequences of the cholera toxin B subunit
sufficient to confer immunogenicity, and sequences
encoding a product which confers resistance to heavy
metals is also described. This third non-Ol V. cholerae
mutant of the invention further is a recombination minus
strain additionally having a deletion wherein the product
of the recA locus is inactivated or absent.
This third method of the invention comprises the
steps of:
(a) providing a first plasmid comprising DNA of the
Vibrio cholerae cholera toxin core region and flanking
sequences of sufficient length to promote detectable in
vivo recombination, ligated to a gene encoding a first
selectable marker of foreign origin which confers
resistance to a selective agent, wherein said first
plasmid is incapable of replicating extrachromosomally in
V. cholerae;
(b) mating a virulent strain of Vibrio cholerae of
a non-O1 serogroup with a first microorganism carrying
the first plasmid;
(c) selecting for and isolating Vibrio cholerae
expressing the first selectable marker;
(d) growing the V. cholerae isolated in step (c) in
the absence of said selective agent;
WO95/10300 217 3 ~ ~ ~ PCT~S94/11424
zl ~
(e) screening the V. cholerae of step (d) for the
loss of expression of said first selectable marker;
(f) providing a second plasmid comprising Vibrio
cholerae non-Ol chromosomal sequences which flank the
cholera toxin locus, deleted of DNA of the cholera toxin
core and RS1 sequences, ligated to a second selectable
marker of foreign origin which confers resistance to a
second selective agent;
(g) mating the screened product of step (e) with a
second microorganism carrying said second plasmid; and
(h) selecting for Vibrio cholerae which express the
second selectable marker;
(g) growing the selected product of step (h) in the
absence of the second selective agent;
(h) screening the V. cholerae of step (g) for the
loss of said second selectable marker;
(i) isolating the screened product of step (h);
(j) providing a third plasmid comprising V.
cholerae chromosomal sequences of sufficient length to
promote detectable in vivo recombination flanking
sequences of the cholera toxin B subunit sufficient to
confer immunogenicity, and sequences encoding a product
which confers resistance to heavy metals, and ligated to
a third selectable marker of foreign origin, wherein said
third plasmid is incapable of replicating
extrachromosomally in V. cholerae;
(k) mating the screened product of step (h) with a
third microorganism carrying said third plasmid;
(1) selecting for Vibrio cholerae which express the
third selectable marker;
(m) isolating the selected product of step (l);
(n) providing a fourth plasmid comprising flanking
sequences sufficient in length to promote detectable
recombination at the V. cholerae recA locus, flanking
recA gene sequences deleted to inactivate the recA gene
product, ligated to a fourth selectable marker, wherein
WO95/10300 2 ~ 0 6 PCT~S9~/11424 -
said fourth plasmid is incapable of replicating
extrachromosomally in V. cholerae;
(n) mating the isolated product of step (m) with a
fourth microorganism carrying said fourth plasmid;
(o) selecting for Vlbrio cholerae which express the
fourth selectable marker; and
(p) isolating the selected product of step (o).
The Vibrio cholerae deletion mutants of this
invention are useful for vaccination to protect against
the symptoms of cholera in response to non-Ol V.
cholerae, as well as in methods for producing cholera
vacclnes.
One Vibrio cholerae strain of the present invention,
designated CVD112, confers substantial protection in
humans against the symptoms of cholera upon subsequent
exposure to a strain of a similar non-O1 serotype. Other
Vi~rio cholerae strains of the present invention,
designated by the third culture, designated CVD112 RM,
can confer substantial protection in humans against the
symptoms of cholera when challenged with a strain of a
similar non-Ol serotype, and also is incapable of recA-
mediated homologous recombination. Another Vibrio
cholerae strain disclosed is designated CVDlll. Strain
V. cholerae CVD111 contains sequences of the cholera
toxin B subunit sufficient to confer immunogenicity as
well as the useful selectable marker of mercury
resistance.
~ BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1. V. cholerae N16961 (pJBK55) (Apr)
Figure 2. Processes of crossing-over and conjugal
gene transfer to construct V. cholerae JBK56.
Figure 3. V. cholerae JBK56.
Figure 4. Scheme for construction of JBK21.
Figure 5. Scheme for construction of pJBK54.
2~73~ . 7
IPEA/US 2 9 SEP 1995
Figure 6. Scheme for construction of V. cholerae
JBK56.
Figure 7. Recombination in vivo by cross over and
elimination of ctx gene.
Figure 8. Scheme for construction of pJBK51.
Figure 9. Scheme for construction of pCVD14 and
pCVD15.
Figure 10. Scheme for construction of pJBK108.
Figure 11. Scheme for construction of pJBK107.
n ~i~ure ~ A sequence of ~top) the XbaI and ClaI
sltes, wh~ch determlne the ends ~ th~ ~ele~Qd Xbal-Cla~
550bp fragment of the A subunit in Ogawa 395, and for
(bottom) the junction in CVD101 after deletion of this
fragment and insertion of a XbaI linker.
Figures 13A and 13B. Effect of V. cholerae culture
supernatant on ileal short circuit current (Isc) and
tissue ionic conductance (Gt). Values are means for 6
animals at each time-point; brackets are 1 standard error
a, Effect of V. cholerae 395 supernatants on Isc (solid
lines) and Gt (dashed lines). b, Effect of V. cholerae
395 (solid Line), CVD101 (long dashed line) and 395N1
(dotted line) supernatants on Gt. Medium control (short
dashed line) consisted of un-inoculated culture medium.
AMENDED SHEEt
21736~6
IPEA/US 2 9 SEP 1995
Figure 14. Quantitation of ZO complexity in tissues
exposed to culture supernatants or broth control.
Figure 15. Reversibility of Gt variations induced by
V. cholerae 395 supernatant. Culture supernatants of V.
cholerae (triangles) and uninoculated medium (squares)
were added and removed at the time indicated by arrows.
Figure 16. Scheme for construction of CVDlO9. The
zot and ctx genes are adjacent to each other on the V.
cholerae chromosome and are in a region of the chromosome
which contains multiple C~p(es ~ 70G S~q4-e~c~ C~
RS1 (repetitive ses~ue~t ~`~ ~ are 0~- S~t h -
sides of zot and ctx genes in virulent V. cholerae strain
E7946 (E1 Tor biotype, Ogawa serotype). The zot and ctx
genes are shown by a large open or hash-marked arrow.
RS1 sequences are shown by a smaller, solid arrow.
Figure 17 (pages 1 and 2). DNA sequence of the zot
gene for zonula occludens toxin from nucleotides number 1
to 1428. Letters above the DNA sequence indicate the
predicted amino acid sequence of the ZOT protein encoded
by the zot gene.
Figure 18. Scheme for construction of plasmid
pCVD621 and plasmid pCVD622.2B.
Figure 19. Scheme for construction of CVDllO.
Abbreviations for restriction endonuclease sites in
the drawings are as follows:
A = AccI restriction endonuclease site
B = BglII restriction endonuclease site
C = ClaI restriction endonuclease site
E = EcoRI restriction endonuclease site
H = HindIII restriction endonuclease site
P = PstI restriction endonuclease site
S = SalI restriction endonuclease site
X = XbaI restriction endonuclease site
K = KpnI restriction endonuclease site
~ 2173B~ PCT~ 9 ~ ~79~
Other abbreviations in the drawings and elsewhere
include:
Ap = Ampicillin resistance gene
Apr = Ampicillin resistance phenotype
Aps = Ampicillin sensitive phenotype
Chrom = Chromosome
Cm = Chloramphenicol resistance gene
CT = Cholera toxin
ctx = gene for cholera toxin
CTA = A subunit of cholera toxin
ctxA = gene for A subunit of cholera toxin
CTB = B subunit of cholera toxin
ctxB = gene for B subunit of cholera toxin
hylA = gene for hemolysin
kb = Kilobases
mer = gene for mercury resistance
p = plasmid
Su = Sulfonamide
Sur = Sulfonamide resistance phenotype
Tc = tetracycline
Tcs = tetracycline sensitive phenotype
Tp = Trimethoprin
zot = gene for zonula occludens toxin
ace = gene for cholera toxin locus accessory protein
cep = potential coding sequence for putative, as yet
unsubstantiated, core encoded pilin
Figure 20. Map showing the relative positions of
the ctx, zot and ace genes, OrfU and the RS1 flanking
sequences. (RS1 is shown by large arrows). The two
boxes with the vertical stripes correspond to the two
open reading frames in which ACE activity was initially
localized. It is now thought that ACE activity is
localized to the open reading frame adjacent to the zot
gene. The fragment contained in the clone pCVD630 is
shown.
Figure 21. Ussing chamber activity of V. cholera
strains CVD110 and CVD110 containing pCVD630. Panel on
event that occurs at the V. cholerae recA locus in the
_ _
2~ ~3
PCT/l~S 9 4 ~
/US 29 SEP l99S
the left Figure 21A shows changes induced in short
circuit current (ISC) and panel on right Figure 21B shows
changes in potential difference (PD).
Figure 22. DNA sequence of the 2.9 kb EcoRV
fragment containing ACE activity (sequence GATATC at
beginning and end of sequence is the EcoRV site). The
complete DNA sequence for classical strain 395 is shown
(SEQ ID No.: 1). Below the 395 sequence is shown the
sequence for this region from El Tor strain E7946 (SEQ ID
No.: 4) - only those bases of E7946 which differ from
those in 395 are shown. Where the sequence is identical
for the two strains, only the 395 sequence is shown.
(Dashed lines at bases 236-239 show that E7946 has a 3
base insert (AGT) which is not present in 395). Above
the primary DNA sequence line is shown the amino acid
sequence (in single letter code) predicted from the 395
sequence. The two ORFs are translated; classical 395
OrfU spans bases 1034 to 2218 (SEQ ID No.: 3); classical
395 ace spans bases 2221 to 2508 (SEQ ID No.: 2); El Tor
20 OrfU spans bases 1037 to 2221 (SEQ ID No.: 6); and, El
Tor ace spans bases 2224 to 2511 (SEQ ID No.: 5).
Figure 23. A representation of the arrangement of
the cholera toxin chromosomal locus of V. cholerae non-O1
strain 1837.
Figure 24. Representation of the arrangement of the
cholera toxin locus of V. cholerae strain 1837 (first
line), strain 1837.1 (second line), strain 1837.2 (third
line) and strain CVD112 (fourth line).
- Figure 25. Representation of plasmids pLC13, pLC14,
pLC15 and pLC16.
Figure 26. Scheme for the insertion of cholera
toxin B subunit encoding sequences and mercury resistance
encoding sequences into the V. cholerae 0139 strain
1837.2 chromosome at the hylA locus. -
Figure 27. Representation of the recombination
event that occurs at the V. cholerae recA locus in the
generation of strain CVD112RM.
