MXPA99001366A - Anti-chlamydial methods and materials - Google Patents

Abstract

The present invention relates to methods for treating chlamydial infection comprising administering to a subject suffering from a chlamydial infection a bactericidal/permeability-inducing (BPI) protein product.

Description

"ANTI-CLAMIDIA METHODS AND MATERIALS" BACKGROUND OF THE INVENTION The present invention relates generally to methods for treating chlamydial infections, by administration of bactericidal / permeability enhancement (BP1) protein products. BPl is a protein isolated from the granules of mammalian polymorphonuclear leukocytes (PMN or neutrophils), which are essential blood cells in the defense against invading microorganisms. Human BPl protein has been isolated from PMN by acid extraction combined with either ion exchange chromatography [Elsbach, J. Biol. Chem., 254: 11000 (1979)] or affinity chromatography E. coli [eiss, et al., Blood, 69: 652 (1987)]. The BPl obtained in this manner is referred to herein as natural BPl and has been shown to have potent bactericidal activity against a broad spectrum of gram-negative bacteria. The molecular weight of human BPl is approximately 55,000 daltons (55 kD). The amino acid sequence of the entire human BP1 protein and the DNA nucleic acid sequence encoding the protein have been disclosed in Figure 1 of the article by Gray et al., J. Biol. Chem., 264: 9505 (1989), which is incorporated herein by reference. The amino acid sequence of Gray et al. Is set forth in SEQ ID NO: 1 herein. BPl is an intensely cationic protein. Half of the N terminal of BPl is responsible for the high net positive charge; half of terminal C of the molecule has a net charge of -3. [Elsbach and iss (1981), supra. ] A fragment of the N-proteolytic terminal of BPl having a molecular weight of approximately 25 kD has an amphipathic character, containing alternative hydrophobic and hydrophilic regions. This fragment of the N-terminal of the BPl possesses the antibacterial efficacy of the holoprotein of human BPl derived naturally from 55 kD. [Ooi et al., J. Bio. Chem., 262: 14891-14894 (1987)]. In contrast to the N-terminal portion, the C-terminal region of the isolated human BPl protein exhibits only slightly detectable antibacterial activity against gram-negative organisms. [Ooi et al., J. Exp. Med., 1 74: 649 (1991).] A fragment of N-terminal BPl of approximately 23 kD, referred to as "rBPl23," has been produced by recombinant means and it also retains anti-bacterial activity against gram-negative organisms. Gazzano-Santoro and others, Infecí. Immun. 60: 4754-4761 (1992). The bactericidal effect of BPl that has been reported to be highly specific for the gram-negative species, eg, in the article by Elsbach and eiss, Inflammation: Basic Principles and Clinical Correlates, editors, Gallin et al., Chapter 30, Raven Press , Ltd. (1992). This disclosed specificity of leptocytes is believed to be the result of intense attraction of BPl to lipopolysaccharide (LPS), which is unique to the outer membrane (or envelope) of gram-negative organisms. Even though BPJ was commonly believed to be non-toxic to the other microorganisms, including yeast and to higher eukaryotic cells, it has recently been discovered that BPl protein products, as designated infra, exhibit activity against gram-positive bacteria, microplasma, icobacteria, fungi, and protozoa. [See co-pending North American patent application, co-owned admitted Serial No. 08 / 372,783 filed on January 13, 1995, the expositions of which - are incorporated herein by reference; the co-owned co-pending US Patent Application Serial Number 08 / 626,646, the disclosures of which are incorporated herein by reference; co-pending US Patent Application Serial No. 08 / 372,105, the disclosures of which are incorporated herein by reference; and the copending North American Patent Application, co-owned Serial Number 08 / 273,470, the expositions of which are incorporated herein by reference.] It has also been discovered that BPl protein products have the ability to improve the activity of antibiotics against bacteria. [See U.S. Patent No. 5,523,288, the teachings of which are incorporated herein by reference in the co-pending co-pending Co-Applicant US Patent Application Serial No. 08 / 372,783.] The precise mechanism by which the BPl kills or kills Gram-negative bacteria have not been fully elucidated, but it is believed that BPl must first bind to the surface of bacteria through electrostatic and hydrophobic interactions between the cationic BPl protein and the negatively charged sites in LPS. Reference has been made to LPS as "endotoxin" due to the potent inflammatory response it stimulates, ie, the release of mediators by host inflammatory cells that may ultimately result in irreversible endotoxic shock. BPl is linked to lipid A, which is known to be the most toxic and biologically active component of LPS. In susceptible gram-negative bacteria, the binding of BPl is thought to break the structure of LPS, leading to the activation of bacterial enzymes that degrade phospholipids and peptidoglycans, altering the permeability of the cell's outer membrane, and initiating events that ultimately lead to cell death. [Elsbach and Weiss (1992). supra]. The BPl is believed to act in two stages. The first is a sublethal stage that is characterized by immediate growth arrest, permeabilization of the outer membrane and selective activation of bacterial enzymes that hydrolyze phospholipids and peptidoglycans. Bacteria in this stage can be rescued by growth in media supplemented with serum albumin [Mannion et al., J. Clin. Invest., 85: 853-860 (1990)]. The second stage, defined by inhibition of growth that can not be reversed by serum albumin, occurs after prolonged exposure of the bacteria to BP1 and is characterized by extensive physiological and structural changes, including apparent damage to the internal cytoplasmic membrane. The initial linkage of BPl to LPS leads to organizational changes that likely result in binding to the anionic LPS groups, which normally stabilize the outer membrane through the binding of Mg and Ca. The fixation of BPl to the outer membrane of gram-negative bacteria produces rapid permeabilization of the outer membrane to hydrophobic agents such as actinomycin D. The binding of BPl and subsequent gram-negative bacterial death depends, at least in part, on the chain length of the LPS polysaccharide, with "smooth" organisms carrying the long O-chain being more resistant to the bactericidal effects of BPl than the "coarse" organisms carrying the short chain [Weiss et al., J. Clin. Invest. 65: 619-628 (1980)]. The first stage of BPl action, the permeabilization of the gram-negative outer envelope, is reversible during the dissociation of BPl, a process that requires high concentration of the divalent cations and the synthesis of the new LPS [Weiss et al., J. Immunol. 132: 3109-3115 (1984)]. The loss of gram-negative bacterial viability, however, is not reversed by processes that restore the integrity of the envelope, suggesting that the bactericidal action is mediated by additional lesions induced in the target organism that can be located in the cytoplasmic membrane. (Mannion et al., J. Clin Invest. 86: 631-641 (1990)). Specific research of this possibility has shown that a molar-based BP1 is at least as inhibitory of cytoplasmic membrane vesicle function as polymyxin B (In't Veld et al., Infection and Immuni ty 56: 1203-1208 (1988)) but the exact mechanism as well as the importance of these vesicles to the studies of intact organisms has not yet been clarified.

