WO1994024275A1 - Model for gonococcal infection - Google Patents
Model for gonococcal infection Download PDFInfo
- Publication number
- WO1994024275A1 WO1994024275A1 PCT/US1994/003811 US9403811W WO9424275A1 WO 1994024275 A1 WO1994024275 A1 WO 1994024275A1 US 9403811 W US9403811 W US 9403811W WO 9424275 A1 WO9424275 A1 WO 9424275A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- clq
- gonorrhoeae
- gonococcal
- infection
- human
- Prior art date
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
- C07K16/1203—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria
- C07K16/1217—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria from Neisseriaceae (F)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Definitions
- 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.
- Neisseria gonorrhoeae 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.
- PID pelvic inflammatory disease
- 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 gonococcal 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 enters the peritoneal cavity.
- DGI disseminated gonococcal infection
- DGI disseminated gonococcal infection
- 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 infection even worse, a history of previous gonorrhoea is a risk factor for gonorrhoea. Therefore, development of a gonococcal 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.
- Clq a component of human blood glycoprotein Clq may be a host factor that increases attachment of GC to tissues and cells in vitro (10,11).
- Clq is a glycoprotein, structurally similar to collagen in terms of protein and carbohydrate moieties (12) .
- 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 epithelial cells from representative portions of the entire human genitourinary tract. This correlates with the observation that urogenital epithelium from human and chimpanzees are the only species susceptible to gonococcal adherence in vitro.
- mice of the strains C3H, CBA, BALB/C, TO and ICR are resistant to gonococcal infection.
- Models on other animals, including implanted chambers were also proposed but were limited to the study of GC im unobiology (50,51,52,53,54,55,56).
- the experimental model for other related species N. meningitidis was developed on newborn rats. Meningococci survived only for six hours. Despite the short time of survival, this model allowed others to study the vaccine (57) .
- the present invention involves provision of an animal having an infection with an organism requiring 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 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 chimpanzees normally experience a low rate and degree of infection.
- the animal is non- primate, in particular, the most preferred animal is a rat.
- the organism may be a microbe such as bacteria or fungi or may be viral.
- 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 Immunodeficiency Virus having a protein which binds Clq.
- 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 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 includes an antibody or N. gonorrhoeae peptides having binding 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 peptides may be outer membrane peptides such as those having approximate molecular weights of 17kDa, 31kDa, 35kDa, and 48kDa.
- 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, 31kDa, 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, diaphragm jelly or an emollient.
- Clq synthetic analogs may be analogs or fragments of Clq retaining binding specificity for N. gonorrhoeae but lacking the binding specificity for the eukaryotic host.
- a coating of Clq analog on N. gonorrhoeae would prevent native Clq from binding and would, thereby, decrease N. gonorrhoeae pathogenicity.
- Such an analog would be most effectively 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 binding specificity for N. gonorrhoeae and without binding specificity for eukaryotic cells.
- a Clq synthetic analog having binding specificity for N. gonorrhoeae and without binding specificity for eukaryotic cells.
- 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.
- PCR polymerase chain reaction
- Figure 1 shows the dose dependent binding of Clq to N. gonorrhoeae JCl.
- Figure 2 shows that gonococcal attachment to leukocytes is enhanced by complement Clq.
- Figure 3 is a hypothetical model of attachment of W. 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 absence of Clq (B) . Notice in 4(B), lack of attachment.
- 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 standards in kilodaltons on the left. Solid 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 Clq 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 standard unit area at 400 x magnification (8A) . The mean values and standard errors are shown for each group of data. Schematic diagrams ( Figures 8B and 8C) represent proposed mechanisms.
- Figures 9A-9C relate to the Clq protection of GC from the bactericidal effect of newborn rat serum (in vitro) .
- Clq dose-dependent 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 standard error values for each group of data ( Figure 9A) .
- Schematic diagrams representing proposed mechanisms are shown in Figures 9B and 9C.
- Figure 10 shows the inhibitory effect of anti-Clq- IgG on the development of gonococcal 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 1:400 was statistically significant. (p ⁇ . 0.05).
- Figures 11A and 11B relate to the effect of Clq on the attachment of Neisseria gonorrhoeae to the human PMNs.