AMENDED SJl~r
21736~
P~J~iJ
2 ~ S
Z7~
Figures 28A, 28B, 28C, and 28D. Wheat germ
agglutinin - horseradish peroxidase (WGA-HRP)
permeability assay on rabbit ileal tissues exposed to
culture supernatants of various V. cholerae strains. a,
medium control; b, V. cholerae 395; c, V. cholerae 395Nl;
d, V. cholerae CVD101.
Figures 29A and 29B. Freeze-fracture studies of
rabbit ileal tissue exposed to culture supernatants of V.
cholerae a, An intact ZO with numerous intersections
(arrowheads~ between junctional strands M, microvilli. b,
` ~~~n~ec~ea-Zo ~o-~r~L~-tis~e ~x~-~sed ~ ~ ~ e
395; the reticulum appears simplified due to greatly
decreased incidence of strand intersections.
. . .
~1~3~
WO 95/10300 PCT/US94/11424
~1 r
DET~TT~ DESCRIPTION OF THE lNvls~ ION
The present invention comprises V. cholerae strains,
- their method of production and their methods of use. The
strains of Vibrio cholerae of the invention are useful as
5 vaccine strains and are specifically altered through
recombinant DNA technology to render them avirulent,
without substantially affecting other components
necessary for immunity. This attenuation was
accomplished by restriction endonuclease digestion of
10 plasmids carrying appropriate V. cholera sequences, to
specifically delete the genes coding for cholera toxin,
or portion thereof. Conjugal gene transfer of these
plasmids carrying deleted cholera toxin genes into
virulent host V. cholera, followed by selection for in
15 vivo recombinants, resulted in strains without the toxin
genes or portion thereof. It will be understood that the
methods of the present invention are applicable to the
isolation of other deletion mutants of virulent V.
cholerae, or to the isolation of strains having all or
20 part of such deleted sequences reintroduced into the V.
cholerae cell.
The starting material for the vaccines were the
toxigenic Vibrio cholerae 01 N16961. Strain N16961 has
been demonstrated to produce in both typical diarrheal
25 disease and strong, protective immunity to subsequent
infection [Levine, M.M. et al., Acute enteric, supra,
1981]. The region of the bacterial chromosome which was
found to be responsible for production of cholera toxin
was cloned into the plasmid cloning vehicle pBR325, after
30 screening HindIII digest of V. cholerae DNA with an E.
coli heat-labile enterotoxin gene probe [Kaper et al.
Amer. Soc., supra; Kaper et al. SYmposium, supra]. A V.
cholerae HindIII chromosomal fragment was found to
contain all genes necessary for toxin production. Next,
35 this chromosomal region was analyzed and mapped for the
exact portions containing the toxin genes [Kaper, J.B. et
al. Lancet II, 1162 (1981)]. Restriction enzymes were
WO95/l0300 PCT~S94/11424 -
2~ 73~ z~
employed to cut out the DNA fragments containing these
genes and a DNA fragment encoding a selectable marker
(e.g., resistance to ampicillin) was inserted by
ligation. The ampicillin resistance gene and the
flanking vibrio DNA were then cloned in a derivative of
pRK290, which can be used to transfer DNA from E. coli to
V. cholerae. The resulting plasmid, pJBK55, was
transferred from E. coli K-12 to V. cholerae N16961 by
conjugation.
The resulting strain, V. cholerae N16961 (pJBK55)
(Apr) contained a region in its chromosome having intact
toxin genes and, in an extrachromosomal state, a plasmid
containing this same region with the toxin genes deleted
and a gene for ampicillin substituted. (See Figure 1.)
At a low frequency, perhaps one in 1o6 to one in 108, the
identical regions flanking the chromosomal toxin genes
and the extrachromosomal (plasmid) ampicillin resistance
gene will exchanged or "crossed over" or undergo in vivo
recombination so that the region of DNA containing the
resistance gene displaces the toxin gene on the
chromosome (Figure 2). This rare event is selected by
testing a mixture of mutated and non-mutated cells for
individual cells which are able to serve as host for an
incoming incompatible plasmid [Ruvkun, G.B. et al. Nature
289, 85 (1981)]. Plasmids are divided into groups
designated A through W, the members of which cannot
stably coexist with each other. For example, a plasmid
of incompatibility group P cannot be stably maintained in
the same cell as another P group (Inc P) plasmid. Thus,
Inc P plasmids, such as R702, which specify resistance to
sulfonamide, cannot be maintained in a cell which has
another Inc P. plasmid such as PRK 290, pJBK45, or
pJBK55. Therefore, R702 can be maintained in a strain in
which the ampicillin resistance has recombined into the
chromosome but not one in which an Inc P Plasmid (e.g.
pJBK55) is replicating extrachromosomally. By mating an
E. coli strain containing Inc P R702 (sulfonamide
WO95/10300 2 ~ ~ ~ 6 ~ ~ PCT~S94/l1424
Z~
resistant) and V. cholerae pJBK55 (ampicillin resistant)
and selecting for V. cholerae which are resistant to both
ampicillin and sulfonamide, colonies are isolated in
which the sulfonamide resistance is mediated
extrachromosomally by p702 and the ampicillin resistance
is mediated chromosomally through substitution of the
ampicillin resistance gene for the toxin gene (Figure 3).
One such strain, designated V. cholerae JBK56 was
isolated and when tested for toxin production was found
to be nontoxinogenic.
The final version of the vaccine strain, JBK70, was
produced by substituting resistance to ampicillin, a
therapeutically useful antibiotic, with resistance to
mercury. This substitution was accomplished by cloning a
gene for mercury resistance directly into the ampicillin
resistant gene of pJBK55, thereby inactivating ampicillin
resistance and conferring mercury resistance. The
resulting plasmid, pJBK66 was also incompatible with R702
and was transferred to V. cholerae JBK56. A mutant in
which the mercury resistance was recombined into the
chromosome was selected using the Inc P plasmid R702 and
selecting for V. cholerae which were ampicillin
sensitive, mercury resistant, and sulfonamide resistant.
A spontaneous derivative was later selected which was
cured of pR702. The final mutant, JBK70, was
nontoxinogenic and resistant to mercury only.
The vaccine strain V. cholerae JBK70 is one of the
Inaba serotype. The other major serotype of V. cholerae
is the Ogawa serotype. It is expected that a vaccine
prepared from one serotype will protect against the other
serotype (34). In the event that this is not the case, a
live vaccine strain can be prepared from an Ogawa
serotype and protection in volunteers [Levine, M.M. et
al. Acute enteric, supra (1981)]. The exact mutation
created in strain V. cholerae Inaba JBK56 was recreated
in strain E7946 by directly transferring the region of
the chromosome containing the ampicillin resistance in
WO 95/10300 2 ~ PCT~S94/11424 -
~G
place of the toxin gene in JBK56 into E7946 through
genetic recombination mediated by P, the sex factor of V.
cholerae [Parker, C. et al., supra]. The P factor, which
is distinct from Inc P plasmid, was transferred into
JBK56 and was then mated with a rifampin resistant mutant
of E7946. By selection of a mutant which was resistant
to both ampicillin and rifampin, a vaccine strain was
isolated which was of the Ogawa serotype with the toxin
genes completely deleted.
If antibacterial immunity is insufficient for
protection, then an antitoxic component can be added by
adding back the genes for production of cholera toxin B
but not A subunit. This has been accomplished by cloning
the B subunit gene into the cloning vector pMS9. The
resulting plasmid, pJBK51, produces high levels of B
subunit and was reintroduced into the nontoxic vaccine
strain V. cholerae JBK70 to make an attenuated vaccine
strain JBK70 (pJBK51) which fails to produce the A
subunit.
The vaccine strains of the present invention are
derived inter alia from V. cholerae N16961 having the
serotype Inaba. It will be understood that other strains
or other biotypes and serotypes can be used to substitute
for N16961 to produce vaccine strains having specific
deletions in the ctx gene or genes, or in other locations
along the V. cholerae chromosome. Since the object of
isolating such vaccine strains is to mimic the infection
process without associated pathological phenomena, site-
directed mutagenesis of virulent strains, as described in
this application, produces substantial possibilities in
the prophylactic vaccination against cholera.
For example, applicants have produced another V.
cholerae vaccine strain CVD101, characterized by a
deletion of most of the A subunit gene in 2 copies of the
ctx genes. Construction of CVD101 followed in general
the principles outlined supra, e.g. the construction of
JBK70, except that the resulting CVD101 had no resistance
WO95/10300 ~ 1~ 3 6 ~ ~ PCT~S94/11424
3i
gene that needed curing. The final step in isolating the
second and find i~ vivo recombinant included a scheme for
- selecting sensitivity to an antibiotic e.g. tetracycline
sensitivity, whereas the parent strain had inserted at
the location of the A gene of CT a tetracycline
resistance gene. It will be understood that such
antibiotic sensitivity is another example of a selectable
marker.
Production of vaccine strains can be performed by a
variety of methods, including the following: Vibrio
cholerae is subcultured from stock cultures into brain/-
heart infusion agar (BHIA) and grown at 37C overnight.
Identity is tested with group-and type-specific antisera
and twenty to thirty colonies are suspended in BHI broth.
Preincubated BHIA plates are inoculated with BHI
suspension. After incubation for five to six hours, each
plate is harvested with 5 ml of sterile saline buffered
to pH 7.2 + O.l. Harvested organisms are centrifuged in
the cold at 750 g for ten minutes, resuspended and washed
twice in four-times the original volume. The suspension
is standardized spectrophotometrically and diluted to
approximate the number of organisms required for
vaccination (ca lO6, which varies depending on the
results of volunteer studies). Replicate, pour-plate
quantitative cultures are made of the inocula before and
after challenge to confirm inoculum size. The final
inoculum is eX~m; ned with Gram's stain and agglutinated
with homologous antiserum prior to feeding.
The Vibrio cholerae strains of the present invention
can be administered by the oral route. Two grams of
NaHCO3 are dissolved in five ounces of distilled water.
Volunteers drink four ounces of the NaHCO3/water; one
minute later the volunteers ingest the vibrios suspended
in the remaining one ounce of NaHCO3/water. Volunteers
are NPO ninety minutes pre- and post-inoculation.
With regard to safety, the major concern is that the
vaccine strain does not revert to toxigenicity (i.e.,
.
WO95/10300 PCT~S9~/11424 -
~ 6 ~ ~ 3~'
.
produce intact cholera toxin) which could cause disease.
The two major assays for testing toxin are the Y-1
adrenal cell assay tSack, D.A. et al. Infect. Immun. 11,
334 (1975)] and the enzyme-linked immunosorbent assay
(ELISA) [Sack, D.A. et al. J. Clin. Micro. 11, 35
(1980)]. The vaccine strain (JBK70) has been repeatedly
tested in these two assays and found to be negative each
time. Far more important, however, are the genetic
assays performed for the presence of toxin genes. The
DNA for cholera toxin genes can be radioactively labeled
and used as a specific probe to identify other cholera
toxin genes in the strain, according to the method of
Southern, E.M. J. Mol. Bio. 98, 503 (1975). When tested
by this method, the vaccine strain described in the
invention possesses no detectable genetic material that
can enclose cholera toxin. The vaccine has also been
tested in an infant mouse model, according to Baselski,
V. et al. Infect. Immun. 15, 704 (1977). After repeated
(ten in all) serial passages, no fluid accumulation
(i.e., evidence of disease has been found. As expected,
JBK70 was found to colonize the infant mouse intestine.