Chlamydia are non-mobile, gram-negative, obligate intracellular bacteria that have unusual biological properties that distinguish them phylogenetically from other families of bacteria. Chlamydiae are currently placed in their own order, the Chlamydiales, family Chlamydiaceae, with a genus Chlamydia. [Schachter and Stamm, Chlamydia, in Manual of Clinical Microbiology, pages 669-611, American Society for Microbiology, Washington, DC (1995).] There are four species, Chlamydia trachoma tis, C. pneumoniae, C. psi ttaci and C. pecorum, which cause a broad spectrum of human diseases. In developed countries, C. trachomatis causes trachoma, the world's leading cause of avoidable blindness. More than 150 million children have active trachoma, and more than 6 million people are currently blind to this disease. In industrialized countries, C. trachomatis is the most prevalent sexually transmitted disease, causing urethritis, cervicitis, epididymitis, ectopic pregnancy and pelvic inflammatory disease. In the last year alone, an estimate of 300 million people contracted sexually transmitted chlamydial infections. Among the 250,000 cases of pelvic inflammatory disease per year in the United States, approximately 25,000 women became infertile each year. Neonatal C. trachomatis infections, contracted at birth from infected mothers, cause hundreds of thousands of cases of conjunctivitis per year, of which approximately half of these infected infants develop pneumonia. Recently, C. pneumoniae has been implicated as a common cause of epidemic human pneumonitis. Members of the genus are not only important human pathogens, but also cause significant morbidity in other mammals and birds. Therefore, chlamydiae are one of the most ubiquitous pathogens at all sites in the animal kingdom. [Zhang et al., Cell, 69: 861-869 (1992).] Their unique developmental cycle differentiates them from all other microorganisms. They are obligate intracellular parasites that are unable to synthesize ATP, and therefore depend on the energy of the host cells to survive. Unlike viruses, they always contain both DNA and RNA, divide by binary fission, contain ribosomes, and can synthesize proteins. Chlamydias have cell walls similar in structure to those of gram-negative bacteria and all members of the genus carry an antigen -like LPS singular, called the complementary fixation antigen (CF), which may be analogous to LPS in certain gram-negative bacteria. [Schac ter and Stam, supra. Chlamydiae also carry a major outer membrane protein (MOMP) that contains specific antigens from both the species and the sub-species. The infectious form of chlamydia is the elementary body (EB), which infects the mammalian cells by attaching to the host cell and entering a phagocytic visicula of the host derivative (endosome), within which the entire growth cycle is completed. The target cell host in vivo is typically the columnar epithelial cell, and the primary input mode is believed to be endocytosis mediated with a receptor, Once the EB has found in the cell, it is rearranged into a reticulated body (RB) that is larger than the EB and metabolically active, synthesizing DNA, RNA and proteins. EBs are specifically adapted for extracellular survival while metabolically active RBs do not survive outside the host cell and appear to be adapted for an intracellular environment. After about 8 hours, the RBs begin to divide by binary fission. As they duplicate within the endorsements of host cells, they form characteristic intracellular inclusions that can be seen by light microscopy. After a period of growth and division, the RB reorganize and condense to form infectious EB. The development cycle is completed when the host cell lysis or chlamydial exocytosis occurs, releasing the EB to start another cycle of infection. The length of the full development cycle as studied in cell culture models is 48 to 72 hours and varies as a function of the infection strain, the host cell and the environmental conditions. [Beatty et al., Microbiol. Rev., 58 (4): 686-699 (1994).] It has been shown, at least for C. trachoma tis, that the fixation of the chlamydia organism in the host cells is mediated by a glycosaminoglycan (GAG) similar to a heparan sulfate, present on the surface of chlamydia. Treatment of chlamydia with either purified heparin, heparin sulfate or heparin receptor analogues (such as platelet factor 4 and fibronectin, both of which are known to bind to heparin sulfate), inhibited heparin binding and infectivity. chlamydia to the host cells. Inhibition is not seen with GAG that is not heparin, such as hyaluronate, chondroitin sulfate, or keratin sulfate. The treatment of C. trachomatis with reduced binding heparitinase and infectivity by more than 90 percent; Subsequent treatment with exogenous heparan sulfate was able to re-establish the ability of the treated organisms to bind to the host cells in a manner that depends on the dose. Other GAGs such as hyaluronate, chondroitin sulfate, or keratin sulfate did not restore the binding capacity. These data suggest that a GAG similar to heparin sulfate mediates the binding of chlamydia to host cells by connecting the mutual GAG receptors on the surface of the host cell and on the surface of the outer membrane of chlamydia. [Zhang and otrtos, Cell, 69: 861-869 (1992).] C. trachomatis is almost exclusively a human pathogen, and is responsible for trachoma, inclusion conjunctivitis, lymphogranuloma venereum (LGV), and genital pathway diseases. [Schachter and Stamm, supra. ] Within this species, serotypes A, B, Ba and C have been associated with endemic trachoma, the most common avoidable form of blindness in the world. Trachoma is a chronic inflammation of the conjunctiva and the cornea, which is not transmitted sexually. Sequelae of potentially blinding trachoma include eyelid distortion, trichiasis (erroneous eyelash management), and entropy (Inward deformation of the margin of the eyelid). These can cause ulceration of the cornea followed by loss of vision. Serotypes Ll, L2 and L3 of C. trachomatis are associated with LGV. The untreated Lymphogranuloma venereum advances through three stages, each more serious than the previous one. The main lesion, if present, appears on the genitals. The second stage is a bubonic state marked by regional lymphadenopathy, during which buboes can ooze and develop festering fistulas. The rectal strictures and the lymphatic obstruction can appear in the tertiary stage. Lymphogranuloma venereum is a common problem in developing countries with tropical or subtropical climates, especially among the lower socioeconomic groups. C. trachomai ts is also the most common agent of the sexually transmitted disease. In men, serotypes D to K are the major identifiable causes of non-gonococcal urethritis, and also cause epididymitis, Reiter's syndrome and proctitis. Chlamydial infections are not easily identified in men by clinical symptoms alone, because the infection can be asymptomatic and because other pathogens cause similar symptoms. Chlamydial urethritis occurs twice as often as urethritis (gonorrhea) in some populations, and its incidence is increasing. Even though N. gonorrhea is shown as being present, urethritis may be due to a double or multiple infection that involves a second organism. Infections of C. trachomatis and N.

Simultaneous gonorrhea have been reported in approximately 25 percent of men with gonorrhea. Epididymitis is the most important complication of chlamydial urethritis in men. C. trachomatis causes one in two cases of epididymitis in younger men in the United States, with infertility as a possible result. Reiter's syndrome is another manifestation of chlamydial infection in men. It is a painful systematic disease that classically includes symptoms of urethritis, conjunctivitis and arthritis. Urethritis and arthritis are by far the most frequent combination; It seems that the urethral infection of chlamydia can introduce arthritis. C. trachomatis can also cause proctitis (anal inflammation), particularly in homosexual men. In women, chlamydial infection results in serotypes sexually transmitted in cervicitis, urethritis, endo-sttritis, salpingitis and proctitis; serious sequelae of salpingitis include tube scarring, infertility, and ectopic pregnancy. Chlamydia infections not recognized in women are common. Approximately 50 percent of women infected with chlamydia are asymptomatic. C. trachomatis causes mucopurulent cervicitis and urethral syndrome, as well as endometritis and salpingitis.

These chlamydia infections of the upper genital tract can cause sterility or predispose to ectopic pregnancies and the most serious complications of chlamydial infections in women. Ten percent of all maternal deaths are due to ectopic pregnancies. C. trachomatis causes more than 30 percent of cases of ucopurulenta cervicitis. As many as half of women with gonococcal cervicitis have concomitant chlamydial infection. If the gonococcal infection is treated with penicillin, concomitant chlamydial cervicitis will continue to be undetected and untreated, and may progress to pelvic inflammatory disease (salpingitis), which can lead to infertility and ectopic pregnancies. C. trachoma tis is a cause of urethral syndrom in women. Chlamydial infections can ascend from the cervix to the endometrium, where C. trachomatis has been found in the epithelial lining of the uterine cavity. It is estimated that approximately half of all women with cervicitis have endometritis. Salpingitis, a leading cause of ectopic pregnancies and infertility, is the most serious complication of female genital infections. Upper abdominal pain is the predominant symptom of perihepatitis. Both C. trachomatis and N. gonorrhea can cause perihepatitis. This condition occurs almost exclusively in women where infection organisms disperse to the surface of the liver of inflamed fallopian tubes. Women infected with C. trachomatis can also transmit the disease to their newborn as it passes through the infected birth canal. These newborns often develop inclusion conjunctivitis or chlamydial pneumonia, but they can also develop vaginal, pharyngeal, or enteric infections. Even when it does not produce blindness, the conjunctivitis of inclusion can become chronic, causing slight scarring and formulation of keratolysis or cloth if left untreated. During their passage through the birth canal, up to two thirds of infants born to mothers with chlamydial genital infections will also become infected. With as many as one in ten pregnant women who have chlamydial cervicitis in some parts of the world, the risk to newborns is considerable. Chlamydia pneumonia occurs in 10 percent to 20 percent of infants born to infected mothers. C. trachomatis is responsible for 20 percent to 60 percent of all pneumonias during the first 6 months of life.

