Note: Descriptions are shown in the official language in which they were submitted.
~ W094/~275' 2 1 6~ 1 5 5 PCT~S94/03811
MODELS FOR
G~NO~OCr~T lNr~ lON
FTELD OF T~E l~.v~ ON
The present invention is generally related to
developing an animal model having an infection with an
organism requiring human factor Clq binding for
pathogenicity~ More particularly, the present invention
is related to providing an in vitro and in vivo rat model
for testing prophylactic and therapeutic agents for
gonorrhea and gonorrhea related disease states.
~CR~ROUND OF TH~ l~.v~ ON
Neisseria gonorrhoeae (GC) is an obligate human
pathogen. Gonorrhoea is currently the most frequently
reported bacterial infectious disease in the United
States (about 2 million cases annually) (6). Gonorrhoea
is a sexually transmitted disease of the young population
of reproductive age. Approximately 15~ of patients with
gonorrhoea develop severe complications and sequelae such
as, pelvic inflammatory disease (PID), infertility due to
fallopian tube adhesion, ectopic pregnancy, and
epididymitis. Frequent complications of PID are
perihepatitis and peritonitis. Peritonitis may occur due
to the menstrual blood refluxes through the fallopian
tubes to the peritoneal cavity (7,8). Clinical
observations strongly suggest that the peritoneal route
is a dominating one in disseminated gonococc~l infection
(DGI) in women. Dissemination of N. gonorrhoeae from
local peritonitis to the blood stream following pelvic
inflammatory disease occurs in a significant portion of
cases of gonorrhoea (8). Association between pelvic pain
and menstruation in PID may suggest that N. gonorrhoeae
ascend at the time of the menses (containing Clq) and
2 2l7~ 01 55 ~ ; PCT~S94/03811
--2--
enters the peritoneal cavity. Approximately 75% of
individuals who develop disseminated gonococcal infection
are women. DGI is typically characterized by arthritis-
dermatitis and/or endocarditis, myopericarditis,
hepatitis, with functional abnormalities of the liver,
and meningitis.
Approximately 10% of pregnant women in many
developing countries may be infected with gonorrhoea (9).
Gonorrhoea during pregnancy is a risk factor for
premature delivery and may be transmitted to the neonate,
thus causing ophthalmic neonatorum. Gonococcal
ophthalmic neonatorum may be lead to blindness. There is
an apparent lack of protective immunity following natural
lS infection, even worse, a history of previous gonorrhoea
is a risk factor for gonorrhoea. Therefore, development
of a gonococc~l vaccine is needed for the prevention of
millions of cases in young people annually, including at
least two million young Americans, and may result in the
savings of millions of U.S. dollars. Years of previous
studies suggest that it is almost impossible to develop
successful gonococcal vaccine in the absence of an
experimental animal model.
Previous experiments suggest that a component of
human blood glycoprotein Clq may be a host factor that
increases attachment of GC to tissues and cells in vitro
(lO,ll). Clq is a glycoprotein, structurally similar to
collagen in terms of protein and carbohydrate moieties
(12). As a first component of complement, Clq present in
human blood initiates activation of the classical pathway
in the presence of antibody as well as in an antibody
independent manner. Clq is synthesized by epithel;~l
cells from representative portions of the entire human
genitourinary tract. This correlates with the
observation that urogenital epithelium from human and
~ WOg4/~75 2 ~ 6 0 1~ ~ PCT~S94/03811
-3-
chimpanzees are the only species susceptible to
gonococcal adherence in vitro.
Development of an animal model of gonorrhoea has
been hampered because only the human develops persistent
colonization and infection. Since the gonococcal
phenotype is influenced, not only by the organism itself,
but also by the host, it seems to be important to
preserve these interrelationships. This would require
continued development and evaluation of models that mimic
human gonococcal infection. Gonococcal urethritis has
been produced experimentally in male chimpanzees (46).
Attempts to infect the genital tract of other animal
species, including baboons, pig-tailed macaques, rhesus
monkeys, squirrel monkeys, owl monkeys, capuchin monkeys,
guinea pigs, and different strains of mice (47,48) have
been unsuccessful. In addition, inoculation of gonococci
into either the respiratory tract or mammary gland failed
to produce infection (49). Kita et al., (1981) reported
gonococcal infection of the genital tract of mice of the
ddY strain (112). This strain from this single report is
unavailable , no one has been able to use these animals
for infection. Johnson et al., (1989) reported that mice
of the strains C3H, CBA, BALB/C, TO and ICR are resistant
to gonococc~l infection. Models on other animals,
including implanted chambers were also proposed but were
limited to the study of GC immunobiology
(50,51,52,53,54,55,56). The experimental model for other
related species N. meningitidis was developed on newborn
rats. Me~inqococci survived only for six hours. Despite
the short time of survival, this model allowed others to
study the vaccine (57).
.
~UMNARY OF THE l~.V~ lON
35
The present invention involves provision of an
animal having an infection with an organism requiring
~ 4_ PCT~S94/03811
complement Clq binding for effective pathogenicity. The
animal is produced by administration of the organism
combined or coated with human factor Clq. The animal is
normally resistant to the organism in question or r
5 experiences infection with only low frequency. The
animal is, however, more susceptible to infection when
the administered organism is combined or coated with
human factor Clq. The animal may be a human or
chimpanzee, for example, human male volunteers and
10 ~h; -n~eeS normally experience a low rate and degree of
infection. Preferably, however, the animal is non-
primate, in particular, the most preferred animal is a
rat.
- 15 The organism may be a microbe such as bacteria or
fungi or may be viral. In particular, the bacteria may
be N. gonorrhoeae, Treponema pallidum or any gram
negative bacteria that produce cardiolipin. The bacteria
may be Chlamydia. A viral organism may be Human
20 Immunodeficiency Virus having a protein which binds Clq.
By "coated" we mean the organism is preincubated with Clq
or, alternatively, Clq is administered first and the
organism is administered in a later, separate, injection.
This animal model is useful for the disease states caused
25 by gonorrhea infection included pelvic inflammatory
disease and disseminated gonococcal infection.
The present invention also provides a prophylactic
or therapeutic composition for intravaginal use which
30 includes an antibody or N. gonorrhoeae peptides having
bin~;ng specificity for human factor Clq. The
composition is useful to prevent or treat gonococcal
infection and may be in the form of a salve, douche,
diaphragm jelly or an emollient. The N. gonorrhoeae
35 peptides may be outer membrane peptides such as those
having approximate molecular weights of 17kDa, 3lkDa,
35kDa, and 48kDa.