In order to avoid undesirable side effects of the
vaccine strains, such as diarrhea and nausea, cramping,
and other symptoms, the vaccine strains may further
comprise a second restriction endonuclease fragment of
DNA coding for zonula occludens toxin (ZOT) deleted.
A culture of Vibrio cholerae comprises a Vibrio
cholerae strain having a first restriction endonuclease
fragment of DNA deleted to confer avirulence and retain
capacity to colonize the intestine of a host animal and
having a second restriction endonuclease fragment of DNA
coding for zonula occludens toxin (ZOT) deleted to reduce
residual diarrhea in the host animal. The first DNA
fragment deleted may code for the V. cholerae toxin or
portions thereof such as the Al subunit. One isolated
deletion mutant encompasses a deletion in the ctx gene,
as defined by AccI restriction endonuclease sites, and a
WO95/10300 ~ 17 3 6 3 6 PCT~S94/11424
deletion in the zot gene. Another isolated deletion
mutant encompasses a deletion in the ctx gene, as defined
by XbaI and ClaI restriction endonuclease sites, and a
deletion in the zot gene, as defined by StuI and AccI
A 5 restriction endonuclease sites
A method of isolating such deletion mutants of
Vibrio cholerae comprises the steps of
(a) constructing a first plasmid comprising Vibrio
cholerae flanking sequences of one or more deleted
lO restriction endonuclease fragments and a gene for a
selectable marker of foreign origin ligated to said
flanking sequences to substitute for and to be in place
of said deleted fragment, wherein said sequences are of
sufficient length to promote detectable in vivo
15 recombination;
(b) mating a virulent strain of Vibrio cholerae
with a first microorganism carrying the first plasmid;
(c) selecting for Vibrio cholerae e,xpressing the
first selectable marker;
(d) mating the selected product of step (c) with a
second microorganism carrying a second plasmid with a
second selectable marker, said second plasmid being
incompatible with the first plasmid;
(e) selecting for Vibrio cholerae expressing both
the first selectable marker and the second selectable
marker;
(f) constructing a third plasmid comprising Vibrio
cholerae flanking sequences of one or more deleted
restriction endonuclease fragments homologous to those
described in step (a) but differing in the absence of a
selectable marker of foreign origin;
(g) mating the selected product of step (e) with a
third microorganism carrying a third plasmid described in
step (f); and
(h) selecting for Vibrio cholerae which no longer
expresses the first selectable marker.
WO95/10300 PCT~S94/11424 -
2~ 73~0~ 3~
This method may be used for ZOT minus only strains
or for making a ZOT minus derivative of a strain which is
already deleted for cholera toxin genes.
Another culture of Vibrio cholerae comprises a
Vibrio cholerae strain having a region of the chromosomal
DNA coding for cholera toxin and zonula occludens toxin
(ZOT) deleted. A method of isolating such deletion
mutants of Vibrio cholerae comprises the steps of
(a) constructing a plasmid comprising Vibrio
cholerae sequences coding for cholera toxin and zonula
occludens toxin and a gene for a selectable marker of
foreign origin, wherein said plasmid is incapable of
replicating extrachromosomally in Vibrio cholerae;
(b) mating a microorganism carrying said plasmid
with a virulent strain of Vibrio cholerae containing said
sequences to promote detectable in vivo recombination;
(c) selecting for Vibrio cholerae expressing said
selectable marker;
(d) growing the selected product of (c) in the
absence of the selective agent;
(e) selecting for Vibrio cholerae which no longer
express the selective marker; and therefore have a region
of the chromosomal DNA coding for cholera toxin and
zonula occludens toxin deleted. Step (b) may comprise:
(b) mating a microorganism carrying said plasmid with a
virulent strain of Vibrio cholerae containing said
sequences inserted between flanking identical copies of a
second sequence such as RS1 elements of sufficient length
to promote detectable in vivo recombination.
The Vibrio cholerae deletion mutants of this
invention are useful in vaccination against cholera.
Herein reported is a new toxic factor elaborated by
V. cholerae which increases the permeability of the small
mucosa by affecting the structure of the intercellular
tight junctions or zonula occludens (Zo) (the
paracellular pathway of ion transport). Production of
this factor by V. cholerae correlates with
WO95/10300 2 ~ 7 ~ ~ a ~ PCT~S9~/11424
diarrheagenicity in volunteers. By disturbing the normal
absorptive processes of the small intestine via the
paracellular pathway, this factor could be responsible
for the residual diarrhea induced by ctx deletion mutants
of V. cholerae and may contribute to the severe diarrhea
that distinguishes cholera from other diarrheal diseases.
Changes in intestinal function induced by three
strains of V. cholerae, one wild type and two attenuated
vaccine strains, were examined. V. cholerae strain 395,
classical biotype, Ogawa serotype, is a highly virulent
strain which has been extensively characterized in
volunteer studies conducted at the Center for Vaccine
Development. This strain induces diarrhea with a mean
stool volume of 5.5 liters (range of 0.3 to 44 1) in
greater than 90% of volunteers ingesting 106 organisms
[Levine, M.M. et al, Infect. Immun. 56, 161-167 (1988)];
tLevine, M.M., Cholera and Related Diarrheas, 195-203]
(Karger, Basel, 1980). Cholera diarrhea is principally
due to the enzymatic effects of the A subunit of CT on
intestinal mucosa. The CT A subunit, encoded by ctx,
stimulates adenylate cyclase and results in net secretion
of fluid into the intestinal lumen. Gill, D.M. Adv.
Cyclic Nucleotide res. 8, 85-118 (1977). V. cholerae
vaccine strain CVD101 is a ctx deletion mutant of 395 in
which 94% of the sequences encoding the A1 peptide of CT
have been removed. Surprisingly, although CVD101 no
longer produces active CT, this strain caused mild to
moderate diarrhea (mean stool volume of 0.9 l with a
range of 0.3 to 2.1 l) in 54% of volunteers ingesting
this organism. A second derivative of 395, vaccine
strain 395N1, constructed by Mekalanos, et al.,Nature
306, 551-557 (1983), lacks ca. 77% of the sequences
encoding the A1 peptide by applicants' calculation. In
contrast to CVD101, 395N1 induced very mild diarrhea (0.3
l stool volume) in only 1 of 21 volunteers (P=0.002
compared to 13 of 24 volunteers with diarrhea after
ingestion of CVD101). [Herrington, D.A. et al. J. Exp.
WO95/l0300 PCT~S94/11424 -
3~ F
~.73~
Med. 168, 1487-1492 (1982)]. Since these strains were
similar in their ability to colonize the intestine,
applicants hypothesize that CVD101 produces a
secretogenic factor which is expressed weakly or not at
all by 395N1 and that this factor is responsible for the
diarrhea seen in volunteers ingesting CVD101.
These strains were studied using rabbit intestinal
tissue mounted in Ussing chambers, a classic technique
for studying the transport process across intestinal
tissue. Supernatants of V. cholerae cultures were added
to the chambers and potential difference (PD) and short
circuit current (Isc) were measured. PD is the
difference in voltage measured on the mucosal side vs.
the serosal side of the tissue and Isc is the amount of
current needed to nullify the PD. From these
measurements, tissue conductance (Gt) was calculated
using Ohm's law: Isc = PD x Gt. Applicants first studied
the effect of supernatants of the wild type strain 395 on
these parameters using uninoculated culture media added
to matched ileal tissue from the same animal as a
negative control. Fig. 13A shows the Isc and Gt
variations obtained. The initial peaks in Isc and PD
that occurred in both negative controls and test samples
were most likely due to the cotransport of Na and
nutrients present in the media. In the negative control,
Isc and PD returned to baseline values after
approximately one hour and subsequently Isc, PD and Gt
remained unchanged for the rest of the experiment. In
contrast, tissues exposed to strain 395 supernatant
exhibited a significant increase in Gt, reaching a
maximum value after 2 hrs of incubation. In such
samples, the Isc never returned to the baseline, but a
steady state period for Isc was noted between 40 and 60
minutes. Since Isc is equivalent to PD x Gt and the
observed PD after 60 min. was similar to the initial
value (data not shown), the significant increase in Isc
in 395-treated tissues at that time point can only be due
WO95/10300 ~ 1 7 3 6 0 ~ PCT~S94/11424
37
to an increase in Gt (see Fig. 13A time 60 Min.) (12).
After 60 min., Isc began to rise again along with PD in
395-treated tissues. This second phase probably reflects
the effect of cholera toxin on ion fluxes since purified
CT increases Isc in rabbit ileal tissue only after a lag
time of at least 40 minutes. These data suggest that
there are two factors expressed by V. cholerae 395 that
can alter ion transport in Ussing chambers. One factor,
cholera toxin, indùces an increase in Isc and PD
beginning ca. 60 minutes after addition of culture
supernatant while a second factor induces an immediate
increase in tissue conductance which is observable within
20 minutes after addition of culture supernatant.
Gt variation induced by culture supernatants of the
attenuated V. cholerae strains CVD101 and 395N1 was next
studied. CVD101 induced an immediate increase in Gt
which was indistinguishable from that seen with 395 (Fig.
13B). In contrast, 395N1 induced no immediate increase
in Gt; Gt variation in 395N1-treated tissues was similar
to the negative broth control and significantly lower
than that seen with 395 and CVDlO1 for almost 100 min of
incubation. After this period, Gt modification in
tissues exposed to 395, CVD101 and 395N1 were similar.
These results suggest that 395N1 produces lower amounts
or a less active form of the factor responsible for this
increase in Gt.