- Figure 12 shows the inhibitory effect of anti-Clq- IgG on the attachment of GC to PMNs. Open square D attachment of Clq treated GC to PMNs in presence of antibodies. 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 1:500 was statistically significant (p ⁇ 0.05).
- 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 reproducible.
- gonorrhoeae strain JCl 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 JCl.
- Neisseria gonorrhoeae JCl was isolated from the blood of a female patient. Deposition of Clq on bacterial cells or purified outer membrane proteins of JCl was analyzed.
- 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 (101) . Strain JCl was grown on gonococcal base agar with Kellogg supplements GCK (Difco Laboratories, Detroit, Michigan) in 5% C0 2 at 37°C (102) .
- 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 1:1000 dilution was made, and the gonococcal dilution was incubated with decomplemented normal human serum (heated for 30 minutes to 56°C) at 37°C for 15 minutes. Then normal human serum was added to a final concentration of 25% and incubation was continued for 30 minutes at 37°C.
- CFU colony forming units
- Outer membranes were isolated by differential solubilization in 1% sodium lauryl sarcosinate and centrifuged at 100,000 x g for 1 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) .
- the outer membrane protein fraction of N. gonorrhoeae JCl was incubated at 100° in the presence of 1.5% sodium dodecyl sulfate (SDS) and 2.5% 2- mercaptoethanol for 2, 5, and 10 minutes, layered onto 5% polyacrylamide stacking gels, and fractionated in 12% polyacrylamide in the presence of 0.1% SDS (106). Two hundred ug of outer membrane proteins was added to each gel. Polyacrylamide gel electrophoresis was performed at room temperature using 30 mA per slab gel. Immunological characterization of the fractions was accomplished after transfer by electrophoresis at 150 mA for 1-1/2 hours at 22°C to a nylon membrane (107) .
- SDS sodium dodecyl sulfate
- 2- mercaptoethanol 2- mercaptoethanol
- 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 (1:300) diluted in 0.1% 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 37°C in 10% HSA diluted in VBS to block nonspecific binding. The localization of antibodies 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 standards in kilodaltons on the left.
- Solid circles show common bands of OMP reacted with complement-Clq as well as with anti-CL IgG.
- Nylon membrane replicas of PAGE gels or nylon membrane dots were incubated for 2 hours with phospholipase C (0.5 units/ml) diluted in 1% BSA Tris saline pH 7.3 activated with 0.001 M CaCl 2 .
- 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 37°C with 10% HSA in VBS to block nonspecific binding.
- Control nylon membrane replicas or dot blots were incubated without phospholipase C. stainin ⁇ of GC Gels;
- Binding 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 staining as described above.
- Anti-cardiolipin antibody 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.
- applicants suggest that the nondenatured cardiolipin-peptides molecules on intact gonococcal cells may be able to bind Clq.
- 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.
- 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 disease, all devastating syndromes associated with gonococcal infections.
- 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.
- gonorrhoeae JCl from disseminated gonococcal infection is resistant to killing by human defense mechanism.
- 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 -70°C 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) (Fig. 2) . In the control fewer attached gonococcal cells (0-4 per leukocyte) were observed (Fig. 2) . Similarly gonococcal cells preincubated with Clq displayed increased binding to leukocytes (10-20 gonococcal cells per leukocytes)
- N. gonorrhoeae Attachment of N. gonorrhoeae to human cells is a necessary 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 disease (PID) and infertility.
- PID pelvic inflammatory disease
- 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 mechanisms (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) .
- Clq appears to recognize receptors on gonococcal (GC) cells; the present inventors have found outer membrane peptides of GC that interact with Clq without opsonizing antibody (40) . Conversely, Clq appears to recognize receptors on leukocytes and, therefore, bridge the bacteria and the host cell (10) . Receptors for Clq are distributed in different tissues as well, therefore, Clq-promoted binding occurs also in other sites exposed to gonococcal infection.
- GC gonococcal
- Cryostat sections of snap frozen human ovarian tissue were prepared and stored at -70°C 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.
- Figure 1 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.
- Clq is a host factor that increases attachment of N. gonorrhoeae to human ovarian tissue. This attachment may be 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.
- the present example describes a rat model of gonococcal infection.
- 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% C0 2 at 37°C (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.