~ W094/~275 2 1 6 ~ 15 ~ PCT~S94/03811
--5--
A further embodiment of the present invention is a
method of developing a treatment regimen for gonococcal
infection. This method includes infecting non-primate
animals with N. gonorrhoeae coated with Clq and
subjecting the animals to a test therapy while monitoring
viable N. gonorrhoeae cells in the animal. The treatment
regimen may involve the use of a therapeutic drug such as
an antibiotic, for example.
A preferred embodiment of the present invention is a
method for formulating a vaccine for protection against
gonococcAl infection. The method includes the steps of
i) administering gonococcal antigens to an animal, ii)
infecting the animal with Clq coated N. gonorrhoeae, and
iii) monitoring viable N. gonorrhoeae cells developing in
said animal to determine an antigen resulting in
inhibited developments of N. gonorrhoeae cells. The
gonococcal antigens may be outer membrane peptides such
as those having approximate molecular weights of 17kDa,
3lkDa, 35kDa and 48kDa.
The present invention provides for a prophylactic or
therapeutic composition for intravaginal use including a
Clq synthetic analog having binding specificity for N.
gonorrhoeae and without binding specificity for
eukaryotic cells. The composition is useful to prevent
or treat gonococcal infection and may be in the form of a
douche, ~iAphragm jelly or an emollient.
Clg synthetic analogs may be analogs or fragments of
Clq retAining binding specificity for N. gonorrhoeae but
lacking the binding specificity for the eukaryotic host.
one skilled in the art understands that a coating of Clq
analog on N. gonorrhoeae would prevent native Clq from
b;n~ing and would, thereby, decrease N. gonorrhoeae
pathogenicity. Such an analog would be most effectively
2ifi015~
W094/~27s PCT~S94/03811
--6--
used as a topical agent similar to the administration of
anti-Clq antibody.
A further embodiment of the present invention is a
method of treatment for gonococcal infection comprising
the administration of a Clq synthetic analog having
b;n~;ng specificity for N. gonorrhoeae and without
b;n~;ng specificity for eukaryotic cells. Likewise any
way of inhibiting the bridging function of Clq for tissue
and GC is now taught to be therapeutic and/or
prophylactic.
One skilled in the art understands that the animal
model of the present invention may provide a diagnostic
method for using polymerase chain reaction (PCR) with
appropriate primers to detect gonococcal cellular DNA in
blood. Current methods exist for using PCR to detect GC
in urine and vaginal fluid, however, methods do not exist
for PCR detection of GC in blood, cerebrospinal fluid,
joint fluid or amniotic fluid.
BRIEF DE8CRIPTION OF T~ DRAWING~
Figure l shows the dose dependent bin~; n~ of Clq to
N. gonorrhoeae JCl.
Figure 2 shows that gonocorcAl attachment to
leukocytes is enhanced by complement Clq.
Figure 3 is a hypothetical model of attachment of N.
gonorrhoeae to human tissue in the presence of Clq.
Figures 4A and 4B show the attachment of N.
gonorrhoeae JCl to ovarian tissue in the presence of Clq
(A) and in the Ah~ e of Clq (B). Notice in 4(B), lack
of attachment.
~WO 94/~275 ~1 6~5 ~ PCT~S94/03811
--7--
Figure 5 shows a Western blot of outer membrane
protein of N. gonorrhoeae JCl probed with human Clq (line
A) and with anti-cardiolipin IgG (line B). Molecular
mass stAn~rds in kilodaltons (on the left). Solid
S circles show common bands of OMP reacted with complement-
Clq as well as with anti-CL IgG.
Figure 6 shows common and type specific structure of
phospholipids tested for Cl~ deposition and anti-CL IgG
binding; arrow indicates cleavage site for phospholipase
C.
Figure 7 shows the protective effect of anti-Clq IgG
on the development of gonococcAl bacteremia on newborn
rats. Clq coated GC were used for i.p. inoculation.
Figures 8A-8C relate to the effect of Clq on the
attachment of Neisseria gonorrhoeae to the genital
tissues. Clq-enhanced attachment of GC to rat and human
tissues but not to that of rabbits and only a lesser
extend in mice is shown by number of GC binding per
stAn~rd unit area at 400 x magnification (8A). The mean
values and stAn~rd errors are shown for each group of
data. Schematic diagrams (Figures 8B and 8C) represent
proposed m~chAn;sms.
Figures 9A-9C relate to the Clq protection of GC
from the bactericidal effect of newborn rat serum (in
vitro). Clq dose-~PpPn~ent protection from the killing
effect of newborn rat serum on GC was represented by the
number of CFU per ml. Each bar represents the mean value
of 3-5 independent experiments and stAn~Ard error values
for each group of data (Figure 9A). Schematic diagrams
representing proposed m~ch~ni~ms are shown in Figures 9B
and 9C.
~1~0155
W094/~27s ~ PCT~S94103811
-8-
Figure lO shows the inhibitory effect of anti-Clq-
IgG on the development of gonococ~Al bacteremia. The
dose-dependent protective effect of monospecific anti-
Clq-IgG was represented by number of CFU per ml of tested
blood. Protection observed at dilution l:400 was
statistically significant. (p< 0.05).
Figures llA and llB relate to the effect of Clq on
the attachment of Neisseria gonorrhoeae to the human
10 PMNs.
Figure 12 shows the inhibitory effect of anti-Clq-
IgG on the attachment of GC to PMNs. Open square
attachment of Clq treated GC to PMNs in presence of
antihoA;es.- Closed square attachment of GC to Clq
treated PMNs in presence of antibodies. The dose-
dependent protective effect of monospecific anti-Clq-IgG
was represented by number of GC per PMNs. Protection
observed at dilution l:500 was statistically significant
(p<0.05).
~ETATT~D ~ESCRIPTION OF THB PREFERRED EMBODl~.. o
The present invention provides an animal model for
testing vaccines and therapeutic agents against
gonorrhea. The model, in a preferred embodiment,
comprises live rats methodically infected with N.
gonorrhoeae coated with human factor Clq. This rat model
has less limitations than the human male volunteer model
whose most severe limitation is the fact that the
infection is localized to the male urethra. The rat
model of the present invention allows the use of females
to study the pathogenesis of DGI and PID, the most
serious consequences of gonorrhea. The routes of entry
and dissemination are equivalent to that in man. Clq
coated N. gonorrhoeae is pathogenic for this animal
model, the model is inexpensive and the illness is
~ W094/~275 ~ 1 6 ~ PCT~S94/03811
_g_
reproducible. Although N. gonorrhoeae strain JC1 is
primarily used in the provided examples, three additional
Neisseria gonorrhoeae strains isolated from patients with
gonococcal infection (from OB/GYN Clinic, UTMB,
Galveston, TX) were used on our new experimental animal
model. All of them developed gonococcal bacteremia
similarly to strain JC1.