Variation in transepithelial conductance reflects
modification of tissue permeability through the
intercellular space, since plasma membrane resistances
are relatively high. Since ZO represents the major
barrier in this paracellular pathway and variation in Gt
is the most sensitive measure of ZO function,
morphological modifications of ZO induced by V. cholerae
395, CVD101 and 395N1 supernatants were examined. If a
low-molecular weight electron-dense marker such as wheat
germ agglutinin - horseradish peroxidase (WGA-HRP) is
added to the mucosal side of an epithelial sheet, it will
. . ~
21736~g
~r
3~ 3
usually not pass beyond to Zo [Alberts, B. et al.,
Molecular Biolo~y of the Cell 2nd ed (1989)]. WGA-HRP
was added to the mucosal side of intestinal tissue
treated with culture supernatants of 395, CVD101 , 395N1
or uninoculated broth control for 60 minutes. Tissues
treated with uninoculated culture medium were not
permeable to WGA-HRP, while 395 and CVD101 -treated
tissues showed the entry of the stain into the
paracellular space. Tissues exposed to 395N1~W~ - ~ S~r~r ndtant~ ~a*~ec-teJ~ 3s~4ck a~ h~
intercellular space remained tight enough to exclude the
passage of WGA-HRP (Figure 14). These results were
confirmed and extended using freeze-fracture electron
microscopy wherein the number of strands lying in
parallel at the Z0 correlates with transepithelial
electrical conductance. Tissues exposed to culture
supernatants showed a mixture of unaltered Z0 and altered
Z0 with decreased strand complexity. Strands lying
perpendicular to the long axis of the Zo appeared to be
preferentially lost, resulting in a decreased number of
strand intersections. The complexity of the Z0 exposed
to each strain supernatant was quantified by measuring
the density of strand intersections. As seen in Figure
14, tissues treated with culture supernatants of 395 or
CVD101 showed a significant decrease in the number of
strands and in the complexity of the reticulum of the Z0
when compared to tissues treated with uninoculated broth
or supernatants of 395N1.
The alterations of ZO morphology induced by 395 and
CVD101 parallel the increased tissue conductance induced
by these strains. The function of intestinal Z0 is to
regulate the paracellular pathway and restrict or prevent
the diffusion of water-soluble molecules through the
intercellular space back into the lumen. This diffusion
is driven by concentration gradients created by the
transepithelial transport processes. As a consequence of
AMENDED~H~
2~73~
PC~JS 94/11 424
,~ IPEA/US ~ g SE~ ~g~
alteration of the paracellular pathway, intestinal mucosa
becomes more permeable and water, Na and Cl leak into the
lumen, resulting in diarrhea. The alteration of the
paracellular pathway induced by V. cholerae 395 and
5 CVD101 is specific for the small intestine; substitution
of rabbit cecal tissue for ileal tissue resulted in no
variation in Gt induced by 395 supernatant (data not
shown). This is the first report of a bacterial factor
which is capable of l~Gsen;n~ nctions in intact
10 intestinal tissue a~
bacterial diarrhea. Clostridium difficile toxin A,
influenza, and vesicular stomatitis (VSV) viruses have
been shown to loosen tight junctions in tissue culture
monolayers but such activity in intact tissue or
15 correlation with diarrhea have not been reported.
Thus, V. cholerae 395 and CVD101 produce a
factor which may be responsible for diarrhea seen in
volunteers ingesting ctx deletion mutants of V. cholerae.
The diarrhea induced by these ct~x mutants is equivalent
20 to that seen with many strains of enterotoxigenic E.
coli. This secretogenic factor, which applicants have
termed ZOT for zonula occludens toxin, induces an early
increase in Isc an~ tissue c-onductance which is not
related to the effects of CT on ion fluxes. This
25 increase in Gt is associated with loosening of the tight
junctions, an effect which was quickly reversed upon
removal of the supernatant (Figure 15). The quick
reversal of this effect is in contrast to the long-
lasting effect of CT. These results do not account for
30 previously unexplained observations of Fields, et al., J.
Clin. Invest. 51, 796-804 (1972) who noted an immediate
increase in Isc induced by crude, but not purified CT
preparations, and may account for Nishibuchi et al.,
Infect. Immun. 40, 1083-1091 (1983) who noted an early
35 fluid accumulation (FA) unrelated to the delayed CT-
induced FA in suckling mice fed V. cholerae. The ability
of CT-negative V. cholerae to induce diarrhea in
AMENDED SH~ET
WO95/10300 PCT~S94/11424 -
2 1 7 3 ~ t; r
volunteers correlates with production of ZOT by two
attenuated strains derived from the same parent straini
CVD101 (diarrheagenic) produces ZOT while 395N1 (non-
diarrheagenic) produces little or no ZOT activity.
Another culture of Vibrio cholerae comprises a
Vibrio cholerae strain having a region of chromosomal DNA
coding for cholera toxin and zonula occludens toxin
deleted, and having inserted a mercury resistance gene
and DNA coding for B subunit of Vibrio cholerae toxin. A
method of isolating such deletion mutants is also
described comprising the steps of:
(a) constructing a plasmid comprising Vibrio
cholerae sequences coding for cholera toxin and zonula
occludens toxin and a gene for a selectable marker of
foreign origin, wherein said plasmid is incapable of
replicating extrachromosomally in Vibrio cholerae;
(b) mating a microorganism carrying said plasmid
with a virulent strain of Vibrio cholerae containing said
sequences coding for cholera toxin and zonula occludens
toxin inserted between flanking identical copies of a
second sequence of sufficient length to promote
detectable in vivo recombination;
(c) selecting for Vibrio cholerae expressing said
selectable marker;
(d) growing the selected product of (c) in the
absence of the selective agent;
(e) selecting for Vibrio cholerae which no longer
express the selective marker, and therefore have a region
of the chromosomal DNA coding for cholera toxin and
zonula occludens toxin deleted;
(f) constructing a second plasmid comprising a
mercury resistance gene and DNA coding for B subunit of
Vibrio cholerae toxin and a gene for a second selectable
marker of foreign origin wherein said plasmid is
incapable of replicating extrachromosomally in Vibrio
cholerae, and wherein sequences of sufficient length to
promote detectable in vivo recombination flank said
WO95/10300 2 1 7 3 ~ ~ ~ PCT~S94/l1424
mercury resistance gene and DNA coding for B subunit of
Vibrio cholerae toxin;
(g) mating a microorganism carrying said second
plasmid with said Vibrio cholerae recited in step (e)
containing sequences homologous to said sequences of
sufficient length to promote detectable in vivo
recombination;
(h) selecting for Vibrio cholerae expressing said
second selectable marker;
(i) growing the selected product of step (h) in the
absence of the second selective agent;
(j) selecting for Vibrio cholerae which no longer
express the second selective marker; and
(k) screening said Vibrio cholerae recited in step
(j) for Vibrio cholerae that have a mercury resistance
gene and DNA coding for B subunit of Vibrio cholerae
toxin and have a region of chromosomal DNA coding for
cholera toxin and zonula occludens toxin deleted.
This method for isolating deletion mutants of Vibrio
cholerae having a region of chromosomal DNA coding for
cholera toxin and zonula occludens toxin deleted, and
having inserted a mercury resistance gene and DNA coding
for B subunit of Vibrio cholerae may use in step (f)
flanking sequences of sufficient length comprising a gene
that can be disrupted without affecting colonization and
immunity of Vibrio cholerae. An example is the hemolysin
gene. V. cholerae CVD110 and CVDlll were constructed
according to this method, and have a region of
chromosomal DNA coding for A and B subunits of cholera
toxin and zonula occludens toxin deleted, and have a
mercury resistance gene and DNA coding for B subunit of
Vibrio cholerae toxin inserted at the site of hemolysin
gene. Other examples of sequences of sufficient length
comprise the his gene (Hone, Microbial Pathoqenesis 5,
pp. 407-478 (1989)) and the nanH gene (Vimr, J. Bacter.,
170, pp. 1495-1504 (1988)).
WO95/10300 PCT~S9~/11424 -
~3~
The invention also relates to strains, vaccines and
method of making these strains and vaccines which
comprise V. cholerae of a non-O1 serogroup. One of these
strains, CVD112, has a deletion entire cholera toxin core
region, which comprises cep, ace, zot, orfU, and ctxAB as
well as deletion of the RS1 elements and attRS1 sites
[Pearson et al., Proc. Natl. Acad. Sci. (USA), 90, pp.
3750-3754, 1993] with the ctx chromosomal locus. In
addition, these strains have sequences encoding a
sufficient part of the cholera toxin B subunit to confer
immunogenicity re-inserted into the vibrio chromosome.
Addition inserted into the chromosome are sequences which
confer resistance to heavy metals, such as mercury.
Another V. cholerae non-O1 strain of the invention,
namely CVD112 RM has the identifying characteristics of
the above-mentioned non-01 strain, and additionally has a
deletion in the Vibrio cholerae recA locus such that the
resultant strain is deficient in homologous
recombination.
In the examples that follow, any of the techni~ues,
reactions, and separation procedures are already well
known in the art. All enzymes, unless otherwise stated,
are available from one or more commercial sources, such
as New England BioLabs--Beverly, Massachusetts;
Collaborative Research--Waltham, Massachusetts; Miles
Laboratories--Elkhart, Indiana; Boehringer Biochemicals
Inc.--Indianapolis, Indiana; and Bethesda Research
Laboratory--Rockville, Maryland, to mention a
representative few. Buffers and reaction conditions for
restriction enzyme digestion are used according to
recommendations supplied by the manufacturer for each
enzyme, unless indicated otherwise. Partial digestions
with restriction enzymes are carried out using a reduced
enzyme concentration which must be predetermined from
preliminary experiments for each enzyme batch. Standard
methodology for other enzyme reactions, gel
electrophoresis separations, and E. coli transformation
WO95110300 2 ~ 7 ~ PCT~S94/11424
~3
may be found in Methods in Enz~mology Volume 68, Ray Wu,
editor, Academic Press (1979). Another standard
reference is Maniatis, T. et al. Molecular Cloninq, Cold
Spring Harbor (1982). Bacteria were grown according to
procedures generally described in Miller, Experiments in
Molecular Genetics, Cold Spring Harbor Laboratory (1972)
Vibrio cholerae were propagated according to procedures
generally described in Lennett, E.A. et al., eds., Manual
of Clinical Microbiology 3rd Edition, American Society of
Microbioloy, Washington (1980). E. coli and V. cholerae
were mated according to procedures generally described in
Johnson, Steven R. et al. J. Bact. 137, 531 (1979); and
Yokata, T. et al. J. Bact. 109, 440 (1972).
The strains of this invention have been deposited at
the American Type Culture Collection, located in
Rockville, Maryland, prior to execution of the present
application. The strains deposited are V. cholerae
JBK56, V. cholerae JBK70, V. cholerae N1696, V. cholerae
JVK70 (pJBK51), V. cholerae Ogawa 395, CVD101, CVD109, V.
cholerae E7946, and E. coli SM10 lambda pir pCVD51, V.
cholerae CVD110, and E. coli SY327 lambda pir
(pCVD622.2B), which have ATCC accession numbers 39,317,
39,318, 39,315, 39,316, 39,541, 39,540, 55,057,
(deposited June 4th, 1990), 55,056 (deposited June 4th,
1990), 68,335 (deposited June 5th, 1990), 55188
(deposited June 3rd, 1991), and 68630 (deposited June
3rd, 1991, respectively.
ExamPle 1
Construction of a Plasmid Havinq a Selectable Marker Gene
Inserted to Replace the Toxin Genes
The plasmid JBK16 contains a 4 kb PstI-BglII
fragment of the chromosome containing the toxin genes.