Strains of C. trachoma tis are sensitive to the action of tetracyclines, macrolides and sulfonamides and produce a glycogen-like material within the inclusion vacuole that is stained with iodine. C. psi ttaci strains infect many avian species and mammals, producing diseases such as psittacosis, ornithosis, feline pneumonitis and bovine abortion. [Schachter and Stamm, supra] C. psi ttaci is ubiquitous among avian species, and infection in birds usually involves the intestinal tract. The organism is poured into fecal matter, pollutes the environment, and is dispersed by aerosol. C. psi ttaci is also common in domestic mammals. In some parts of the world, these infections have important economic consequences, since C. psi ttaci is a cause of a number of systematic and debilitating diseases in domestic mammals and, more importantly, can cause abortions. Human chlamydial infections of this agent usually result from exposure to an infected avian species, but can also occur after exposure to infected domestic mammals. This species is resistant to the action of sulfonamides and produces inclusion that does not stain with iodine. C. pneumoniae has less than 10 percent connexity with DNA for other species and has a pear shape instead of round elemental bodies (EBs). Like C. trachomatis, it seems to be exclusively a human pathogen without an animal deposit. C. pneumoniae has been identified as the cause of a variety of respiratory diseases and is distributed worldwide. [Schachter and Stamm, supra. ] Infections appear to be commonly acquired in late childhood, adolescence, and early adulthood, resulting in seroprevalence of 40 percent to 50 percent in people aged 30 to 40 years. Manifestations of infection include pharyngitis, bronchitis and mild pneumonia, and transmission is mainly through respiratory secretions. In seroepidemiological studies, these infections have been linked to coronary artery disease, and their role in atherosclerosis is currently under intense scrutiny. The role of C. pecorum as a pathogen is unclear, and specialized reatives are required for its identification. The recommended procedure for the primary isolation of chlamydia is cell culture. Chlamydia will grow in the sac of the embryonic chicken egg yolk, as well as in cell culture (with some variability). C. trachomatis can infect several cell lines such as murine de-li heteroploid cells.

McCoy, HeLa 229 cells, BHK-21 cells, or L-929 cells. HL cells and Hep-2 cells may be more sensitive for the recovery of C. pneumoniae. The most common technique involves the inoculation of clinical specimens in McCoy cells treated with cycloheximide. The basic principle involves the centrifugation of the inoculum towards the monolayer of the cell, the incubation of the monolayers for 48 to 72 hours, and the demonstration of typical intracytoplasmic inclusions by appropriate immunofluorescence staining procedures, iodine or Gie sa. Cell culture usually requires two to six days to complete due to the required incubation time. Chlamydia can also be detected in samples by a direct fluorescent antibody test (DFA), wherein the slide slides are incubated with monoclonal antibodies conjugated with fluorescein, and the fluorescent elementary bodies are detected using a fluorescent microscope. This test has approximately 80 percent to 90 percent sensitivity and 98 percent to 99 percent specificity compared to cell cultures when both tests are carried out under ideal circumstances. [Schachter and Stamm, supra. ] A number of commercially available products can detect chlamydial antigens in clinical specimens using enzyme inhibition (EIA) procedures. Most of these products detect Chlamydia LPS, which is more soluble than the MOMP. Without confirmation, the tests have a specificity in the order of 97 percent. [Schachter and Stamm, supra. ] Several nucleic acid probes can also be obtained commercially. A commercially available probe probe (GenProbe) uses DNA-RNA hybridization in an effort to increase sensitivity by detecting chlamydial RNA. The complementary fixation test (CF) is the most frequently performed serological test, and measures the serum level of the complementary fixation antibody. (antibody for the CF antigen group). It is useful for diagnosing psittacosis, where sera in acute- and convalescent phase pairs frequently show increases in titre of four times or greater. The same seems to be the case for many C. pneumoniae infections. Approximately 50 percent of these infections are CF-positive, even though they may require 24 weeks to detect seroconversion. The CF test can also be useful for diagnosing LGV, where single-point assessments greater than 1:64 are highly evident in this clinical diagnosis. [Schachter and Stamm, supra. ] Elevated titers of complementary fixation antibodies are not found in chlamydialis or genital duct conjunctivitis infections, and therefore are not sensitive to these infections. The microimmunofluorescence method (micro-IF) is a more sensitive procedure for measuring anti-cla idiasis antibodies. This indirect fluorescent antibody technique uses antigens prepared by infecting the yolk sacs of the fertile chicken embryos with each chlamydial serotype. Serial dilutions of the patient's serum are added to the prepared antigens, and the level of the antibody in the blood sample is determined by the use of immunoforescence. Trachoma, inclusion conjunctivitis, and genital duct infections can be diagnosed using the micro-IF technique and sera can be obtained in appropriately synchronized pairs but the procedure is of limited clinical utility because the diagnosis requires demonstration of a four-fold change or higher in the antibody titration in specimens in pairs, and because patients with superficial genital infections, such as urethritis may not have a change in the evaluation. However, an evaluation of elevated antibody in a single serum specimen of a patient with Reiter's syndrome and a high evaluation of IgM in the serum of an infant with pneumonia, are useful to establish a diagnosis. Variation from strain to strain in antimicrobial susceptibility profiles and resistance to the newly acquired drug are both uncommon among chlamydia. Among the most active in vitro drugs against C. trachomatis, C. pneumoniae, and C. psi ttaci are the tetracyclines, such as tetracycline and doxycycline, the macrolides, such as erythromycin and azithromycin, the quinolones, such as ciprofloxacin and ofloxacin, chloroamphenicol , rifampin, clindamycin and the sulfonamides. Tetracyclines and macrolides have generally been the therapy supports for infections due to chlamydia. [Schachter and Stamm, supra; Goodman and Gilman, The Pharmacological Basis of Therapeutics, Ninth Edition, McGraw-Hill, New York, NY (1996).] The antimicrobial susceptibility test is performed infrequently for chlamydial infections but can be carried out as follows . Test organisms are grown for at least two transmissions in cells cultured in antibiotic-free media before being harvested. An adjusted inoculum of -100 inclusion forming units per microassay well is then used to infect the monolayers of the antibiotic-free cell. After centrifugation of the inoculum to the monolayer, serial dilutions of the test antibiotic can be added either immediately or at different time intervals over the next 24 hours. After 48 hours, monoclonal antibodies conjugated with fluorescein are used to identify the minimum inhibitory concentration (MIC), ie, the highest antibiotic dilution that inhibits the formation of intracellular inclusion. In general, the monolayers are also broken and also passed to define the minimum bactericidal concentration (MBC), that is, the highest antibiotic dilution that prevents viable chlamydia from being detected in the passage (MBC).

COMPENDIUM OF THE INVENTION The present invention provides methods for treating a patient suffering from chlamydial infection by administering a therapeutically effective amount of the BPl protein product. This is based on the surprising discoveries that BPl protein products inhibit the infectivity of chlamydia and inhibit the proliferation of chlamydia in established intracellular infection. The BPl protein products can be administered alone or together with other well-known anticholidiasis agents. When the subject matter of the adjunctive therapy is made, administration of the BPl protein products may reduce the amount of anti-chlamydial agent that is not of BPl necessary for effective therapy, thereby limiting the potential toxic response and / or the high cost of treatment. The administration of BPl protein products can also improve the effect of these agents, accelerate the effect of these agents, or reverse chlamydia resistance to these agents. In addition, the invention provides a method for killing or inhibiting the growth of chlamydia comprising contacting chlamydia with a BPl protein product. This method can be practiced in vivo or in a variety of in vitro uses such as its use to decontaminate fluids and surfaces and to sterilize surgical equipment and other medical equipment and implantation devices including prosthetic joints and invasive devices. introduced. A further aspect of the invention involves the use of a BPl protein product for the manufacture of a medicament for the treatment of chlamydial infection. The medicament may include, in addition to a BPl protein product, other chemotherapeutic agents such as anti-chlamydial agents that do not contain BPl. Numerous additional aspects and advantages of the invention will be apparent to those skilled in the art when taking into account the following detailed description of the invention, which describes the modalities thereof currently preferred.