Unless defined otherwise, all te~hn;cal and
scientific terms used herein have the same meAn;ng as
commonly understood by one of ordinary skill in the art
to which this invention pertains. Although any methods
and materials similar or equivalent to those described
herein can be used in the practice or testing of the
present invention, the preferred methods and materials
are now described. All publications mentioned hereunder
are incorporated herein by reference. Unless mentioned
otherwise, the techn;ques employed herein are stA~rd
methodologies well known to one of ordinary skill in the
art.
EXAMPL~ I
ID~ CATION OF Clg-BINDING NOLBCULE8
IN OUTER M~MRp~F~ OF NRT.C~ RTA GONOl~R~07;~AR
GonoroccAl strains appear to deposit and activate
complement, however, such strains survive in the human
host. The exact molecules on which strains of N.
gonorrhoeae from disseminated gonococcal infection
deposit complement were heretofore unknown. Neisseria
~onorrhoeae JCl was isolated from the blood of a female
patient. Deposition of Clq on bacterial cells or
purified outer membrane proteins of JC1 was analyzed.
35 Human Clq reacted with four polypeptides of
approximate molecular weights 17kDa, 3lkDa, 35kDa and
48kDa. Three bands (17kDa, 31kDa, 35kDa) which bind Clq
W094/~275 2 1 ~ ~ 1 S 5 PCT~S94/03811 ~
.--10--
; '
reacted with anti-car~iolipin IgG. The reactivity of
outer membrane proteins with Clq and anti-cardiolipin IgG
was abolished after phospholipase C treatment. This
example demonstrates that yonococcal outer membrane
peptides serve as a bin~;ng target for human Clq. The
antibody independent bi n~; ~g of Clq appears to take place
on cardiolipin molecules associate with such peptides.
Mat~rial~ and Metho~s
Patients, Bacteria and Growth Conditions
N. gonorrhoeae JCl was isolated from the blood of a
female patient with disseminated gonococcal infection.
Strain JCl was resistant to killing by normal human serum
in vivo and in vitro (lOl). Strain JCl was grown on
gonococcal base agar with Kellogg supplements GCK (Difco
Laboratories, Detroit, Michigan) in 5% C02 at 37C (102).
~uman ~erum
Whole human blood was obtained by venipuncture from
healthy donors who had no history of gonorrhoea. The
blood was allowed to coagulate for one hour at room
temperature, and then centrifuged for 5 minutes at 2,500
x g. The serum was then divided and stored in plastic
tubes at -70C.
8~rum Bactericidal As3ays
The serum bactericidal assay utilized was a
modification of the procedure described previously
(103,104). To determine the resistance of strain JCl to
complement mediated killing by normal human serum,
gonococci were grown for 18 - 20 hours on gonococcAl
based agar, then suspended in gonococcal base broth to an
optical density at 595 nm of 0.2. A l:lO00 dilution was
made, and the gonococcal dilution was incubated with
WOg4/~275 ~ PCT~S94/03811
decomplemented normal human serum (heated for 30 minutes
to 56OC) at 37OC for 15 minutes. Then normal human serum
was added to a final concentration of 25% and incubation
r was continued for 30 minutes at 37C. From the assay
5 mixture, l0-fold and l00-fold dilutions were made, and 50
t ~1 was spread over the gonococcal base agar. The numbers
of colony forming units (CFU) were determined after
overnight incubation at 37C in 5% CO2.
10 Outer Membrane Preparation IOMP)
Cells of N. Gonorrhoeae JCl grown for 20 hours were
removed from the plates, washed in 50 Mm Tris
hydrochloride buffer (p~ 8.0), and suspended in 20%
l5 sucrose in the same buffer. Lysozyme in a concentration
of l mg per ml was added and the cells i~cllhAted for 30
minutes at 4C. The cells were diluted l:5 in 50 mM Tris
hydrochloride buffer (pH 8.0), and were subjected to
sonication in two bursts of 30 seconds each. Cellular
debris was removed by centrifugation at 5,000 x g for l0
minutes, and gonococcal membranes were isolated by
centrifugation at lO0,000 x g for l hour. Outer
membranes were isolated by differential solubilization in
1% sodium lauryl sarcosinate and centrifuged at l00,000 x
g for l hour. The pellet contained the outer membrane
proteins were washed with ethanol and suspended in
sterile water (105). Peptide patterns of purified OMP
was clearly different from that of CMP. Protein I, II
and III characteristic for OMP were present in the OMP
preparation (not shown).
PolYacrylamide Gel ElectroPhoresis and ~r~nsfer of
Fractionated Proteins to Nylon Nembrane
35 The outer membrane protein fraction of N.
gonorrhoeae JCl was incubated at l00 in the presence of
l.5% sodium dodecyl sulfate (SDS) and 2.5% 2-
Wog4/~27~ 2 ~ PCT~S94/03Sll
-12-
mercaptoethanol for 2, 5, and 10 minutes, layered onto 5%
polyacrylamide stacking gels, and fractionated in 12%
polyacrylamide in the pr~cenc~ of 0.1% SDS (106). Two
hundred ug of outer membrane proteins was added to each
5 gel. Polyacrylamide gel electrophoresis was performed at
room temperature using 30 mA per slab gel. Immunological r
characterization of the fractions was accomplished after
transfer by electrophoresis at 150 mA for 1-1/2 hours at
22C to a nylon membrane (107).
Reaqents
Human isolated Clq and polyclonal goat antisera to
human Clq (Cytotech, San Diego, CA) were used.
15 Horseradish peroxidase-conjugated rabbit anti-goat IgG,
phospholipase C and phospholipids;
phosphatidylethanolamine, phosphatidylinositol,
phosphatidylserine and diphosphatidyIglycerol
(cardiolipin), (Sigma Chemical, St. Louis, M0) were also
20 utilized as reagents. Identification of complement (Clq)
binding macromolecules in outer membrane proteins of N.
gonorrhoeae was performed utilizing nylon membrane
strips. They were incubated for 2 hours at 37 in 10%
human serum albumin (HSA) diluted in veronal buffer
25 saline (VBS) to block nonspecific protein bin~;ng sites.