The toxin genes are flanked by AccI sites and contain an
internal AccI site. JBK16 was digested to completion
with AccI and the AccI ~ragments containing the toxin
genes were separated from the rest of the plasmid. The
WO95/10300 PCT~S94/11~24 -
2~7~6 ~
remaining overlapping or "sticky" AccI ends were made
blunt-ended by "filling in" with the Klenow fragment of
E . coli polymerase (i.e., the single-stranded DNA
remaining after AccI digestion were made double-stranded
with flush ends). A gene encoding ampicillin resistance
was purified from the plasmid pREG153 (pREG153 is a
derivative of pREG151 [Weiss, A. et al. J. Bact. 152,
549-552] altered by substitution of ampicillin resistance
for trimethoprin resistance and addition of cos
sequences) and the "sticky" ends "filled in" as above.
This fragment was then ligated to the vibrio DNA so that
the Ap resistance genes were in exactly the same place as
the now-deleted toxin genes, flanked by the same vibrio
sequences. The resulting plasmid was designed pJBK21
(Figure 4) containing the deletion toxin region and the
Ap resistance gene.
Example 2
Addition of Flankinq Homologenous Sequences Followed bY
Coniuqal Gene Transfer into V. Cholerae
To insure the specific insertion into the chromosome
of the deletion in pJBK21, approximately 7,000 bp of
additional DNA was added to each end of the PstI-BglII
fragment from pJBK21. (The probability of the homologous
recombination event occurring increases with increasing
length of flanking homologous sequences.) To achieve
this, an approximately 18 kb fragment was cloned from the
chromosome of N16961. This clone was designated pJBK44
and contains the 4 kb PstI-BglII tox gene fragment
flanked by approximately 7kb of DNA on each side (see
Figure 5). The plasmid pJBK21 was partially digested
with PstI so that only one of the Pst sites would be cut
(an additional Pst site was added within the ampicillin
resistance gene) followed by digestion with BglII to
isolate the 4 kb PstI-BglII fragment containing the
deletion toxin region and the Ap resistance region. The
plasmid pJBK44 containing the ca. 18 kb Vibrio fragment
2~7~
WO95/10300 PCT~S94/ll424
~5
was partially digested with BglII so that only one of the
4 BglII sites present would be cut. This partial
digestion was followed by complete digestion with PstI
and the resulting fragments separated by electrophoresis
through 0.3% agarose. The separated fragments were then
purified and analyzed and one fragment was found which
contained all of the sequences of pJBK44 except for the 4
kb. PstI-BglII tox gene fragment (see Figure 5.). This
fragment representing the flanking DNA was then mixed
ligated to the PstI-BglII fragment from pJBK21 containing
the ampicillin resistance. The resulting plasmid,
pJBK54, contained approximately 17 kb of Vibrio
chromosomal DNA with an ampicillin resistance gene
substituted for the deleted toxin genes.
The modified chromosomal region was then cloned into
a plasmid which can be readily mobilized in V. cholerae.
The plasmid pRK290 [Ditta, G. et al. Proc. Nat. Acad.
Sci. 77, 7347 (1980)] belongs to the plasmid
incompatibility group P and possesses a single EcoRI site
into which pJBK54 was cloned (Figure 6). The resulting
plasmid pJBK55 was then mated into V. cholerae N16961
using the conjugative plasmid pRK2013, yielding V.
cholerae N16961 (pJBK55) (Apr).
Example 3
Recombination in vivo
The mutant toxin genes, after conjugal gene transfer
as described in Example 2, now existed extrachromosomally
in V. cholerae strain N16961 (see Figure 1). At a very
30 low frequency (perhaps 10-6 to 10-8) the homologous
flanking sequences base pair and cross over into the
chromosome (see Figure 7). This rare event will result
in the substitution of the deleted toxin region on the
plasmid for the ctx genes on the chromosome. To select
for this rare event, the plasmid incompatibility
phenomenon was exploited [Ruvkin, G.B., supra ] . Plasmids
can be divided into incompatibility groups, designated A
WO95/10300 2 ~ 7 3 ~ ~ ~ PCT~S94/11424 -
through W, on the basis of their ability to be stably
maintained together in the same cell. If two plasmids
cannot be stably maintained together in the same cell,
they are incompatible and belong to the same
incompatibility group presumably because they utilize the
same replication mechanism in the cell. By selectively
using an antibiotic resistance present on one plasmid but
not on the other, it is possible to select which of two
incompatible plasmids will be maintained The plasmid
pJBK55, because of its pRK290 origin, belongs to the
(Inc) group P. The plasmid R702 also belongs to the Inc
P group and encodes resistance to kanamycin,
tetracycline, sulfonamide, and streptomycin, but not
ampicillin. By mating pR702 (SuR) into
N16961(pJBK55)(ApR) and selecting on media containing
both ampicillin and sulfonamide, selection was made for
cells in which the ampicillin resistance had been
incorporated into the chromosome and sulfonamide
resistance remains on the plasmid R702, since pR702 and
pJBK55 are incompatible (see ~igure 2). The resultant
strain JBK56 (Figure 3) was ampicillin resistant, and
toxin negative when tested in Y-l adrenal cells and by
Gml ELISA. ~urthermore, when chromosomal DNA was
hybridized to DNA probes containing clone cholera toxin
(CT) genes, JBK56 was negative, suggesting that the toxin
genes were completely deleted.
The antibiotic resistance encoded on R702 was elimi-
nated by selecting a spontaneously cured derivative
lacking the plasmid (this occurred at a fre~uency of
about 1 in 2,000).
Example 4
Elimination of the Selectable Marker of Example 1
To eliminate the ampicillin resistance, a
derivative of pJBK55 was constructed in which genes
encoding resistance to mercury tHgr) from R100 were
cloned into the PstI site of the Ap gene, thereby
WO9~/10300 ~ 7 3 ~ ~ ~ PCT~S94/11424
insertionally inactivating the ampicilIin resistance.
This derivative was then mated into V. cholerae JBK56,
followed by pR702 and selection made as above for Hgr,
Aps V. cholerae. The final strain, V. cholerae JBK70, is
sensitive to all antibiotics tested, resistant to
mercury, and phenotypically toxin negative. Its
chromosomal DNA did not detectably hybridize to DNA
probes containing CT genes. Short of sequencing the DNA
for the entire chromosome, JBK70 appears to be unaltered
from the parent strain N16961 except for the deletion of
the toxin genes and insertion of mercury resistance and
inactive ampicillin resistance genes. Such a strain
cannot revert ~o toxigenicity because the toxin genes are
not merely mutated but are completely deleted.
Example 5
Coniugal Gene Transfer to Confer Antitoxic Immunity
If both antibacterial immunity and antitoxic
immunity are desired for synergy, a derivative of JBK70
can be made to produce the B su;bunit of cholera toxin
only. To accomplish this end, a toxin derivative was
made that produces B only and lacks the genes for A
(Figure 8). A HpaII fragment from pJBK16 containing the
B structural gene was cloned into a phage cloning vector,
M13mp7 placing a BamHI and an EcoRI site on either side
of the gene (Figure 8). The fragment, now flanked by
BamHI sites was cloned into pMS 9 which~contains the very
strong trp promoter. The placing of the B genes under
the transcriptional control of a strong promoter insures
high production of B antigen. Of the clones examined,
approximately 50~ produced no antigen. This finding
reflects the two possible orientations for the cloned
insert--one forward, one backward. One derivative,
pJBK51, which produced B subunit was mated into Vibrio
cholerae JBK70 and found to produce even more B antigen
then the parent strain N16961, yielding JBK70 (pJBK51).
Other B-only mutants have been created using different
~ S ~.~
2173~
r~ 9 4 / l ~ ~ J ~i
~8 IPEA/US 2 ~ SEP ~9~3
promoters, including the P~ promoter and these can be
evaluated in appropriate models for any significant in
vivo expression differences.
Example 6
Colonization of Infant Mouse Intestine with JBK70 without
Reversion to Toxi~enicity
Suckling mice (2.0-3.5g.) were removed from their
mothers and starved for 3 to 8 hours. Four of them were
then inoculated on day 1 per os to stomach using a 22g
animal feeding needle. The inoculum was about 108 CFU
(colony-forming units)/mouse of JBK70 in a volume of
between 0.05 ml and 0.1 ml. The inoculum was prepared in
BHI broth essentially as described in Baselski, V. et al,
supra. The inoculum contained about 0.01% Evans blue
dye. The presence of this dye in the stomach, seen
through the abdominal wall, indicated proper delivery of
the inoculum. Addition of Evans blue dye was
discontinued after day 1, to avoid inhibition of JBK70.
Subsequent inoculations involved mouse-to-mouse
(MXM), or alternatively, mouse-to-plate-to-mouse (MXPXM),
but required different procedures to prepare the inoculum
compared to the Baselski protocol for the inoculation on
day 1.
To prepare MXM inoculum, the gut was dissected from
stomach to anus under sterile precautions. The gut was
weighed, placed in a glass homogenizer tube, and about
0.5 ml BHI broth added. The mixture was homogenized
briefly with a Teflon pestle until tissue was liquified.
The resulting suspension was used to inoculate about 108
CFU into each infant mouse. It was checked for purity by
streaking on MEA (meat extract agar) plates. No Evans
blue dye was added.
To prepare MXPXM inoculum, a sterile loop was used
to transfer cells from an MEA plate to BHI broth. About
1011 CFU/ml were added to about 1 ml of BHI so that a
tD ~r ~
WO95tl0300 ~ 17 ~ PCT~S94/11424
dense suspension was formed. The mixture was vortexed to
homogeneity, and 0.05-0.1 ml. (about lo10 CFU) inoculated
per os into each infant mouse. No Evans blue dye was
added.
For all inoculations, mice were held in beakers at
room temperature of 73-76F. Beakers were placed in a
plastic box which was loosely covered in order to
maintain the mice at slightly above ambient temperature,
about 78F.
As the results in Table I indicated, there were
sufficient cells in the intestine to inoculate the next
animal, as checked by streaking on MEA plates. The
Vibrio cholerae JBK70 therefore colonized the gut of
infant mice. Furthermore, the fluid accumulation levels
did not increase since there were no substantial
increases in the FA ration (an FA ratio greater than or
equal to 0.065 is a positive fluid accumulation).
Evidence of reversion to toxigenicity would have
indicated otherwise.
Example 7
Construction of V. cholerae strain CVD101 having a
Restriction Fraqment Deletion within the Gene coding
for the A Subunit
Another classical strain chosen for attenuation was
Vibrio cholerae Ogawa 395 (alternatively designated
"395") which, like N16961, has been extensively studied
in volunteers and confers solid immunity [Levine, M.M.
"Immunity to cholera as evaluated in volunteers," in
Cholera and Related Diarrheas: 43rd Nobel Symposium,
Stockholm 1978. (O. Ouchterlong & J. Holmgren, eds.)
Basel: S. Karger, pp. 195-2-3 (1980); Levine, M.M et al.