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to the surprising discovery that a BPl protein product can be administered to treat patients suffering from chlamydial infection, and provides methods to prophylactically or therapeutically treat these infections. Unexpectedly, BPl protein products were shown to have anti-chlamydial activities, as measured, for example, by a reduction in the number of reproductive bodies seen in host cells. They can be treated, according to the invention, a variety of chlamydial infections caused by C. trachomatis, C. pneumoniae, C. psittaci and C. pecorum. The term "treat" or "treatment" as used herein encompasses both prophylactic and therapeutic treatment.

The BPl protein product can be administered systematically or topically. Systematic routes of administration include oral, intravenous, intramuscular or subcutaneous injection (including depots for long-term release), intraocular or retrobulbar, intrathecal, intraperitoneal (e.g. by intraperitoneal lavage), transpulmonary using aerosol or nebulized or transdermal drug. Topical routes include administration in the form of ointments, creams, jellies, ophthalmic drops or ophthalmic ointments, ear drops, suppositories such as vaginal suppositories or irrigation fluids (for e.g. wound irrigation). When administered parenterally, the compositions of the BPl protein product are usually injected in doses ranging from 1 microgram per kilogram to 100 milligrams per kilogram per day, preferably ranging from 0.1 milligram per kilogram to 20 milligrams per kilogram per kilogram. day, and more preferably, doses that vary from 1 to 20 milligrams per kilogram per day. The treatment can continue by continuous infusion or intermittent injection or infusion, or a combination thereof at the same reduced or increased dose per day as long as it is so determined by the physician carrying out the treatment. When administered topically, the compositions of the BPl protein product are usually applied in unit doses ranging from 1 microgram per milliliter to 1 gram per milliliter, and preferably in doses ranging from 1 microgram per milliliter to 100 milligrams per milliliter. . Those skilled in the art can optimally carry the effective doses and monotherapeutic or concurrent administration regimens for the BPl protein product and / or the other anti-chlamydial agents, as determined by good medical practice and the condition clinic of the individual patient. The BPl protein product can be administered together with other anti-chlamydial agents currently known to be effective. Preferred anti-chlamydial agents for this purpose include tetracyclines such as tetracycline and oxycycline, macrolides such as erythromycin and azithromycin, quinolones such as ciprofloxacin and ofloxacin, chloramphenicol, rifampin, clindamycin and sulfonamides. The simultaneous administration of BPl protein product with anti-chlamydial agents is expected to improve the therapeutic efficacy or anti-chlamydial agents. This can occur through reducing the concentration of the anti-chlamydial agent that is required to eradicate or inhibit the growth of chlamydia, e.g. the duplication. Because the use of some agents is limited by their systematic toxicity or prohibitive cost, decreasing the concentration of the preferred anti-chlamydial agent for therapeutic efficacy reduces the toxicity and / or the cost of treatment and therefore allows the most extensive use of the agent. The concomitant administration of the BPl protein product and another anti-chlamydial agent can produce a bactericidal or bacteriostatic effect faster or more completely than could be achieved with any agent alone. The administration of the BPl protein product can reverse chlamydial resistance to anti-chlamydial agents. The administration of the BPl protein product can also convert a bacteriostatic agent into a bactericidal agent. An advantage of the present invention is that the broad spectrum of activity of the BPl protein products against a variety of organisms, and the use of the BPl protein products as adjunctive therapy to improve the activity of the antibodies, makes the BPl protein products are an excellent selection to treat double or multiple infections with chlamydia or another organism, such as the gram-negative bacterium N. gonorrhea. Therefore, BPl protein products can be especially useful for inhibiting the transmission of sexually transmitted diseases that frequently involve double gonococcal / chlamydial infection. It is therefore proposed that the BPl protein products be incorporated into contraceptive compositions and devices, e.g. included in spermicidal creams or jellies or coated on the surface of condoms. Another advantage is the ability to treat chlamydia that has acquired resistance to known anti-chlamydial agents. An additional advantage of concomitant administration of BPl with an anti-chlamydial agent having undesirable side effects is the ability to reduce the amount of anti-chlamydial agent necessary for effective therapy. The present invention can also provide quality of life benefits due to e.g. decreased therapy ratio, reduced permanence in intensive care units or total reduced permanence in the hospital, with the reduced concomitant risk of serious nosocomial infections (acquired in the hospital). The "concomitant administration" as used herein includes the administration of the agents together or before or after each other. Protein PBI products and anti-chlamydial agents can be administered by different routes. For example, the BPl protein product can be administered intravenously while the anti-chlamydial agents are administered intramuscularly, intravenously, subcutaneously, orally or intraperitoneally. Alternatively, the BPl protein product can be administered intraperitoneally, while the anti-chlamydial agents are administered intraperitoneally or intravenously, or the protein product BPl can be administered in a nebulized aerosol form while the anti-chlamydial agents are administered. are administered vg intravenously The protein product Rk BPl and the anti-chlamydial agents can both When administered intravenously, the BPl protein product and the anti-chlamydial agents can be administered in sequence in the same intravenous line, after an intermediate wash or they can be administered in different intravenous lines. The BPl protein product and the anti-chlamydial agents can be administered simultaneously or sequentially, as long as they are administered in a manner sufficient to allow both agents to achieve effective concentrations at the site of infection. 20 The concomitant administration of the protein product of BPl and the antibiotic is expected to provide more effective treatment of chlamydial infections. Concomitant administration of the two agents may provide greater therapeutic effects in the one that provides any agent, when administered individually. For example, concomitant administration may allow a reduction in the dose of one or both agents to achieve a similar therapeutic effect. Alternatively, concomitant administration may produce a bactericidal / bacteriostatic effect faster or more completely than could be achieved with any agent alone. Therapeutic efficacy is based on satisfactory clinical performance and does not require that the anti-chlamydial agent or anti-chlamydial agents kill or kill 100 percent of the organisms involved in the infection. Success depends on achieving a level of anti-chlamydial activity at the site of infection that is sufficient to inhibit chlamydia in a manner that includes balance in favor of the host. When the host's defenses are effective to the maximum, the required anti-chlamydial effect will be minimal. Reducing the burden of the organism by a logarithm (a factor of 10) can allow the host's own defenses to control the infection. In addition, increasing an early bactericidal / bacteriostatic effect may be more important than a long-term bactericidal / bacteriostatic effect. These early events are a significant and critical part of therapeutic success because it allows time for the host defense mechanisms to activate. The BPl protein product is thought to interact with a variety of host defense elements present in whole blood or serum, including the PL5 and LBP complement and other cells and components of the immune system. These interactions can result in the potentiation of the activities of the BPl protein product. Because of these interactions, BPl protein products can be expected to exert even greater activity in vivo than in vitro. Therefore, even when in vi tro tests can predict in vivo utility, the absence of in vi tro activity does not necessarily indicate the absence of in vivo activity. For example, BPl has been found to have a greater bactericidal effect in gram-negative bacteria in whole blood or plasma assays in trials using conventional media. [Weiss et al., J. Clin. Invest. 90: 1122-1130 (1992)]. This may be due to the fact that conventional in vi tro systems lack blood elements that facilitate or reinforce the function of BPl in vi ve, or due to conventional means that contain higher concentrations of the physiological concentrations of magnesium and calcium, which are inhibitors typically of the activity of BPl protein products. In addition, in the host, the BPl protein product is available to neutralize the translocation of gram-negative bacteria the concomitant release of endotoxin, an additional clinical benefit that is not seen in nor can be predicted by in vitro testing. It is also proposed that the BPl protein product can be administered with other products that enhance the activity of the BPl protein products including the anti-chlamydial activity of the BPl protein products. For example, the serum complement reinforces the gram-negative bactericidal activity of the BPl protein products; the combination of the BPl protein product and the serum complement provides synergistic bactericidal / growth inhibitory effects. See e.g. Ooi et al. J. Biol. Chem. 265: 15956 (1990) and Levy et al. J. Biol. Chem, 268: 6038-6083 (1993) which focus on the 15 kD proteins that occur naturally reinforcing the antibacterial activity of BPl. See also the co-pending PCT Application Number US94 / 07834 filed July 13, 1994 corresponding to US Patent Application Serial No. 08 / 274,303 filed July 11, 1994 as a continuation-in-part of US Patent Application 08 / 093,201 filed July 14, 1993. These applications, which are all incorporated herein by reference, describe the methods for enhancing the gram-negative bactericidal activity of the BPl protein products, by administering proteins from lipopolysaccharide binding (LBP) and LBP protein products. Protein derivatives of LBP and derived hybrids lacking immunostimulatory properties of CD-14 are described in PCT Application Number US94 / 06931 filed June 17, 1994, which corresponds to the co-pending