Subsequently, the nylon membrane strips were ;ncllhAted
with 70 ug/ml of isolated Clg diluted in VBS with 15 x 10-
5M Mg 2+ for 1 hour at 37. The concentration of Clq in
human serum is about 70 ug/ml. Localization of human Clq
30 in specific zones in these strips was demonstrated with
horseradish peroxidase-conjugated polyclonal goat
antisera to human Clq. After three additional washes,
localization of the peroxidase enzyme was visualized with
3.3'-diaminobenzidine in the presence of appropriate
35 co~ntrations of hydrogen peroxide. The nylon membrane
strips were washed three times for 15 minutes each
between steps in 0.1% HSA in VBS in room temperature. To
~wo g4/~27S 2 1 ~ ~ 1 55 PCT~S94/03811
-13-
identify molecules that might cause nonspecific binding
of these antibodies, control nylon membrane replicas were
in~llh~ted with human Clq which had been heat inactivated
at 56 for 30 minutes.
Identification of anti-cardiolipin (anti-CL) IgG
binding macromolecules in the outer membrane proteins of
N. gonorrhoeae was performed using nylon membrane
replicas of polyacrylamide gel (PAGE). They were
incubated with rabbit anti-cardiolipin IgG (l:300)
diluted in O.l~ HSA in VBS with 0.05% Tween 20 in room
temperature. Before exposure to these antisera, the
nylon membrane strips were incubated 2 hours in 37C in
10% HSA diluted in VBS to block nonspecific binding. The
localization of ant; ho~ i es to specific components in the
transblots were identified using horseradish peroxidase-
conjugated goat anti-rabbit IgG in a dilution of 1:300.
Figure 5 shows a Western blot of outer membrane protein
of N . gonorrhoeae JCl probed with human Clq (line A) and
with anti-cardiolipin IgG (line B). Molecular mass
st~n~Ards in kilodaltons (on the left). Solid circles
show common bands of OMP reacted with complement-Clq as
well as with anti-CL IgG.
Pho~PholiPase C Treatment
Nylon membrane replicas of PAGE gels or nylon
membrane dots were ;nctlhAted for 2 hours with
phospholipase C (0.5 units/ml) diluted in 1% BSA Tris
saline pH 7.3 activated with O.OOl M CaCl2. Before
exposure to human Clq, the nylon membrane strips were
washed three times for 15 minutes each in 0.2% sodium
dodecyl sulfate (to stop reaction), and three times in
0.1% HSA in VBS, and incubated 2 hours in 37C with 10%
35 HSA in VBS to block nonspecific binding. Control nylon
membrane replicas or dot blots were incubated without
phospholipase C.
WOs4/~7s 216~1~5 -1.- rCT~S94/~811 ~
Btainina of GC Gels:
.
B; n~ i ng of Clq to GC cells was done by indirect
fluorescence using as first antibody goat anti-Clq-IgG
and second fluorochrome conjugated rabbit anti-goat IgG.
GC cells were also immobilized on nylon membranes and
stained according to the procedure used for OMP st~;ning
as described above.
Results ~ Di3cussion
In this Example, the deposition of Clq on intact
bacterial cells was tested by two alternative approaches.
First GC cells were incubated with Clq and immunostained
with anti-Clq rabbit IgG and a second antibody to rabbit
IgG conjugated with fluorochrome. Staining of cells
in~-1h~ted with Clq was found in the fluorescence
microscope. In the second approach, dot blot analysis of
whole cells immobilized on nitrocellulose membrane was
performed. Strong stA;ning was also observed by this
method indicating that molecules present on the bacterial
surface binds Clq without an antibody intermediary (Table
1) .
T~ble l. Reactivity of GC cells and purified OMP** with
Clg ~n~ ~nti-CL IgG
Reactivity Before PLC* Treatment After PLC Treatment
with
Clq Anti-CL Clq Anti-CL IgG
IgG
GC Cells + + NT NT
OMP + +
Note: (+) indicates positive reaction
(-) indicates no reaction
* PLC = phospholipase C
NT = not tested
** OMP = GC outer membrane preparation
~ W094l~7S 2 1 6 ~ 1 5 ~ PCT~S94/03811
-15-
Deposition of complement Clq on purified outer
membrane proteins of N. gonorrhoeae JC1 immobilized on
nylon membrane was tested by dot blot analysis. Antibody
against Clq reacted with immobilized proteins that were
exposed to Clq (Table 1). Specific molecules which
exhibited this function were identified in SDS-PAGE
fractionated outer membrane proteins of JC1 strain, which
were transferred to a nylon membrane ;ncllhAted with human
Clq. Antibody against human Clq reacted with four
polypeptides including 17kDa, 31kDa, 35kDa, and 48kDa
(Fig. lA). Our studies on human mitochondria proteins
showed that a number of proteins are associated with
cardiolipin. The peptide cardiolipin complexes of
mitochondria origin were found to bind Clq (108).
Therefore, antibody against cardiolipin was utilized to
determine if the outer membrane-preparation of N.
gonorrhoeae JC1 contained diphosphatidylglycerol
(cardiolipin). Table 1 shows the results of a dot blot
of outer membrane protein of N. gonorrhoeae JC1 probed
with anti-cardiolipin IgG. Anti-cardiolipin IgG reacted
with outer membrane proteins of JCl. Further analysis of
cardiolipin-cont~;ning molecules in the outer membrane
proteins of N. gonorrhoeae, were performed on resolved
proteins transferred to nylon membranes and probed with
rabbit anti-cardiolipin IgG. Six molecules having
estimated molecular weights of 17kDa, 28kDa, 3lkDa,
35kDa, 50kDa and 55kDa were detected by anti-cardiolipin
IgG (Fig. lB). Three bands (17kDa, 31kDa, 35kDa) which
bind Clq reacted with anti-cardiolipin IgG. Anti-
cardiolipin IgG recognized three additional molecules
(28kDa, 50kDa, 55kDa) which did not bind Clq. In order
to test if the anti-cardiolipin IgG utilized in these
experiments, bind specifically to cardiolipin, the nylon
membrane dot blots of purified selected phospholipids
were i~cl~hAted with anti-cardiolipin IgG (Table 2). Five
different concentrations were used for all phospholipids
-
W094/24275 2 1 ~ ~ 1 5 S PCT~S94/03811 ~
-16-
(5 mg, lO mg, 20 mg, 40 mg, 80 mg per l ml) and 5
different dilutions of ~nti-cardiolipin IgG (1:50; l:lO0;
1:200; 1:300; 1:500). Anti-cardiolipin IgG reacted with
cardiolipin, but did not r2act with
phosphatidylethanolamine, phosphatidylinositol, or
phosphatidylserine (Table 2 and see structures of
phospholipids in Fig. 6).