Acute Enteric, supra (1981)]. The procedure employed in
the attenuation of 395 was not substantially different
from that employed for N16961 (as described in Examples
1-5).
WO95/10300 PCT/USg~/11424
Sc~ ,
The first step involved the cloning and mapping of
the two toxin gene copies of 395. Southern blot analysis
revealed two HindIII fragments of about 16 and about 12
kb in length, both of which hybridized with cloned
5 cholera toxin genes. These fragments were purified by
agarose gel electrophoresis and cloned into alkaline
phosphates treated HindIII digested pBR325 (Figure 9).
The resulting recombinant plasmids containing the toxin
genes were designated pCVD14 and pCVD15.
Plasmids pCVD14 and pCVD15 were then mapped with
restriction endonucleases. A XbaI-ClaI fragment of about
550 bp was found, containing the entire base sequence of
the A1 subunit with the exception of codons for the first
10 amino acid residues of A1. This XbaI-ClaI fragment
15 was deleted in vitro from both pCVD14 and pCVD15 in a
series of steps as shown in Figure 10 for pCVD15. First,
partial digestion with Cl aI yielded a population of
linear molecules in which only one of five ClaI sites was
cut. Next, the ends of the linear molecules were made
20 blunt-ended by filling in with DNA polymerase. XbaI
linkers were ligated onto the blunt-ended ClaI sites
yielding a collection of molecules to which a XbaI enzyme
was added to trim the linker, and a tetracycline
resistance gene on a XbaI fragment was added and
25 ligated. After transformation into E. coli K-12 and
selection on tetracycline, the plasmid content of a
number of transformants was e~m;ned. A variety of
deletion mutations were found in which one or more XbaI-
ClaI fragments were deleted. One deletion mutant was
30 chosen which lacked only the 550 bp XbaI- ClaI fragment
containing the A1 gene. This deletion mutant, designated
pCVD25 was purified, digested with XbaI and religated to
delete the tetracycline resistance gene. The resulting
clone, pCVD30, was negative for holotoxin as measured in
35 Y-l adrenal assay [Sack, D.A. et al. supra (1975)], but
positive for production of B subunit, as measured by
ELISA [Sack, D.A. et al. supra (1980)], and lacked the
WO 95/10300 ~ ~ 7 ~ PCT/US94/11424
r
genes for A1, as shown by DNA hybridization using labeled
A1 probe. The HindIII fragment of pCVD30 containing the
toxin deletion mutation was then cloned into pJBK85, a Tc
sensitive, Cm resistant derivative of pJBK108. The
resulting plasmid was designated pJBK108.
The lack of selectable marker in the toxin deletion
mutation in PJBK108 necessitated a modification of the
method previously used to attenuate El Tor N16961. To
accomplish the deletion of the A1 genes from 395, the
10 HindIII fragment from pCVD15 was cloned into PJBK85,
resulting in pJBK88 (Figure 11). The tetracycline
resistance gene on a XbaI fragment wa then cloned into
the XbaI site within the A1 gene of PJBK88, yielding
pJBK107. This tetracycline resistance gene was then
recombined into the chromosome of 395 as previously done
for V. cholerae pJBK56. PJBK107 (Tcr, Cmr) was mobilized
into 395 and a second Inc P plasmid, pR751 (Tpr) was
introduced. Selection of Tcr, Tpr Cms colonies resulted
in V. cholerae JBK113, which contained tetracycline
resistance genes in both chromosomal toxin gene copies.
pJBK108, containing the deletion mutation, was then
mobilized into V. cholerae JBK113. Homologous recom-
bination of the deletion mutation into the chromosome
will result in the loss of the A1 gene sequences, an
event which can be detected by loss of tetracycline
resistance. Because the recombination even occurs at a
very low frequency, an enrichment procedure for
tetracycline sensitive cells in a population of
tetracycline resistant cells was employed. This
enrichment procedure exploited the fact that tetracycline
is a bacteriostatic antibiotic whereas ampicillin and D-
cyclo-serine are bactericidal. Therefore, a culture of
V. cholerae JBK113 containing pJBK108 was grown for 3 hr
at 37 in L-broth containing 2 micro g/ml tetracycline,
50 micro g/ml ampicillin and 50 micro g/ml D-cycloserine.
At the end of 3 hours, most of the tetracycline resistant
cells were killed, and tetracycline sensitive cells were
WO95/10300 PCT~S94tll424 -
~173~ 5~
detected by plating onto L-agar and replica plating onto
L-agar with tetracycline. Tetracycline sensitive
colonies were probed for the presence of A1 genes by DNA
hybridization. One tetracycline sensitive strain having
deletions for both gene copies of the A1 subunit was
designated V. cholerae CVD101 and tested for production
of B subunit by ELISA [Sack, supra]. V. cholerae CVD101
was found to produce B subunit antigen at levels
substantially equivalent to the toxigenic parent V.
cholerae 395.
Exam~le 8
DNA sequence of the Toxin Genes
The entire DNA sequence of the toxin genes of V.
cholerae Inaba 62746 has been determined, part of which
has been reported by Lockman, et al., J. Biol. Chem. 258,
13722 (1983). The restriction endonuclease mapping of
pCVD14 and pCVD15 indicates that the se~uences found in
strain 62746 are also present in the toxin genes of 395.
The predicted junction after deletion of the 550 bp XbaI-
ClaI fragment, but with addition of a XbaI linker
sequence, is shown in Figure 12. The XbaI site of the
cholera toxin sequence span amino acid residues 10 and 11
of the A1 structural gene (not counting the 18 amino acid
leader sequence for A1.
Example g
Construction of a V. Cholerae Strain Having a
Zonal Occludens Toxin Deletion in CVD101
A zot deletion mutant of V. cholerae is prepared in
the same way as the CVD101 cholera toxin deletion mutant
described in Example 7. The zot gene is contained in the
recombinant plasmid pBB68. pBB68 consists of an EcoRI-
PbrI chromosomal DNA fragment from V. cholerae 569B which
contains the zot gene and the ctx genes which have a
deletion of a 550 bp XbaI-ClaI fragment. A StuI-AccI
restriction fragment of 575 base pairs is deleted in
~ 2~73~
WO 95/10300 PCT/US94/11424
53 r
vitro from pBB68 by digesting with the restriction
enzymes StuI and AccI, and making the ends of the
molecules blunt-ended by filling in with DNA polymerase.
(This will remove 48% of the 1199 base par zot gene).
One half of the sample is ligated to a tetracycline
resistance gene (of foreign origin), these giving a
selectable marker.
The zot deletion mutant constructed in vitro above
is introduced into the chromosome of V. cholerae CVD101
as previously described for the construction of the ctx
deletion mutant of CVD101. The tetracycline resistant
clone derived above is cloned into the Inc P plasmid
pJBK85. This plasmid (Tcr, Cmr) is mobilized into
CVD101, selecting for Tcr. A second Inc P plasmid, pR751
(Tpr) is introduced. Selection of Tcr, Tpr, CmS colonies
result in V. cholerae strains in which the Tcr gene has
recombined into the zot gene.
The plasmid containing the StuI-AccI deletion mutant
without the Tcr gene is then mobilized into the Tcr V.
cholerae strain. Homologous recombination of the
deletion mutant into the chromosome results in the loss
of the zot gene sequences, an event which can be detected
by loss of Tcr. Tcs colonies are selected and screened
for loss of zot sequences by DNA hybridization using the
2 5 StuI-AccI fragment as a probe.
ExamPle 10
Construction of CVD109- a V. Cholera StrainDeletions of
Sequences coding for V. Cholera Toxins and for Zonula
Occludens Toxin
The construction of attenuated V. cholerae ~strain
CVD109 (ATCC# 55057) involves in introduction of cloned
Vibrio sequences along with sequences encoding a
selectable marker into the chromosome of a virulent V.
cholerae strain. An initial in vivo recombination event
of homologous sequences from the recombinant plasmid into
the chromosome provides a selectable marker at this site.
2173S~
W U ~ 4 s ~ q ~ !
~ IPEAJUs 2 9 SEP 19~5
A second in vivo recombination event between homologous
flanking sequences results in excision of proficient
genes from the chromosome with the end product being a
deletion mutation.
Figure 16 illustrates the construction of CVD109.
The zot and ctx genes are adjacent to each other on the
V. cholerae chromosome. Multiple copies of a 2700 base
pair DNA sequence called RSl (for repetitive sequence)
are on both sides of the zot and ctx genes in virulent V.
cholerae strain E7946 (El Tor biotype, Ogawa serotype).
In Figure 16A, the zot and ctx sequences are shown by a
large open or hashmarked arrow. RS1 sequences are shown
by a smaller, solid arrow.
The recombinant plasmid, pCVD51 (Figure 16A),
contains cloned zot/ctx sequences (open arrow) which are
homologous to the chromosomal zot/ctx sequences (shown by
hash-marked arrow in Figure 16A) and contains a
selectable marker, ampicillin resistance (Apr). The
plasmid vector into which the Vibrio sequences were
cloned is pGP704 (Miller and Mekalanos J. Bact. 170,
2575-2583 (1988)). This plasmid cannot replicate
extrachromosomally in V. cholerae but can replicate in
permissive E. coli strains. pCVD51 was mated from E.
coli to V. cholerae E7946. Since this plasmid cannot
replicate extrachromosomally in V. cholerae, selection of
Apr colonies yielded strains in which the entire plasmid,
with the Apr marker, was homologously recombined into the
chromosome at the site of the zot/ctx sequences. (The
exact site of recombination, whether in zot or ctx gene,
is not known.) The result of this single cross over (not
double cross over) event is termed a "cointegrate"
structure and is depicted in Figure 16B.
The RS1 sequences flanking the zot/ctx region are of
sufficient length to provide detectable in vivo
recombination; intra-molecular recombination between the
homologous RSl elements results in the loss of all
sequences between them. The Apr V. cholerae with the
217~
~,~ IP~ S 29 SEP 1995
integrated plasmid was grown in the absence of ampicillin
and the Ap sensitive (Aps) colonies were selected.
Recombination of the RS1 elements flanking ctx and zot
resulted in the loss of the intervening zot and ctx
sequences along with the plasmid vector containing Apr
(Figure 16C).
The Aps V. cholerae colonies resulting from the
above steps were screened by DNA hybridization for zot
sequences. The DNA probe consisted of a 575 base pair
? 0 Stu~-~ccI restriction fragment derived from the cloned
zot gene. ColonLes whi~did~ ~ a~lze to th1s~pro~e
were selected and probed for the presence of ctx genes by
DNA hybridization using a ctx gene probe. The
hybridization results confirmed the loss of both the zot
and ctx genes. One representative strain was saved and
designed CVD109. Figure 16D depicts the chromosome of
CVD109 which is deleted of zot and ctx sequences but
retains one copy of the RS1 element. (The plasmid shown
in Figure 16D is not retained in the final Aps strain but
is depicted only to illustrate the outcome of the second
cross-over event. This transient product is
spontaneously lost since the plasmid cannot replicate
extrachromosomally'in V. cholerae.)