US Pat. co-owned Serial Number 08 / 261,660, filed June 17, 1994 as a continuation in part of the US Patent Application Serial Number 08 / 079,510 filed on June 17, 1993, the expositions of which are incorporated in the present by reference. It has also been observed that poloxamer surfactants enhance the antibacterial activity of BPl protein products, as described in Lambert's US Patent Application Serial No. 08 / 586,133 filed January 12, 1996, which is a continuation in part of US Patent Application Serial No. 08 / 530,599 filed September 19, 1995, which is a continuation in part of US Patent Application Serial No. 08 / 372,104 filed January 13, 1995, all of which correspond to the PCT Application Number PCT / US96 / 01095; The poloxamer surfactants can also improve the activity of the anti-chlamydial agents. In addition, the invention provides a method for killing or killing or inhibiting the growth of chlamydia comprising contacting chlamydia with a BPl protein product. This method can be practiced in vivo with a variety of in vitro uses such as to decontaminate fluids and surfaces or to sterilize surgical equipment or other medical equipment and implantable devices, including prostheses and intrauterine devices. These methods can also be used for the sterilization of introduced invasive devices such as intravenous lines and probes, which are often pockets of infection. A further aspect of the invention involves the use of a BPl protein product for the manufacture of a medicament for the treatment of chlamydial infection. The medicament may include, in addition to a BPl protein product, other chemotherapeutic agents such as anti-chlamydial agents. The medicament may optionally comprise a pharmaceutically acceptable diluent, adjuvant or carrier. As used herein, the term "BPl protein product" includes naturally produced and recombinantly produced BPl protein; recombinant biologically active polypeptide fragments of the BP1 protein; biologically active polypeptide variants of BPl protein or fragments thereof including hybrid and dimer fusion proteins; biologically active polypeptide analogues of BPl protein or fragments or variants thereof, including cysteine-substituted analogs and BPl-derived peptides. BPl protein products administered in accordance with this invention can be generated and / or isolated by any means known in the art. The American Patent Number 5, 198, 551, the disclosure of which is incorporated herein by reference, discloses recombinant gene coding and methods for expression of BPl proteins, including recombinant oligoprotein BPl, which is referred to as rBPI and recombinant fragments of BPl. The co-pending US Patent Application Serial No. 07 / 885,501 and a continuation thereof, the US Patent Application Serial Number Number 08 / 072,063, filed May 19, 1993 and correspondingly PCT Application Number 93/04752, filed May 19, 1993, which is all incorporated herein by reference, discloses novel methods for purification and recombinant BPl protein products expressed in and secreted from host cells of mammal genetically transformed into cultures disclosed how large quantities of suitable recombinant BPl products can be produced to be incorporated into stable homogeneous pharmaceutical preparations. The biologically active fragments of BPl (fragments of BPl) include biologically active molecules having the same or similar amino acid sequence of a holoprotein of natural human BPl, with the exception that the fragment molecule lacks amino-terminal amino acids, amino - internal acids and / or carboxy-terminal amino acids of holoprotein. Non-limiting examples of these fragments include a N-terminal fragment of the native human BPl of approximately 25 kD, which is described in the article by Ooi et al., J. Exp. Med. 174: 649 (1991), and the product of recombinant expression of DNA encoding the N-terminal amino acids from 1 to about 193 to 199 of the natural human BPL described in the article by Gazzano-Santoro et al., Infect. Immun. 60: 4754-4761 (1992) and referred to as rBPl23 >; In this publication, an expression vector was used as a source of DNA encoding a recombinant expression product (rBPl23) having the signal sequence of residue 31 and the first 199 amino acids of the N-terminus of mature human BPl, as noted in Figure 1 of the article by Gray et al., supra, with the exception that valine at position 151 is specified by GTG instead of GTC and residue 185 is glutamic acid (specified by GAG) instead of Usina ( specified by AAG). The recombinant holoprotein (rBPI) has also been produced by obtaining the sequence (SEQ ID NOS: 145 and 146) indicated in Figure 1 of the article by Gray et al., Supra, with the exceptions mentioned for rBPI 23 and with the exception that the residue 417 is alanine (specified by GCT) instead of valine (specified by GTT). Other examples include the numerical forms of the BPl fragments as described in the co-pending US Patent Application, co-owned Serial Number 08 / 212,132, filed on March 11, 1994, and 'the corresponding PCT application. PCT / US95 / 03125, the exhibits of which are incorporated herein by reference. Preferred numerical products include dimeric BPl protein products wherein the monomers are amino-terminal BPl fragments having the N-terminal residues from about 1 to 175 to about 1 to 199 of the BP1 holoprotein. A particularly preferred dimeric product is the dimeric form of the BP1 fragment having terminal N1 to 193 terminal residues, which is designated the rBPl42 dimer. Biologically active variants of BP1 (BP1 variants) include but are not limited to proteins. recombinant hybrid fusion comprising holoprotein of BP1 or the biologically active fragment thereof and at least a portion of at least one other polypeptide and the dimeric forms of the BP1 variants. Examples of these hybrid fusion proteins and dimeric forms are described by Theofan et al., In co-pending US Patent Application Serial No. 07 / 885,911, and a request for continuation in part thereof, US Patent Application Serial Number 08/064, 693 filed May 19, 1993, and the corresponding PCT Application Number US93 / 04754, filed May 19, 1993, which are all incorporated herein by reference and which include hybrid fusion proteins comprising, at the amino terminus, a BP1 protein or a biologically active fragment thereof and, at the carboxy terminal end, at least one constant domain of an immunoglobulin heavy chain or a allelic variant thereof. The biologically active analogues of BPl (BPl analogs) include but are not limited to BPl protein products where one or more of the amino acid residues have been replaced by a different amino acid. For example, the co-pending US Patent Application Serial No. 08 / 013,801 filed on February 2, 1993 and the corresponding PCT Application Serial Number US94 / 01235 filed on February 2, 1994, the expositions of the which are incorporated herein by reference, discloses BPl polypeptide analogs, BPl fragments, wherein a cysteine residue is replaced by a different amino acid. A stable BPl protein product described by this application is the DNA expression product encoding from amino acid 1 to about 193 or 199 of the N-terminal amino acids of the BP1 holoprotein, but where the cysteine in the residue number 132 is replaced by alanine and is designated rBPl2i? Cys or rBPl2i- Other examples include the dimeric forms of the BPl analogues; e.g., the co-pending and co-owned US Patent Application Serial Number 08 / 212,132 filed on March 11, 1994, and the corresponding PCT Application Number PCT / US95 / 03125, the exhibits of which are incorporated herein by reference. Other BPl protein products useful according to the methods of the invention are peptides derived from or based on BPl, produced by recombinant or synthetic means (peptides derived from BPl) such as those described in the co-pending PCT Application and from co-owned US94 / 10427 filed September 15, 1994, corresponding to US Patent Application Serial No. 08 / 306,473 filed September 15, 1994, and PCT Application Number US94 / 02465 filed on November 11, 1994; March 1994, corresponding to US Patent Application Serial No. 08 / 209,762 filed March 11, 1994, which is a continuation in part of the US Patent Application Serial Number 08 / 183,222 filed on January 14, 1994, which is a continuation in part of the US Patent Application Serial Number 08 / 093,202 filed July 15, 1993 (for which, the internal application The corresponding application is PCT Application Number US94 / 02401, filed March 11, 1994), which is a continuation in part of the US Patent Application Serial Number 08 / 030,644, filed on March 12, 1993, the expositions of which are incorporated herein by reference. Currently preferred BPl protein products include N-terminal fragments of recombinantly produced BPl, especially those having a molecular weight of about 21 to 25 kD such as rBPl2i or rBPl23, or the dimeric forms of these N-terminal fragments (eg dimer of rBPl42) • In addition, the preferred BPl protein products include the rBPI peptides or peptides derived from BPl. The administration of BPl protein products is preferably achieved with a pharmaceutical composition comprising a BPl protein product and a pharmaceutically acceptable adjuvant or carrier diluent. The BPl protein product can be administered without or in conjunction with known surfactants, other known chemotherapeutic agents or additional anti-chlamydial agents. A stable pharmaceutical composition containing the BPl protein products (eg rBPI, rBP? 23) comprises the BPl protein product at a concentration of 1 milligram per milliliter in citrate stabilized saline (5 or 20 mM citrate, 150 mM NaCl, pH 5.0) comprising 0.1 weight percent of poloxamer 188 (Pluronic F-68, BASF of Wyandotte, Parsippany, NJ) and 0.002 weight percent of polysorbate 80 (Tween 80, ICI A ericas Inc., of Wilmington, DE). Another stable pharmaceutical composition containing the BPl protein products (eg rBPl2i) comprises the BPl protein product at a concentration of 2 milligrams per milliliter in 5 mM citrate, 150 mM NaCl, 0.2 percent poloxamer 188, and 0.002 percent of polysorbate 80. These preferred combinations are described in the co-pending PCT Application, co-owned US94 / 01239, filed February 2, 1994, which corresponds to US Patent Application Serial No. 08 / 190,869, filed on February 2, 1994, and US Patent Application Serial No. 08 / 012,360, filed on February 2, 1993, the exhibits of which are incorporated herein by reference. Other aspects and advantages of the present invention will be understood by taking into account the following illustrative examples. Example 1 focuses on the use of the BPl protein product to inhibit infection of chlamydial host cells when administered concurrently as a chlamydial challenge. Example 2 focuses on the anti-chlamydial activity of the BPl protein product in chlamydia-infected host cells.