W094/~275 ~ 1 6 0155 PCT~S94tO3811
-17-
Table 2. Reactivity of select~ pho~pholipids w~th
$solated human Clq and with anti-c~rdiolipin
IgG ~nti-CL IgG)
Phospholipid Before PLC* After PLC
Treatment Treatment
Clq Anti-Cl Clq Anti-CL
IgG IgG
Phosphatidylethanol-
amine
Phosphatidylinositol
Phosphatidylserine
Diphosphatidylglycerol + +
Note: (+) indicates positive reaction
(-) indicates no reaction
* PLC = phospholipase C
Purified cardiolipin and dot blots of outer membrane
proteins of N. gonorrhoeae JCl that were treated with
phospholipase C lost reactivity with Clq and anti-
cardiolipin IgG (Tables l and 2). Further investigationof molecules affected by phospholipase C treatment was
performed on nylon membrane replicas of separated outer
membrane proteins ;ncllhAted with the enzyme for 3 hours
at 37C. In control experiments phospholipase C was
replaced with phosphate buffered saline. The reactivity
of all molecules of outer membrane proteins with Clq and
anti-cardiolipin IgG was abolished.
Although some bacterial species have been suggested
to bind Clq without an antibody intermediary, there has
been no such phenomenon described for N. gonorrhoeae .
Moreover, gonococcal macromolecules involved in Clq
bi~; n~ have not been identified yet. Such molecules, if
identified, might be of potential importance to
understand and prevent disorders associated with
gonococcal infections.
W094/~27S PCT~S94/03811
-18-
This example demonstrates that gonococcal cells and
isolated outer membrane peptides may serve as a binding
target for human Clq. Attàchment of Clq to at least four
peptides occurred without antibody intermediary. The
antibody independent binding of Clq appears to take place
on cardiolipin or cardiolipin-like molecules associated
with some OM peptides. The few outer membrane-
cardiolipin-like molecules on N. gonorrhoeae, which
reacted with anti-cardiolipin IgG, did not bind Clq.
This may be explained by Clq activation tests with
vesicles contA; n i ng increasing amounts of cardiolipin
indicated that over 30 mol ~ of cardiolipin is required
to activate Clq (l09).
The majority of previous studies on complement
binding and activation on bacterial surface used an
approach that included i~cllh~tion of intact cells with
complement, solubilization of cells and separation by
SDS-PAGE electrophoresis. With this approach however,
solubilization and SDS treatment may have resulted in
dissociation of bound molecules, so that obt~ine~ results
may be difficult for interpretation. In our study we
have first analyzed Clq binding to the surface of intact
cells. Once binding was confirmed, intact complement
components were used for identification of potential
binding sites on bacterial outer membrane peptides
separated on SDS-PAGE and immobilized on nitrocellulose
or nylon membrane. Although the solubilization process
of OMP in SDS was performed for 2, 5 and l0 minutes, Clq
binding to several OMP was not abolished. Recent
findings demonstrate that peptide-cardiolipin molecules
are present in mitochondria and are resistant to SDS page
treatment (108). Since cardiolipin is known to be
present in gonococcal cells, consequently we considered
the possibility that phospholipid like molecules, such as
cardiolipin, may be involved in Clq deposition. Such
~ W0941~275 2 1 6 0 15 ~ PcT~s94103811
--19--
molecules are found to exist in association with some OM
peptides.
Anti-cardiolipin antibcdy used in the present study
shows high specificity only for diphosphatidylglycerol
(cardiolipin). Diphosphatidylglycerol, one of the cell
membrane phospholipids unique in its structure and
function, possess two negatively charged phosphate group.
In contrast, the other phospholipids carry only one
negatively charged phosphate groups. Results
demonstrated that among tested phospholipids only
cardiolipin may deposit Clq. This finding is in
agreement with other investigations which demonstrated
that Clq bin~i ng on cardiolipin may initiate deposition
lS and activation of classical pathway (108,109). Taken
together, applicants suggest that the nondenatured
cardiolipin-peptides molecules on intact gonococcal cells
may be able to bind Clq. In the light of the above
presented observations we propose that also bacterial
cells of N. gonorrhoeae may deposit and activate the
complement cascade without a requirement for specific
antibody.
Gonococcal septicemia is associated with
multiplication of bacterial cells in the bloodstream
resulting in dissemination into several anatomical sites
including joints, genitourinary tract and other tissues.
Consequently applicants believe that circulating and
tissue-associated bacterial cells may continuously
activate the complement cascade without much harm to the
pathogen. In turn, activated complement or individual C
components may display their biological activities
including increased permeability an infiltration by the
cellular immune system. Activated humoral and cellular
systems may lead to pathogenic sequelae such as
arthritis, dermatitis or perhaps pelvic inflammatory
W094/~275 2 1 6 0 1 5 ~ PCT~S94/03811
-20-
disease, all devastating syndromes associated with
gonococcal infections.
EXANPL~ II
GON~rO~ . AT~ O ~UMAN L~u~G~ I8 ENHANCED
BY CONPLEMENT Clg
Phagocytosis and intracellular survival of Neisseria
gonorrhoeae in leukocytes appear to be an important
feature of gonococcal strains associated with
disseminated infections. Attachment of N. gonorrhoeae to
human cells, including leukocytes, is recognized as an
important first step necessary prior to invasion or
phagocytosis. Example I describes gonococcal outer
membrane receptors that bind purified Clq without
opsonizing antibody. Clq receptors are known to be
present on mammalian cells including leukocytes. Clq
molecules mediate the enhancement of phagocytic cell
functions. Strain of N. gonorrhoeae ~Cl from
disseminated gonococcal infection (DGI) is resistant to
killing by human defense me~hAni~m. This strain was
cultivated on GC medium and used for experiments (optical
density of 0.2 at 600 nm). Human PMNs were freshly
isolated. Microscope slides of PMNs were prepared and
stored at -70C until used. Purified Clq (70 mg/ml) was
preincubated either with bacteria or leukocytes. Washed
bacteria and leukocytes on slides were then used for
experiments. Binding of gonococci to leukocytes was
estimated in phase-contrast microscope. Attachment of
gonococcAl cells to leukocytes pretreated with Clq was
observed (20-30 bacterial cells per leukocyte) tFig. 2).
In the control fewer attached gonococcal cells (0-4 per
leukocyte) were observed (Fig. 2). Similarly gonococcal
cells preincubated with Clq displayed increased b;n~ing
to leukocytes (10-20 gonococcal cells per leukocytes).
The binding of gonococcal cells to its receptors on human
leukocytes was inhibited by monospecific antibody against
~ W094/~275 2 1 ~ ~ lS 5 PCT~S94/03811
-21-
Clq (Fig. 2). Clq, therefore, is a host factor that
increases attachment of N. gonorrhoeae JCl to human
leukocytes (Fig. 3). Therefore, increased binding via
Clq should be considered as a virulence factor of N.