- VBS veronal buffer saline
- Pregnant female Sprague-Dawley rats were housed under standard conditions (25°C; 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.
- 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.
- N. gonorrhoeae JCl (0.1 ml) in concentration of 5xl0 6 /ml was inoculated. Colony types Tl-85% and 0p(+) 80% were used for inoculation.
- GC isolated from all infected tissues showed in all animals one colony form (Tl) : small, domed, highlighted colonies which are known to produce pili (Swanson Tl) 98-100% of all colonies recover from newborn rat tissues were opaque (Op) .
- 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) .
- 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 increased redness and overall they looked ill as compared to the control pups.
- 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) .
- GC not coated with Clq did not develop any symptoms of gonococcal infection in the pups from the same litter.
- GC were not isolated from any pups, organs at any time.
- Several phenomena may have 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.
- 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).
- 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.
- 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 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.
- 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 hybridomas are defined in U.S. Patent 4,595,654, which is incorporated by reference herein.
- 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) .
- 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 10 shows that anti-Clq antibodies protect animals from gonococcal infection by Clq-coated GC in a dose-dependent fashion, demonstrating a causal effect of Clq function.
- Anti-human Clq antibodies used in dilutions 1:800, 1:400, 1:200, 1:100, and 1:50 reduced the number of GC recovered from the blood to 10 3 , 10 2 , 10, and 0 CFU/ml respectively ( Figure 10) .
- Blood from control animals without antibody treatment showed 10 5 CFU/ml ( Figure 10) .
- FIG. 11A shows Clq-enhanced attachment of GC to PMNs is shown by the number of GC binding per PMNs.
- 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 D show attachment of Clq treated GC to PMNs in presence of antibodies.
- 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) .
Abstract
Description
Claims
Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP94912398A EP0694066A4 (en) | 1993-04-08 | 1994-04-07 | Model for gonococcal infection |
AU64984/94A AU6498494A (en) | 1993-04-08 | 1994-04-07 | Model for gonococcal infection |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
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US4549093A | 1993-04-08 | 1993-04-08 | |
US08/045,490 | 1993-04-08 |
Publications (1)
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WO1994024275A1 true WO1994024275A1 (en) | 1994-10-27 |
Family
ID=21938188
Family Applications (1)
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PCT/US1994/003811 WO1994024275A1 (en) | 1993-04-08 | 1994-04-07 | Model for gonococcal infection |
Country Status (4)
Country | Link |
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EP (1) | EP0694066A4 (en) |
AU (1) | AU6498494A (en) |
CA (1) | CA2160155A1 (en) |
WO (1) | WO1994024275A1 (en) |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
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ZW7583A1 (en) * | 1982-04-07 | 1983-06-29 | Baylor College Medicine | Products and methods for treatment of cancer |
US4595654A (en) * | 1983-11-07 | 1986-06-17 | Immunomedics Inc. | Method for detecting immune complexes in serum |
-
1994
- 1994-04-07 EP EP94912398A patent/EP0694066A4/en not_active Withdrawn
- 1994-04-07 AU AU64984/94A patent/AU6498494A/en not_active Abandoned
- 1994-04-07 CA CA002160155A patent/CA2160155A1/en not_active Abandoned
- 1994-04-07 WO PCT/US1994/003811 patent/WO1994024275A1/en not_active Application Discontinuation
Non-Patent Citations (4)
Title |
---|
Clinical Microbiology Reviews, Volume 2, Suppl., issued April 1989, BROOKS et al., "Humoral Immune Response to Gonoccoccal Infections", pages S5-S10, see entire article. * |
Clinical Microbiology Reviews, Volume 2, Suppl., issued April 1989, DENSEN, "Interaction of Complement of Neisseria Meningitidis and Neisseria Gonorrhoeae", pages S11-S17, see entire article. * |
Science, Volume 177, issued 29 September 1972, R.J. ARKO, "Neisseria Gonorrhoeae: Experimental Infection of Laboratory Animals", pages 1200-1201, see entire article. * |
See also references of EP0694066A4 * |
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Publication number | Publication date |
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AU6498494A (en) | 1994-11-08 |
EP0694066A1 (en) | 1996-01-31 |
EP0694066A4 (en) | 1997-01-29 |
CA2160155A1 (en) | 1994-10-27 |
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