EXAMPLE 1 USE OF THE PROTEIN PRODUCT BPl TO INHIBIT THE INFECTION OF CELLS HOSTS WITH CLAMIDIA A. Preparation of the Chlamydia material A serovar L2 material of Chlamydia trachomatis (Ct) was prepared in the following manner. McCoy cells (Accession number of ATCC CRL 1696) were grown overnight in a growth medium [Nutrient Mixtures of Eagles Medium (MEM) M-3786, Sigma, St. Louis, MO] with 1 percent pyruvate sodium (S-8636, Sigma) and 10 percent fetal bovine serum (FBS, A115-L, Hyclone, Logan, VT). The medium was aspirated into a small Ct frit, thawed rapidly and mixed with 30 milliliters of Dulbecco's phosphate-buffered saline (PBS, Sigma) and 7 percent sucrose (DPBS-7). Ten milliliters of the suspension were added to each of 3 T150 flasks and the flasks were incubated up to 37 ° C - while they were oscillated periodically through the next two hours to distribute in inoculum. DPBS-7 was aspirated from the flasks and 50 milliliters of the grown-up growth medium was added to each flask. After incubation for three days at 37 ° C in 5 percent CO 2, the Ct was harvested as follows. The growth medium was aspirated from the flasks and glass beads were added to the flasks to a depth of ~ 6.35 millimeters. Ten milliliters of the MEM Eagles (without FBS) were added to each flask and the beads were oscillated through the monolayer until all the cells had been dislodged. The beads and trash from the cells were collected in 50 milliliter capacity capped tubes with screw cap, the flasks were washed twice with PBS and the washings were added to the bead suspension. Each tube was placed on ice and solidified for 60 seconds to break the cells. The broken cell / bead suspension was centrifuged at low speed (~ 800 revolutions per minute). The supernatant liquid was removed and collected with a 250 milliliter polycarbonate centrifuge bottle and then centrifuged for one hour at high speed (~ 25,000 x g). The granule was resuspended in FBS (40 milliliters) by repeated passage through a # 16 thick needle and syringe. The 1 milliliter aliquots were distributed in small bottles NUNC® (Naperville, IL) and frozen at -70 ° C.

B. Evaluation of Chlamydia Material Three small flasks of the Ct material prepared as described above in Section A, were thawed rapidly at 37 ° C and serially diluted in 10-fold concentrations of the Eagles MEM or DPBS- 7 without serum. The twenty-four well plates with coverslips in each well containing monolayers of the McCoy cell for 24 hours were then prepared. The medium was aspirated, the wells were washed once with PBS, and Eagles or DPBS-7 were duplicated to play the McCoy cells. The plates were incubated at 37 ° C in 5 percent CO2 for 2 hours, the medium was aspirated and 2 milliliters of the growth medium was added. The plates were then reincubated at 27 ° C in 5 percent CO2 for 3 days, fixed in methanol, and stained for 30 minutes in a humid chamber with the mouse monoclonal anti-chlamydial antibody irradiated with FITC Confirmation Test of Trachomatis Cultivation of Chlamydia Syva MicroTrak®). The stained coverslips were washed in water, air dried, inverted in a drop of mounting fluid (50 percent glycerol, 50 percent PBS) and viewed using a Leitz fluorescent microscope with a 25X objective (length excitation wavelength of 480 nm, emission wavelength of 520 nm). The inclusion bodies were counted and comparable results were obtained through the concentration scale of 10 ~ 2 to 10-10 tested in MEM of Eagles and DPBS-7. Dilution at 10 ~ of the material preparation provided an inclusion of 100 to 300 body-forming units per milliliter; this dilution was selected for use in all subsequent studies using this Ct material. Additional environmental studies were carried out using the Eagle Basic Medium (BME), Sigma), MEM Eagles (E-MEM, Sigma), RPMI-1640 with HEPES (Sigma), RPMI-1640 without HEPES (Sigma), F-12 (Gibco) and Nutrient Mixture of Dulbecco's Modified Eagle Medium F -12 Ham (DMEM / F-12, Gibco). DMEM / F-12 without FBS was selected for use in the subsequent Chlamydia infectivity studies. The medium without FBS was selected for use because the addition of 10 percent FBS to the previously tested medium cited inhibited infection of McCoy cells by Ct.

C. Infection by Chlamydia in Presence or Absence of Protein Product BPl The tested BPl protein product was rBPl2i [2 milligrams per milliliter in 5mM sodium citrate, 150mM sodium chloride, pH 5.0, with 0.2 percent PLURONIC® P123 (BASF Wyandotte, of Parsippany, NJ), 0.002 percent polysorbate 80 (TWEEN® 80, ICI Americas Inc., of Wilmington, DE) and 0.05 percent EDTA]. Equal volumes of the formulation stabilizer alone were used [5mM sodium citrate, 150mM sodium chloride, pH 5.0, with 0.2 percent PLURONIC® P123, 0.002 percent polysorbate 80 and 0.05 percent EDTA ], as a control. Serial dilutions of rBPl2i or the formulation stabilizer were prepared with DMEM / F-12 (without FBS) so that when the serial dilutions were added at a ratio of 9: 1 to 1 milliliter of a 10 ~ 4 solution of the Ct material, the final concentration of Ct would be a 10-4 dilution of the material of Ct and the final concentrations of rBPl2i would be 128, 64, 32, 16 and 8 micrograms per milliliter. Comparable formulation stabilizer controls (in volume) were also prepared. The final suspensions were incubated at 37 ° C for 30 minutes in a water bath. McCoy cells in DMEM / F-12/10 percent FBS were contaminated at 2 x 10 ^ cells per well in 24-well tissue culture plates (Corning # 25820), incubated for 24 hours and the medium aspirated . Ct, with and without BPl, was added in 1 milliliter to duplicate the wells at each concentration rBPl2i- The plates were centrifuged at 2500 revolutions per minute for 30 minutes, incubated for 2 hours at 37 ° C in 5 percent CO 2, and the wells were sucked. Each well received 2 milliliters of DMEM / F-12/10 percent FBS and 1 microgram per milliliter of cycloheximide (Sigma) and the plates were re-incubated for 3 days. After the removal of the medium, the wells were washed with a phosphate-stabilized saline solution (PBS), air-dried and fixed with methanol and stained with Gram iodine. The cells may alternatively be stained with anti-chlamydia antibodies irradiated with FITC as described in section B above.