S gonorrhoeae in DGI.
In preliminary studies on animal tissues, the
present inventors have tested attachment of N.
gonorrhoeae JCl to various genital tissues from different
animal species in the pr~ence and absence of Clq and
observed Clq-dependent binding of GC to the tissues from
rats (Table 3). GC did not attach very well to the
various animal tissues from other tested species (Table
3).
Table 3: Atta¢hment of N. gonorrhoeae to ~ifferent
tissuQs u~ing Clg a8 a potential adhesion.
8pecies Tissue N. N.
gonorrhoeae ~ gonorrh~-e
Clq ~ithout Clq
HUMAN Leukocytes +++ +
Uterus ++ +
Ovary +++
Fallopian tubes +++ +++
RATS Uterus ++ +
(one week Liver +++ +
old)
MICE Uterus + +
RABBITS* Uterus - -
Fallopian tubes
Ovary - -
-(0); +(1-4); +(5-l0), ++(10-20); and +++(30-40)
gonococcal cells per unit area.
*Rabbit tissues kindly provided by V. Schnell, M.D.,
~ Department of Obstetrics and Gynecology, The University
of Texas Medical Branch, Galveston, Texas.
5~ Pcrnss4/~3sll ~
--22--
E~aMPLE III
o;~r-~T, ATq'7'~ TO ~ lAN OVARIAN TI~8UE
Attachment of N. gonorrhoeae to human cells is a
n~c~sary first step prior to colonization or invasion.
The majority of gonococcAl attachment studies have been
performed on cells of human fallopian tubes. Very little
is known about the attachment of gonococcal organisms to
ovary cells, although ovarian tissue is often involved in
the infectious process, resulting in, e.g., pelvic
inflammatory ~;~e~e (PID) and infertility. Outer
membrane proteins and fimbriae of N. gonorrhoeae are
known to play an important role in attachment to selected
human tissues or cells, as observed by in vitro
experiments (83,84). However, many tissues or cell types
are not easily recognized by gonococcal cells.
Therefore, there may be alternative attachment mechAni~ms
(10). The inventors' experiments indicate that Clq
serves as a bridging molecule between gonococcal cells
and leukocytes.
Clq, a heat-labile protein present in human blood
and ovarian follicular fluid, is the first component of
complement, initiating activation of the classical
pathway. Clq is a glycoprotein that is structurally
similar to collagen in terms of its protein and
carbohydrate moieties. Clq is involved in different
types of immunoregulatory functions (85).
The molecular m~c-h~nicm associated with the bridging
function of Clq may include the following phenomenon.
These possible mech~nifims are presented to more fully
elucidate an underst~n~ing of the present invention's
importance but should not be viewed to limit the appended
claims. Clq appears to recognize receptors on
gonococcal (GC) cells; the present inventors have found
outer membrane peptides of GC that interact with Clq
W094/~75 2 1~ PCT~S94/03811
-23-
without opsonizing antibody (40). Conversely, Clq
appears to recog~ize receptors on leukocytes and,
therefore, bridge the bacteria and the host cell (lO).
~ Receptors for Clq are distributed in different tissues as
well, therefore, Clq-promoted binding occurs also in
other sites exposed to gonococcal infection.
Cryostat sections of snap frozen human ovarian
tissue were prepared and stored at -70C until used. A
suspension of strain of N. gonorrhoeae JCl isolated from
female blood was prepared (optical density of 0.2 at 600
nm). Cryostat sections were incubated with 40 ~l of GC
suspension in the presence or absence of Clq (20 ~g/ml)
for 30 min in a moist chamber. Samples were washed five
times with PBS pH 7.2 and bacterial binding visualized in
phase-contrast or fluorescence microscope.
Figures 4A and 4B show attachment of N. gonorrhoeae
JCl to ovarian tissue in the presence and absence of Clq.
Attachment to the ovarian tissue is dramatically
increased if either tissue or bacteria are preincubated
with Clq. In the absence of Clq, only few bacterial
cells were found to be attached to the ovary.
The capacity of Clq binding to gonococcal cells
immobilized on microtiter plates was investigated.
Different concentrations of purified Clq and monospecific
antibody (IgG) to Clq were used in an ELISA assay.
Figure l shows a dose-dependent binding of Clq to N.
gonorrhoeae JCl. Clq binding was saturated at about 8
~g/ml; concentration of Clq in human blood is about 70
~g/ml.
In summary, these experiments showed that Clq is a
host factor that increases attachment of N. gonorrhoeae
to human ovarian tissue. This attachment may be
W094/~275 ~ 5 ~ - PCT~S94103811
-24-
explained by a bridging function of Clq that recognize
receptors in both the bacteria and the tissue. Clq
dependent attachment should result in an increased
colonization of the human genital tissue such as ovary,
especially when ovarian follicular fluid contains
complement at concentration similar to the blood.
Therefore, increased binding, via complement, should be
considered as a possible virulence factor of N.
gonorrhoeae in pelvic inflammatory disease or
infertility.
EXAMPL~ IV
GQ ~COC-~T lN~,lON IN NEWBORN RATS
~ITH Clq COATED G~..~O~T C~L~5
The present example describes a rat model of
gonococcal infection.
Baoteria an~ Growth Con~itions
N. gonorrhoeae JCl was isolated from the blood of a
female patient with disseminated gonococcal infection.
Strain JCl is resistant to killing by normal human serum
in vivo and in vitro (36). Strain JCl was grown on
gonococcal base agar with Kellogg supplements GCK (Difco
Laboratories, Detroit, Michigan) in 5% CO2 at 37C
(36,45). A suspension of N. gonorrhoeae in veronal
buffer saline (VBS) was prepared with an optical density
of 0.8 at 595 nm, divided into two tubes and incubated
for 30 min. in 5% CO2 at 37C; one tube in the pres~nce
and the other in absence of Clq (40 ~g/ml) (Cytotech, San
Diego, CA). Gonococcal colonies were visualized with a
dissecting microscope and were phenotyped by the method
of Swanson, et al., (61,62). The difference in size and
color/opacity colony types (TI, T2-piliated and T3, T4
non-piliated in combination with Op+ and Op-) was used
= =
~ W094l~275 2 1 6 0 1 5 5 PCT~S94/03811
-25-
separately for all experiments and the outco~;ng types
quantitated.
Animals and Experimental Infe¢tion
Pregnant female Sprague-Dawley rats were housed
under st~n~rd conditions (25C; relative humidity 40%)
on a 12:12 hours light to dark cycle and given food and
water libitum. The rat pups remained with their mothers
after parturition and were used at the age of three days
(+ 1 day), when they weighed 10 to 15 g.