Example 11
Construction of CVD110-a V. Cholerae Strain Having
Deletions of Sequences Coding For A and B Subunits of V.
Cholerae Toxin and for Zonula Occludens Toxin, and Having
Inserted a Mercury Resistance Gene and DNA Codinq for B
Subunit of V. Cholerae Toxin
CVD110 (ATCC# 55188) was constructed directly from
V. cholerae CVD109, the description of which has already
been provided. V. cholerae CVD109 lacks both the A and B
subunits of cholera toxin (CT) as well as the gene
encoding zot and is sensitive to mercury. A gene
fragment that contains the CT B subunit gene (ctxB) and a
mercury resistance gene was constructed in vitro. This
AME~IDED SHEE~
56
construction was then inserted into the chromosome of
CVD109, specifically into the hemolysin gene. Thus, the
final vaccine strain, CVD110, produces the B but not the
A subunit of cholera toxin, is resistant to mercury and
does not produce wild type HlyA protein (hemolysin).
CT B construction: The ctxB and ctx promoter
sequences were obtained from plasmid pCVD30, which is
described in Example 7. This plasmid pCVD30 contains a
deletion of the ctxA gene. A 1.4 kilobase fragment
containing the ctxB gene and the ctx promoter but not the
zot gene was obtained by digesting pCVD30 with HinP1 and
HaeIII enzymes. The fragment was treated with T4 DNA
polymerase to render the ends of this fragment blunt-
ended and synthetic KpnI linkers were ligated to this
fragment. The fragment was then cloned into the vector
pCVD315 [Galen, et al. Advances in Research on Cholera
and Related Diarrheas, vol. 7 (Sack et al., Eds.) pp.
143-153 (1990)] Vector pCVD315 has no particular
significance for this purpose other than the presence of
a KpnI site. The resulting plasmid containing the ctxB
gene was called pCVD621 (Figure 18).
Mercury resistance genes: The source of the mercury
resistance genes (mer) was the same as that used for V.
cholerae JBK70. A 4.2 kb NcoI- StuI fragment containing
mer was originally derived from pDB7 [Barrineau, et al.
J. Molecular & Applied Genetics (1984) vol. 2, pp.601-
619]. The fragment was treated with DNA polymerase
(Klenow fragment) to render the ends of this fragment
blunt-ended and synthetic KpnI linkers were ligated to
this fragment. The fragment was then cloned into plasmid
pCVD43.2 (unpublished), which is a derivative of pCVD43
[Kaper, et al. Advances in Research on Cholera and
Related Diarrheas, vol. 6 (Ohtomo, et al., Eds.) pp. 161-
167 (1988)]. pCVD43 contains the cloned hemolysin genes
(hlyA) of V. cholerae without a 400 bp HpaI fragment
internal to hlyA. The deletion of the 400 bp HpaI
fragment renders the gene inactive [Kaper, et al.
2~3~
- PCTIU.~ 9 4 ~ L
~ . .
~ ~, J L_ J ~, _; ~ r,,
Advances, etc. vol. 6]. pCVD43.2 is identical to pCVD43
except that a synthetic KpnI linker has been ligated into
the single HpaI site of pCVD43. The combined clone of
the mer genes inserted into the hylA gene is called
5 pCVD43.3.
Insertion of ctxB and mer genes into CVD109: To
introduce these genes into the chromosome of CVD109,
plasmid vector pGP704, which is described in Example 10,
was used. An 8.1 kb ClaI-BglII fragment from pCVD43.3
10 containing mer and hy1 clone~ in ~7Q4 t~ ~Gduce
pJMK12 (Figure 18). ^~ di~ested ~Ith~
KpnI to yield a population of linear molecules in which
only one of 3 RpnI sites was cut. The 1.4 kb fragment of
pCVD621 (described above) containing the ctxB gene was
then ligated to pJMK12 to yield pCVD622.2B. The relative
position and orientation of the inserted genes is shown
in Figure 18.
pCVD622.2B was then introduced into V. cholerae
CVD109 by conjugation from an E. coli host strain. As
described in Example 10, pGP704 cannot replicate
extrachromosomally in V. cholerae but can replicate in
permissive E. coli strains. Since pCVD622.2B cannot
replicate extrachromosomally in V. cholerae, selection of
Apr colonies [pGPio4 contains a gene encoding ampicillin
resistance] yielded strains in which the entire
pCVD622.2B plasmid, with the Apr marker, was homologously
recombined into the chromosome at the site of the hylA
gene. [It could not recombine into the ctx or zot
sequences because CVD109 lacks these genes.] The result
of this single cross-over (not double cross-over) event
is termed a "cointegrate" structure or "merodiploiod"
(these terms are used interchangeably) and is depicted in
Figure l9B.
A second cross-over event can occur between the
homologous hylA sequences flanking the integrated pCVD-
622.2B. This second cross-over event occurs
spontaneously and is detected by selection of colonies
; "-,; . . .
~ 2173~
~PEA/US 2 9 SEP 1995
which have lost the Apr phenotype. This second cross-
over event can have one of two possible outcomes,
depending upon the exact site of recombination. Both
possible outcomes result in the loss of the pGP704
plasmid vector sequences and are depicted in Figures l9C
and l9D. One outcome simply re-generates the original
situation, i.e., a strain identical to CVD109 which lacks
ctx, zot, and mer. The second outcome results in the
lost of pGP704 sequences but the mer and ctx sequences
contained within the hlyA sequences are retained. The
two possible outcomes are readily distinguished by DNA
hybridization using radiolabeled ctx sequences as a
probe. To isolate the desired outcome, a culture of
CVD109 containing the integrated pCVD622.2B was grown up
in L-broth without added antibiotics. This culture was
plated on non-selective L-agar plates and the resulting
colonies were replicated onto Ap containing L-agar
plates. Aps colonies were then hybridized to the ctx
probe and colonies possessing ctx sequences were
isolated. One such colony was designated V. cholerae
CVDllO. This strain was confirmed by DNA hybridization
to contain ctx and mer sequences and to also lack pGP704
sequences and the 400 bp HpaI fragment internal to the
hlyA gene. V. cholerae CVD110 was also confirmed to
produce the B subunit of cholera toxin by ELISA [Sack,
D.A. et al. supra (1980)].
DNA se~uence of inserted genes: The exact DNA
sequences of the inserted ctx and mer genes are known
from the literature. The exact site of the hlyA gene
into which these genes were inserted is also known.
2173~
PCT/lJS 9 4 / 1 1 4 2 4
- ~ IPE4/lJS 2 9 SEP 1995
Example 12
DescriPtion of ACE (Accessory Cholera Enterotoxin)
As previously described, (Example 10, p. 48) the zot
and ctx genes are on a 4.3 kb region of DNA which, in
many El Tor strains, is flanked by copies of the RS1
sequence. For the vaccine strain CVD110, this entire
region is deleted. In addition to the zot and ctx genes,
there are DNA sequences encoding a third toxin, ACE. In
Figure 20, the map of this region is shown. A 2.9 kb
EcoRV fragment (SEQ ID No.: 1) was cloned into the vector
pCVD315 (p. 47, line 289) to produce the clone pCVD630
(shown in Figure 20).
The plasmid pCVD630 was then introduced into V.
cholerae strain CVD110 and the acti~ity of this strain in
Ussing chambers was studied (Ussing chambers as
previously described. Culture supernatants of CVDllO and
CVDllO containing pCVD630 were tested as previously
described. Figure 2lA shows the results of supernatant
fractions which contained molecules less than 10 kDa in
size and fractions which contained molecules greater than
10 kDa. Essentially no Ussing chamber activity was seen
in the <10 kDa fraction. In the >10 kDa fraction, CVDllo
produced some changes in short circuit current (Isc) but
no more than the negative control, PBS (phosphate
buffered saline). However, supernatants of the CVDllO
(pCVD630) gave a significant increase in Isc. This
difference was statistically different (p<0.02) than the
result seen with CVDllO alone. As previously described,
an increase in Isc can be due to either an increase in
potential difference (PD) or tissue conductivity (Gt).
The increase in Isc induced by the genes contained on
pCVD620 is due an increase in PD, as shown in Figure 2lB.
AM~NDED SltEET
217
.,) 9~ 4~ ~
~, IP~ 2 9 S~P ~995
Thus, the 4.3 kb region flanked by the RS1 elements
in El Tor strains contains 3 putative enterotoxins.
(This same 4.3 kb region is present in classical strains
but RS1 sequences are found on only one side.) All three
of these toxins are capable of increasing Isc in Ussing
chambers using rabbit ileal tissue, an activity which
correlates with diarrheagenicity in humans. Two toxins,
cholera toxin and ACE, act by increasing PD while the
third, ZOT acts by increasing tissue conductivity. It is
~0 desirable that all three activities are eliminated from
an a~tenuated V. cholerae vacc ~ Z~in to avoid the
reactogenicity seen with CVD101 and other attenuated
vaccine strains. CVD110 does lack all three activities
and as seen in Figure 21, produces changes in Ussing
chamber comparable to changes induced by PBS (i.e.
essentially no changes).
The DNA sequence of the 2.9 kb EcoRV fragment (SEQ
ID No.: 1) contained in pCVD620 is shown in Figure 22.
There are two open reading frames (ORFs) immediately
upstream of the zot gene. The smaller ORF immediately
upstream of zot is a 297 bp ORF (ACE) which could
potentially encode a protein of 11 kDa and the larger ORF
immediately upstream of the 297 bp ORF is a 1185 bp ORF
(OrfU) which could potentially encode a protein of 44
kDa. Ace activity is thought to be localized to the 297
bp open reading frame.
ExamPle 13
Construction of V. cholerae CVD112 - a V. cholerae
vaccine strain of the 0139 serogrouP
Strains of V. cholerae are classified into multiple
O groups. Strains of O group 1 cause cholera gravis with
the potential for widespread transmission in pandemics.
V. cholerae of other O groups, so-called non-O1 V.
cholerae, cause illness, but until recently, did not
produce pandemic illness. Recently, tens of thousands of
cases of illness due to non-O1 V. cholerae have been
;--r ~ I r
- 2 1 ~ 3 ~ 9 4 ~
~ IPEA/~JS 2 g ~tt~ 1995
reported. It is thought that current 01-based vaccine
strains may not prove effective in protection against the
symptoms of non-01 serogroups. The new non-01 strain
isolated was given the new serogroup designation 0139.
The starting strain for the production of a non-01
V. cholerae vaccine strain is V. cholerae 1837. This
strain is of the 0139 serotype and was isolated from a
patient with cholera in Bangladesh. Figure 23 depicts
the chromosomal arrangement of the cholera toxin locus in
stra in 1837.