Using an inverted microscope, 100 percent of each well was screened for the presence of inclusion bodies, which stain brown with Gram iodine, due to the high concentration of glycogen in the vacuoles produced by the reproductive bodies. The results are shown below in Table 1.

Table 1 Number of inclusion bodies per well to 128 micrograms per mi 0 110 64 micrograms per mi 0 115 32 micrograms per mi 0 115 16 micrograms per mi 1.5 114 8 micrograms per mi 59 124 Positive Control 151 (only Ct) Negative Control (without Ct) These representative results from one of the three studies indicate that rBPl2i can inhibit the infection of the permissible cells.

Example 2 ACTIVITY OF ANTI-CLAMIDIASIS OF THE PROTEIN PRODUCT BPl AGAINST CELLS WHO ARE INFECTED WITH CLAMIDIA The serovar L2 material of Chlamydia trachomatis (Ct) prepared as described in Example 1, diluted to 10-5 with Eagle's Nutrient Medium Blend Modified from Dulbecco F-12 Ham (DMEM / F-12) with 10 percent fetal bovine serum (FBS). McCoy cells in DMEM / F12 / 10 percent of FBS were contaminated at 1 X 105 cells per well in 24-well tissue culture plates (Corning # 25820), incubated for 24 hours, and the medium aspirated. Ct (1 milliliter of the 10-5 material) was added to each well of four plates except for two negative control wells per plate. The plates were centrifuged at 2500 revolutions per minute for 30 minutes, incubated for 24 hours at 37 ° C in 5 percent CO2, and the wells were aspirated. The rBPl2] _ as described in Example 1 was diluted to final concentrations of 128, 64, 32, 16 and 8 micrograms per milliliter in DMEM / F-12 and 1.0 milliliter was added to the appropriate duplicate wells in each plate. The comparable stabilizer controls of the formulation as described in Example 1 were also prepared. The plates were incubated for 2 hours, and 1 milliliter of DMEM / F12 / 20 percent of FBS and 2 micrograms per milliliter of cycloheximide were added to the wells causing the concentration of rB l2 to decrease by a factor of two. The plates were re-incubated for up to 5 days. At 24, 48, 72 and 120 hours, the medium was removed from a single plate, the wells were washed with PBS and air dried, fixed with methanol and stained with Gram iodine. Using an inverted microscope, 100 percent of each well was screened for the presence of inclusion bodies. The results are shown in Table 2, which is presented below.

Table 2 Concentration of Number of Bodies of Inclusion per Pit rBPl2i Initial * to the at 24 hours 48 hours 72 hours 0 285.5 358 335.75 8 194.5 180 108 16 138 140.5 109.5 32 112.5 95 57.5 64 119.5 81 39 128 113 77.5 5 * This initial concentration, which was present during the first two hours of incubation, decreased to half the initial value during the rest of the 5-day incubation.

These representative results from one of two studies show that rBPl2 at initial concentrations ranging from 16 micrograms per milliliter to 128 micrograms per milliliter was able to reduce the number of intracellular inclusion bodies where cells infected with Ct when administered at 24 hours after the challenge with Ct. It is expected that numerous modifications and variations in the practice of the invention will occur for those persons skilled in the art when taking into account the above-described description in the modalities thereof preferred at present. Consequently, the only limitations that should be assigned in the scope of the present invention are those appearing in the appended claims.