For each experiment, about 5 pups from the same
litter were used. The suspension of N. gonorrhoeae was
intraperitoneally injected in 0.1 ml volume. After the
injection, the pups were returned to their mothers.
10 x 108, 5 x 108, 1 x 108, 5 x 107, 1 x 107 and 5 x
106 bacterial cells per ml were used to select the lowest
concentration that may develop infection and the
concentration that may cause death of infected animals.
The kinetics of bacteremia was followed by serial blood
cultures.
~ecoverY an~ Identification of Gonococci from Bloo~ an~
Other Tis~ue of Infecte~ Pups.
Groups of pups were sacrificed after 1, 2, 5 and 6
days. Blood cultures were collected before sacrifice.
Aliquots (0.05 ml) of blood were cultured on GC agar and
in 1 ml of Columbia broth containing sucrose, sodium
polyethane sulfonate, "increased cysteine," and Co2.
~ Gonococci were identified by Gram stain, fluorescent
antibody stain (no. 2361-56; FA-N. gonorrhoeae; Difco,
- 35 Detroit, Mich.), colony morphology, oxidase positivity,
and sugar fermentation pattern. Characterization of
colony types of N. gonorrhoeae isolated from different
-
W094/~275 2 1 ~ 5 PCT~S94/03811
-26-
tissues were analyzed by the method of Swanson (61, 62).
Gram-methyl green-pyronin-light green stain, which was
developed specifically for the purpose of detecting
bacteria in tissue sections, was used for detection of GC
cells as well (63).
Results
Table 4 shows the susceptibility of newborn rats to
gonococcal infection in the presence and absence of human
complement Clq. N. gonorrhoeae was isolated on chocolate
agar plates from the blood of newborn rats 48 hours after
the injection of gonococcal cells coated with Clq. N.
gonorrhoeae was not isolatable from blood of newborn rats
injected with N. gonorrhoeae, uncoated with Clq.
Tabl~ 4 . ~o8t-Tnoc~ 1 r tion recovQry of N. gonorrhoeaQ
from ~ewborn rat bloo~. ~
Time N. gonorrhoeae N. gonorrhoeae
coated with Clq
Hours After Number Number of Number Number of
Inoculations of Animals of Animals
of GC Animals With Animals With
Used Positive Used Positive
Blood Blood
Culture Culture
3 5 0 7 7
6 5 O 7 7
9 5 O 7 7
12 5 0 7 7
24 4 O 4 4
48 2 O 2 2
Total 26 O(O%) 34 34(lO0~)
* Summary of three different experiments except for
the forty-eight hour experiment which was performed once.
~ W094/~U75 2 1 6 0 1 5 5 rCT~594/03811
The kinetics and localization of GC in other tissues
during gonococcal infection in this rat model were tested
in the next series of experiments. The results are shown
in Table 5.
W0 94/24275 ~ PCT/US94/03811
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W094/~27S 2 1 ~ ~1 55 PCT~S94/03811 ~
-30-
N . gonorrhoeae JC1 (0.1 ml) in concentration of
5xl06/ml was inoculated. Colony types T1-85% and Op(+)
80~ were used for inoculation. GC isolated from all
infected tissues showed in all animals one colony form
(T1): small, domed, highlighted colonies which are known
to produce pili (Swanson T1) 98-100% of all colonies
recover from newborn rat tissues were opaque (Op).
These results indicate that GC coated with human Clq
may develop DGI in rat pups. Data were summarized from
experiments on 17 pups from two litters. Tissues of 8
pups from one litter were analyzed on day one and day
five after GC inoculation. Tissues of nine pups from
another litter were analyzed on day one, two and day six
after GC inoculation.
Overall, GC coated with Clq injected i.p. were found
to reach the blood stream and survive in pups blood up to
5-6 days (Table 5). At day 6, GC could be cultured from
some organs, e.g., liver. Microscopic estimation of
tissues changes indicated that the liver was enlarged,
presumably from inflammation by gonococcal infection.
Rat pups inoculated with Clq coated GC were less active,
their skin showed increasèd redness and overall they
looked ill as compared to the control pups.
GC coated with Clq, circulating with blood may
penetrate to the cerebrospinal fluid (CSF), and reach
different tissues. Clq may be a bridging molecule (in
vivo) between GC cells and infected tissue. It is known
that Clq binds to human tissues via Clq receptors
(13,14,15,17,59).
In the control group, GC not coated with Clq did not
develop any symptoms of gonococc~l infection in the pups
from the same litter. GC were not isolated from any
pups, organs at any time. Several phenomena may have
W094/~275 -31- PCT~S94/03811
happened: 1) GC, not coated may not have reached the
blood stream, or were killed immediately by the pups'
serum and/or leukocytes, 2) GC, when not coated, may not
attach to various tissues in vivo and 3) immune response
in pups is efficient to clear infection with GC not
coated with Clq.
Results from Table 4 and Table 5 indicate that 100%
of newborn rats were infected with N. gonorrhoeae
pretreated with Clq. In control experiments, none of the
newborn rats were infected with N. gonorrhoeae when Clq
was not present. These results suggest that human serum
glycoprotein Clq plays an important role in gonococcal
infection and is necessary in the experimental gonococcal
infection model on newborn rats. Isolation of GC cells
from blood suggests that N. gonorrhoeae in the presence
of human complement Clq becomes invasive in vivo and
penetrates from the peritoneal cavity to the blood system
of newborn rats. Clq coated GC circulated in blood
stream may disseminate and reach other tissues and
colonize them.
The present inventors then investigated if anti-Clq
IgG would prevent animals from infection. Dilutions of
anti-Clq IgG (1:50, 1:100, 1:500) were given i.p. 1.5
hours before inoculation of Clq coated or non-coated GC
cells. These experiments showed dose-dependent
protection of newborn rats from GC-Clq promoted infection
(Fig. 7).
UTILITY OF THE RAT MODBL
- The availability of a system for infection of rats
with N. gonorrhoeae can now serve as an experimental tool
for the study of gonococcal infection and can be employed
for numerous purposes, including those listed below.
W094l~275 2 ~ ~ O I ~ 5 PCT~S94/03811 ~
-32-
l. The development of vaccines and other
immunization procedures designed to prevent infection
with N . gonorrhoeae and/or to prevent subsequent
development of gonorrhea in infected humans (e.g.,
procedures designed to maximize neutralizing antibody
production).
2. Development and testing of prophylactic and
therapeutic drugs and procedures including passive
immunization with antibodies to prevent and combat
gonorrhea in humans.