Pla~ pC~O~L ~a5 lnt~oduce~ Int~ s~aln 1~31 a~
described in Example 10. This plasmid recombined into
the bacterial chromosome and a second recombination event
resulted in the loss of one copy of the toxin genes and 3
copies of the RSl elements. The resulting strain, V.
cholerae 1837.1, is shown on the second line of Figure 24
For simplicity, in Figure 24 the cep, orfU, ace, zot,
ctxAB sequences are together referred to as the V.
cholerae toxin "core." The V. cholerae chromosomal locus
comprises the one or more core regions and associated RS1
elements.
The remaining core sequences of strain 1937.1 were
deleted as follows
Chromosomal DNA from strain 1937.1 was prepared and
partially digested with the restriction endonuclease
Sau3a to yield fragments ca. 30 to 40 kb in size. These
fragments were cloned into the cosmid cloning vector
pHC79 (Hohn and Collins, Gene, 11, 291-298, 1980) and the
resultant clones were screened after introduction into E.
coli by DNA hybridization using ctx gene sequences as
probe. A clone containing the cxt genes on a ca. 30 kb
vibrio insert was identified and isolated and the
resultant plasmid partially mapped. The isolated
construct was designated pLC13 Figure 25. This plasmid
was digested with EcoRI and religated onto itself,
thereby reducing the size of the insert to ca. 25 kb.
The resulting plasmid was designated pLC14. pLC14 was
.
2 l 7 3 ~ Ç ~
partially digested with ~indIII and then religated onto
itself to remove an ca. 14.6 kb HindIII fragment
containing the core region, the RS1 se~uences and
approximately 7.4 kb of uncharacterized V. chol erae DNA.
The resulting plasmid, which lacks the core, RSl
sequences was designated pLC15. A similar HindIII
fragment was removed in an attenuated strain construction
described in Pearson et al., P.N.A.S. fUSA), 90, 3750-
3754, 1993. Plasmid pLC15 was then digested with Sal I
and EcoRI and the ~
vector pGP704, describ@d l~ ~xample L0. ~ne resultLng
plasmid was designated pLC16.
The suicide vector pLC16 containing V. chol erae
strain 1837.1 chromosomal sequences which flank the ctx
locus was mobilized into V. cholerae strain 1837.1, by
the method described in Example 10. Homologous
recombination of the vector sequences into the chromosome
was detected by screening for ampicillin resistant
colonies, as ampicillin resistance is encoded by the
vector sequences.
An ampicillin resistant V. cholerae produced by this
method and having the plasmid sequences integrated into
the bacterial chromosome was thereafter grown in the
absence of ampicillin and the ampicillin sensitive
colonies were selected. A second recombination event
resulted in the loss of the ctxA, ctxB, zot, ace, orfU,
cep and RS1 sequences along with the plasmid vector
encoding ampicillin resistance. This resulting strain
was designated 1837.2. The vibrio chromosome in strain
1837.2 is graphically depicted in the third line of
Figure 24.
The gene encoding the B subunit of cholera toxin
along with a gene encoding mercury resistance was added
to strain 1837.2 as described in Example 11 above. The
same suicide vector, pCVD622.2B, was used as was employed
to make strain CVD110. The event resulting in the
incorporation of the B subunit sequences is depicted in
AMENDEDSHEEt
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Figure 26. The final strain, CVD112, is deleted of all
ctx, zot, ace, cep and RS1 sequences, expresses CTB
subunit, and is mercury resistant.
Preparation of CVD112 and Al1837 for vaccine studies.
The V. cholerae CVD112 and Al1837 strains were
stored in TSB containing 18% glycerol at -70C. Before
administration to volunteers, a vial of the appropriate
strain was thawed and streaked onto blood agar, TCBS, and
BI agar. After 24 hours incubation at 37C, colonies
that agglutinate the appropriate sera were chosen and
plated onto BHI, and further incubated at 37C for about
20 hours. The V. cholerae was re-plated, followed by 5
hours incubation at 37C and harvested with sterile
saline. The concentrations were determined by optical
density of the solutions compared to that of known
standards. Further quantitation was made by the replica
plate technique before and after administration of the
organism to volunteers.
Vaccine clinical trials.
Adult volunteers 18 to 40 years of age were
recruited and informed, signed consent was obtained.
Volunteers were carefully screened to ensure that they
were in excellent physical and mental health.
A group of 14 volunteers were admitted to a medical
isolation ward and acclimated to the environment for two
days while medical screening was completed and baseline
samples collected for antibody measurement.
Volunteers were randomized to receive one of the
following, in double-blind manner, on the third day:
(i) CVD112 at 5X108 cfu with buffer (n=6)
(ii) CVD112 at 5X106 cfu buffer (n=6)
(iii) buffer alone
The two unvaccinated volunteers who received buffer
only were kept with the vaccinees to determine whether
person-to-person transmission of the vaccine strain
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occurred in this setting. Vaccinees were closely
observed for 5 days after vaccination and then treated
with a 5 day course of tetracycline.
The volunteers were challenged approximately four
weeks later with the pathogenic V. cholerae 0139 strain
A11837 to access vaccine efficacy against homologous
challenge. the challenge dose comprised approximately
106 cfu of V. cholerae 0139 strain A11837. Volunteers
were closely monitored for about 120 hours following the
challenge. All volunteers received tetracycline (500 mg
every 6 hours for 5 days) beginning 5 days after
vaccination and 4 days after challenge to eradicate
carriage of the vaccine and challenge strains.
Eight vaccinees were challenged, four from the 5X108
group and four from the 5X106 group. Fifteen
unvaccinated controls were also given a challenge dose.
One of the eight vaccinees (13%) and twelve of the
controls (80%) developed diarrhea after challenge, giving
a vaccine efficacy of 84%. The one vaccinee who
developed diarrhea had received thè 5xl06 dose of the
CVD112 vaccine.
Three (38%) of the eight vaccinees and 14 (93%) of
the controls shed the challenge strain, although the peak
excretion was 100-fold less in the vaccinees, indicating
that the vaccine strain had effectively colonized the
vaccinees.
These results indicate that V. cholerae CVD112 is a
safe and effective vaccine for protection against the
symptoms of cholera for non-Ol vibrio strains of the 0139
serotype.
ExamPle 14
Construction of V. cholerae CVD112 RM - An attenuated
recA~ V. cholerae derivative of the 0139 seroqroup.
V. cholerae strain CVD112 is the starting strain for
the construction of a recA deficient, 0139 serogroup,
attenuated V. cholerae vaccine strain. Mutation of the
recA gene is thought to be desirable to lower the already
t -31,.~
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L~ l&~r~ J ~ ~J
low probability that an attenuated strain would become
virulent via recombination with wild-type ctx genes in
vivo. The recA mutant was constructed in a manner
similar to that described in Ketley et al., Infect. and
Immun., 58 (5), 1481-1484, 1980 and Goldberg et al., J.
Bacter., 165 (3), 715-722, 1986.
Plasmid pCVD831, which contains a ca. 7 kb NarI-NheI
fragment having the recA gene of V. cholerae El Tor
strain N16961 cloned into the broad host plasmid pCVD316,
a derivative of the IncP plasmid pRK290 previously
described above. pCVD831 was digested with XbaI and
PvuII to remove a ca. 50 bp fragment internal to the recA
gene coding sequences. The ends of the restricted DNA
were made blunt and the plasmid was religated to itself.
The ca. 50 bp deletion inactivates the recA protein. The
resulting plasmid was designated pCVD832.
Plasmid pCVD832 is mobilized into V. cholerae CVD112
and homologous recombination replaces the native recA
gene with the mutant recA gene. The homologous
recombination event is detected by screening for
sensitivity to methylmethanesulfonate (MMS), essentially
as described by Kettler et al., (1980). supra. The
strain resulting from the abQve procedure is deleted of
all ctx, cep, orfU, ace, and RSl sequences and has an
inactivating mutation in the chromosomal recA locus
(i.e., is a recA~ strain). The recombination event that
occurs at the V. cholerae recA locus in the generation of
strain CVD112 RM is graphically depicted in Figure 27.
V. cholerae strains CVD112 and CVD112 RM are
substantially efficient vaccine strains and afford
substantial protection against virulent 0139 serotype and
other non-01 strains upon subsequent challenge with these
virulent strains.
CVD112 and CVD112RM may also be employed with one or
more V. cholerae vaccine strains, toxoids,
procholeragenoid, etc., in a combination vaccine for
protection against both virulent 01 strains and virulent
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WO95/10300 PCT~S94/11424 ~
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non-O1 strains, for example strains of the Ogawa or Inaba
serotype, of V. cholerae.
Recently, it has been reported that the cep locus,
which encodes the core encoded pilus, might act as an
accessory colonization factor. However, the cep gene
product is not thought to substantially interfere with
the ability of Vibrio cholerae strains under the
invention to colonize the intestines of a host organism,
in particular, the human intestine. However, if
desirable, the sequences comprising the cep, or any other
suitable colonization factor, can be re-inserted into the
chromosome of the V. cholerae of the invention following
the guidance and methods described herein.
As example, in re-inserting DNA encoding the cholera
toxin B subunit and a DNA encoding for resistance to
mercury into the chromosome at the hemolysin locus, one
can conveniently include the gene of interest, operably
linked to expression signal, into the plasmid employed in
the recombination procedure.
The resultant strain of such a recombination event
produces the B subunit of cholera toxin, a protein
conferring resistance to mercury and other heavy metals,
and the additional gene of interest.
ExamPle 15
V. cholerae CVD111 vaccine strain
Another V. cholerae vaccine strain designated V.
cholerae CVD111 (ctxA-, zot~, ace~, mer, ctxB) was
constructed in the same manner as that described above
for vaccine strains CVD109 and CVD110. However, the
starting strain for this additional vaccine was instead
V. cholerae El Tor Ogawa N16117. This strain produces
cholera toxin, ZOT and ACE. However, despite producing
cholera toxin, strain N16117 did not cause diarrhea when
tested in volunteers. Strain N16117 was initially
described in Levine et al. Acute Enteric Infections in
Children New ProsPects for Treatment and Prevention,
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1981, supra. It should be understood that the recA
mutation described above in CVD112RM may also be
generated in the CVD111 V. cholerae by the methods
described if such is desired.
As V. cholerae CVDlll is derived from a strain which
does not cause diarrhea, deletion of the ctxA, zot and
ace genes provides a vaccine strain that has minimal
reactogenicity but, by still expressing the cholera toxin
B subunit, elicits substantial immunogenic response.
V. cholerae CVD111 is administered in dosage
regimens as described above, to produce protection
against the symptoms of cholera upon subsequent exposure
to virulent V. cholerae strains.
While the invention has been described in connection
with specific embodiments thereof, it will be understood
that it is capable of further modification and this
application is intended to cover any variations, uses, or
adaptations of the invention following, in general, the
principles of the invention and including such departures
from the present disclosure as come within known or
customary practice within the art to which the invention
pertains and as may be applied to the essential features
hereinbefore set forth, and as follows in the scope of
the appended claims.