3. Testing various isolates of N. gonorrhoeae in
order to determine whether there are significant
differences in the ability of different strains of N.
gonorrhoeae to cause disease.
4. To determine whether there are synergistic
effects in gonorrhea infection caused by use of certain
drugs or other substances.
It is noted that, given the model system of the
present invention, all of the above-mentioned utilities
can be routinely accomplished without specific
elaboration herein of the procedures involved, it being
pointed out that such procedures are quite st~ rd and
well known to those of ordinary skill in the art to which
this invention pertains. Thus, a method for testing
anti-gonorrhea therapeutic agent, comprises (a)
administering a therapeutic agent to be tested, to live
N. gonorrhoeae infected rats; and (b) determining the
effect of said agent on the progression disseminated of
gonorrhea infection in said rats, an arresting of
gonorrhea infection progression in said rats being
indicative of the efficacy of said agent.
W094/~275 ~ l 6 ~ 15 ~ PCT~S94/03811
-33-
Similarly, a method for testing anti-gonorrhea
vaccine comprises (a) administering an anti-gonorrhea
vaccine to be tested, to a group of live uninfected rats;
and (b) then infecting the rats of step (a) with Clq
coated N. gonorrhoeae while concomitantly infecting a
t control group of unvaccinated rats with the same batch of
Clq coated N. gonorrhoeae, a failure of gonorrhea
infection to develop in the rats of group (a) and the
progression of gonorrhea infection in control group of
rats being indicative of the efficacy of the anti-
gonorrhea vaccine.
EXAMPLB V
U8e of Anti-Clq Antibody or Clg Receptor Fr~gments
Polyclonal antibodies against human Clq were
prepared. Three rabbits were immunized subcutaneously
with purified Clq protein (lOO~g) prepared in incomplete
Freund's adjuvant (50~1). The s~con~ry immunization was
given one week later, and the animals were bled a week
later to test immune response. Subsequent boosts have
been given once every week for four weeks (total 6
weeks). Prior to the first injection, blood has been
collected (2 mL) from each animal to obtain control serum
levels of antibody to the injected preparation.
Commercially available monospecific polyclonal goat
antisera to human Clq was also obtained from Cytotech,
San Diego, CA.
Monoclonal anti-Clq antibodies that prevent the N.
gonorrhoeae-tissue bridging function may also be used.
Anti-human complement Clq monoclonal antibodies are
available from the American Type Culture Collection,
Rockville, Maryland, as ATCC HB 8327 and HB 8328. These
35 hybridomas are defined in U.S. Patent 4,595,654, ~hich is
incorporated by reference herein.
W094/~275 2 1 6 ~ 1 5 ~ PCT~S94/03811 ~
-34-
Experiments have now shown that Clq is a human
factor that not only increased the virulence of
gonococcal cells (GC) in vitro by mediating GC attachment
(Figure 8), but also by increasing GC resistance to
bacteriolytic effect of serum components (Figure 9).
These effects allowed survival and dissemination of GC in
experimental animals. Detection of Clq on GC attached to
human tissues taken from patients with gonococcAl
infection indicates involvement of Clq in pathogenesis of
human gonococcal infection (Nowicki, et al. 1993).
Attachment of GC to host tissues is a first and
necessary step to establish gonococcal infection.
Inhibition of GC attachment (the first step in
pathogenesis) represents an important strategy in new
prophylactic approaches. Anti-Clq antibodies reduced the
virulence function of Clq-coated GC in vitro and in vivo.
Figure lO shows that anti-Clq antiho~ies protect
animals from gonococcal infection by Clq-coated GC in a
dose-dependent fashion, demonstrating a causal effect of
Clq function. Anti-human Clq antiho~ies used in
dilutions l:800, l:400, l:200, l:lO0, and l:50 reduced
the number of GC recovered from the blood to 103, lO2, lO,
and 0 CFU/ml respectively (Figure lO). Blood from control
animals without antibody treatment showed lO5 CFU/ml
(Figure lO).
Anti-Clq inhibits or even abolishes attachment of GC
to human polymorphonuclear leukocytes (PMN) (Figure ll)
and other tissues including endometrium and ovaries (not
shown). Figure llA shows Clq-enhanced attachment of GC
to PMNs is shown by the number of GC binding per PMNs.
A) Binding of prei~cllh~ted with Clq N. gonorrhoeae to
human PMNs. B) Binding of N. ~onorrhoeae to PMNs
preincubated before with human Clq. C) Bi nA; ~g of
preincubated with Clq N. gonorrhoeae to PMNs also
~ W094/~275 2 i 6 01 5~ PCT~S94/03811
-35-
pre;n~lh~ted with Clq. D) Binding of N. gonorrhoeae to
PMNs in the absence of Clq. Values were determined in
triplicate and error bars represent the stAn~Ard
deviation from the means. The binding of N. gonorrhoeae
in presence of Clq was 10 to 30 fold higher than Clq-
independent values. Schematic diagrams represent
proposed mPch~nisms (Figure llB).
Anti-Clq-IgG protects animals from experimental
gonococcal infection. Anti-Clq-IgG abolishes attachment
of GC to host tissues. Anti-Clq-IgG antibodies are
useful in protection against gonococcal infection. Anti-
Clq-IgG antibodies may be used in vaginal cream before
intercourse (especially in groups with high risk for
gonococcal infection).
GC attachment to host tissues that express Clq
receptors is supported by Clq on the surface on GC.
Therefore, to block attachment of GC to host tissues,
synthetic analogues of Clq and/or Clq receptors expressed
in host tissues and/or GC may be used.
Figure 12 shows the inhibitory effect of anti-Clq-
IgG on the attachment of GC to PMNs. Open squares O show
attachment of Clq treated GC to PMNs in presence of
ant; ho~; es. Closed squares show attachment of GC to
Clq treated PMNs in presence of antibodies. The dose-
dependent protective effect of monospecific anti-Clq-IgG
was represented by number of GC per PMN. Protection
observed at dilution 1:500 was statistically significant
p<0.05)-
The structure and partial amino acid sequence of Clq
and Clg receptor are known (Malhotra et al . Immunol ogy,
78, 341, 1993). Components of either the Clq or its
receptor that inhibit Clq interaction with GC or tissue
may be used to prevent or inhibit GC infections.
w094/~275 2 ~ ~ a 13 ~ PCT~S94/03811
-36-
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It is understood that the examples and embodiments
described herein are for illustrative purposes only and
that various modifications in light thereof will be
suggested to persons skilled in the art and are to be
included within the spirit and purview of this
application and scope of the appended claims.