LIST OF SEQUENCES (1) GENERAL INFORMATION: (i) APPLICANT: XOMA Corporation (ii) TITLE OF THE INVENTION: Methods and Materials of Anti-Chlamydia (iii) NUMBER OF SEQUENCES: 2 (iv) ADDRESS FOR CORRESPONDENCE: (A) RECIPIENT: Marshall, O 'Toóle, Gerstein, Murray & Borun (B) STREET: 6300 Sears Tower, 233 South Wacker Drive (C) CITY: Chicago (D) STATE: Illinois (E) COUNTRY: United States of America (F) POSTAL CODE: 60606-6402 (v) READILY FORM COMPUTER: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) SOFTWARE: Patentln Relay # 1.0, Version # 1.25 (vi) APPLICATION DATA CURRENT: (A) APPLICATION NUMBER: (B) DATE OF SUBMISSION: (C) CLASSIFICATION: (vii) DATA FROM THE PREVIOUS APPLICATION: (A) APPLICATION NUMBER: (B) DATE OF PRESENTATION: (C) CLASSIFICATION: ( viii) ATTORNEY / AGENT INFORMATION: (A) NAME: Borun, Michael F. (B) REGISTRATION NUMBER: 25,447 (C) ATTORNEY'S NUMBER OF REFERENCE / TOCA: 27129/33433 (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: 312 / 474-6300 (B) TELEFAX: 312 / 474-0448 (C) TELEX: (2) INFORMATION FOR SEQ ID NO: l: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 1813 base pairs (B) TYPE: nucleic acid ico (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (ix) PARTICULARITY: (A) NAME / KEY: CDS (B) LOCATION: 31..1491 (ix) PARTICULARITY: (A) NAME / KEY: mat_peptide (B) LOCATION: 124..1491 (ix) PARTICULARITY: (A) NAME / KEY: particulcity_miscellaneous (D) OTHER INFORMATION: "rBPI" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: l: CAGGCCTTGA GGTTTTGGCA GCTCTGGAGG ATG AGA GAG AAC ATG GCC AGG GGC 54 Met Arg Glu Asn Met Wing Arg Gly -31 -30 -25 CCT TGC AAC GCG CCG AGA TGG GTG TCC CTG ATG GTG CTC GTC GCC ATA 102 Pro Cys Asn Wing Pro Arg Trp Val Ser Leu Met Val Leu Val Wing -20 -15 -10 GGC ACC GCC GTG ACA GCG GCC GTC AAC CCT GGC GTC GTG GTC AGG ATC 150 Gly Thr Wing Val Thr Wing Wing Val Asn Pro Gly Val Val Val Arg lie -5 1 5 TCC CAG AAG GGC CTG GAC TAC GCC AGC CAG CAG GGG ACG GCC GCT CTG 198 Ser Gln Lys Gly Leu Asp Tyr Ala Ser Gln Gln Gly Thr Ala Ala Leu 10 15 20 25 CAG AAG GAG CTG AAG AGG ATC AAG ATT CCT GAC TAC TAC GAC AGC TTT 2 6 Gln Lys Glu Leu Lys Arg lie Lys lie Pro Asp Tyr Ser Asp Ser Phe 30 35 40 AAG ATC AAG CAT CTT GGG AAG GGG CAT TAT AGC TTC TAC AGC ATG GAC 294 Lys lie Lys His Leu Gly Lys Gly His Tyr Ser Phe Tyr Ser Met Asp 45 50 55 ATC CGT GAA TTC CAG CTT CCC AGT TCC CAG ATA AGC ATG GTG CCC AAT 342 lie Arg Glu Phe Gln Leu Pro Ser Ser Gln He Ser Met Val Pro Asn 60 - 65 70 GTG GGC CTT AAG TTC TCC ATC AGC AAC GCC AAT ATC AAG ATC AGC GGG 390 Val Gly Leu Lys Phe Ser He Ser Asn Wing Asn He Lys He Ser Gly 75 80 85 AAA TGG AAG GCA CAA AAG AGA TTC TTÁ AAA ATG AGC GGC AAT TTT GAC 438 Lys Trp Lys Wing Gln Lys Arg Phe Leu Lys Met Ser Gly Asn Phe Asp 90 95 100 -10--5 CTG AGC ATA GAA GGC ATG TCC ATT TCG GCT GAT CTG AAG CTG GGC AGT 486 Leu Ser He Glu Gly Met Ser He Be wing Asp Leu Lys Leu Gly Ser 110 115 120 AAC CCC ACG TCA GGC AAG CCC ACC ATC ACC TGC TCC AGC TGC AGC AGC '534, Asn Pro Thr Be Gly Lys Pro Thr He Thr Cys Ser Ser Cys Ser Ser i 125 130 135 ( CAC ATC AAC AGT GTC CAC GTG CAC ATC TCA AAG AGC AAA GTC GGG TGG 582! His He Asn Ser Val His Val His He Ser -Lys Ser Lys Val Gly Trp * 3-40 1 5 150 I CTG ATC CAA CTC TTC CAC AAA AAA ATT GAG TCT GCG CTT CGA AAC AAG 630 • Leu lie Gln Leu Phe His Lys' Lys He Glu Be Ala Leu Arg Asn Lys 15 = > 160 165 ATG AAC AGC CAG GTC TGC GAG AAA GTG ACC AAT TCT GTA TCC TCC AAG 678 Met Asn Ser Gln Val Cys Glu Lys Val Thr Asn Ser Val Ser Ser Lys 170 175 180 185 CTG CA CCT TAT TTC CAG ACT CTG CCA GTA ATG ACC AAA ATA GAT TCT 726 Leu Gln Pro Tyr Phe Gln Thr Leu Pro Val Met T r Lys He Asp Ser 190 195 200 GTG GCT GGA ATC AAC TAT GGT CTG GTG GCA CCT CCA GCA ACC ACG GCT 774 Val Wing Gly He Asn Tyr Gly Leu Val Wing Pro Pro Wing Thr Thr Wing 205 210 215 GAG ACC CTG GAT GTA CAG ATG AAG GGG GAG TTT TAC AGT GAG AAC CAC 822 Glu Thr Leu Asp Val Gln Met Lys Gly Glu Phe Tyr Ser Glu Asn His 220 225 230 CAC AAT CCA CCT CCC TTT GCT CCA CCA GTG ATG GAG TTT CCC GCT GCC 870 His Asn Pro Pro Pro Pro Wing Pro Val Met Glu Phe Pro Wing Wing 235 240 245 CAT GAC CGC ATG GTA TAC CTG GGC CTC TAC GAC TAC TTC TTC AAC ACÁ 918 His Asp Arg Met Val Tyr Leu Gly Leu Ser Asp Tyr Phe Phe Asn Thr 250 255 260 265 GCC GGG CTT GTA TAC CAA GAG GCT GGG GTC TTG AAG ATG ACC CTT AGA 966 Wing Gly Leu Val Tyr Gln Glu Wing Gly Val Leu Lys Met Thr Leu Arg 270 275 280 GAT GAC ATG ATT CCA AAG GAG TCC AAA TTT CGA CTG ACA ACC AAG TTC 1014 Asp Asp Met He Pro Lys Glu Ser Lys Phe Arg Leu Thr Thr Lys Phe. 285 290 295 TTT GGA ACC TTC CTA CCT GAG GTG GCC AAG AAG TTT CCC AAC ATG AAG 1062 Phe Gly Thr Phe Leu Pro Glu Val Wing Lys Lys Phe Pro Asn Met Lys 300 305 310 ATA CAG ATC CAT GTC TCA GCC TCC ACC CCG CCA CAC CTG TCT GTG CAG 1110 He Gln He His Val Ser Ala Ser Thr Pro Pro His Leu Ser Val Gln 315 320 325 CCC ACC GGC CTT ACC TTC TAC TCC CCT GCC GTG GAT GTC CAG GCC TTT GCC 1158 Pro Thr Gly Leu Thr Phe Tyr Pro Wing Val Asp Val Gln Wing Ala Phe Wing 330 335 340 345 GTC CTC CCC AAC TCC TCC CTG GCT TCC CTC TTC CTG ATT GGC ATG CAC 12OS Val Leu Pro Asn Ser Ser Leu Ala Ser Leu Phe Leu He Gly Met His 350 355 360 ACÁ ACT GGT TCC ATG GAG GTC AGC GCC GAG TCC AAC AGG CTT GTT GGA 1254 Thr Thr Gly Ser Met Glu Val Ser Wing Glu Ser Asn Arg Leu Val Gly 365 370 375 GAG CTC AAG CTG GAT AGG CTG CTC CTG GAA CTG AAG CAC TCA AAT ATT 1302 Glu Leu Lys Leu Asp Arg Leu Leu Leu Glu Leu Lys His Ser Asn He 380 385 390 GGC CCC TTC CCG GTT GAA TTG CTG CAG GAT ATC ATG AAC TAC ATT GTA 1350 Gly Pro Phe Pro Val Glu Leu Leu Gln Asp He Met Asn Tyr He Val 395 400 405 CCC ATT CTT GTG CTG CCC AGG GTT AAC GAG AAA CTA CAG AAA GGC TTC 1398 Pro He Leu Val Leu Pro Arg Val Asn Glu Lys Leu Gln Lys Gly Phe 410 415 420 425 CCT CTC CCG ACG CCG GCC AGA GTC CAG CTC TAC AAC GTA GTG CTT CAG 1446 Pro Leu Pro Thr Pro Wing Arg Val Gln Leu Tyr Asn Val Val Leu Gln 430 435 440 CCT CAC CAG AAC TTC CTG CTG TTC GGT GCA GAC GTT GTC TAT AAA 1491 Pro His Gln Asn Phe Leu Leu Phe Gly Wing Asp Val Val Tyr Lys 445 450 455 TGAAGGCACC AGGGGTGCCG GGGGCTGTCA GCCGCACCTG TTCCTGATGG GCTGTGGGGC 1551 ACCGGCTGCC TTTCCCCAGG GAATCCTCTC CAGATCTTAA CCAAGAGCCC CTTGCAAACT 1611 TCTTCGACTC AGATTCAGAA ATGATCTAAA CACGAGGAAA CATTATTCAT TGGAAAAGTG 1671 CATGGTGTGT ATTTTAGGGA TTATGAGCTT CTTTCAAGGG CTAAGGCTGC AGAGATATTT 1731 CCTCCAGGAA TCGTGTTTCA ATTGTAACCA AGAAATTTCC ATTTGTGCTT CATGAAAAAA 1791 AACTTCTGGT TTTTTTCATG TG 1813 (2) INFORMATION FOR SEQ ID NO: 2: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 487 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2 Met Arg Glu Asn Met Wing Arg Giy Pro Cys Asn Wing Pro Arg Trp Val -31 -30 -25 -20 Ser Leu Met Val Leu Val Wing He Gly Thr Ala Val Thr Ala Ala Val -15 -10 -5 i Asn Pro Gly Val Val Val Arg He Ser Gln Lys Gly Leu Asp Tyr Wing 5 10 15 Ser Gln Gln Gly Thr Wing Ala Leu Gln Lys Glu Leu Lys Arg He Lys 20 25 30 He Pro Asp Tyr Ser Asp Ser Phe Lys He Lys His Leu Gly Lys Gly 35 40 45 His Tyr Ser Phe Tyr Ser Met Asp He Arg Glu Phe ln Leu Pro Ser 50 55 - 60 65 Ser Gln He Ser Met Val Pro Asn Val Gly Leu Lys Phe Ser He Ser 70 75 80 Asn Wing Asn He Lys He Ser Gly Lys Trp Lys Wing Gln Lys Arg Phe 85 90 95 Leu Lys Met Ser Gly Asn Phe Asp Leu Ser He Glu Gly Met Ser He 100 105 110 Ser Wing Asp Leu Lys Leu Gly Ser Asn Pro Thr Ser Gly Lys Pro Thr 115 120 125 - He Thr Cys Ser Ser Cys Ser Ser His He Asn Ser Val His Val His 130 135 140 145 He Ser Lys Ser Lys Val Gly Trp Leu He Gln Leu Phe Hie Lys Lys 150 155 160 He Glu Be Ala Leu Arg Asn Lys Met Asn Ser Gln Val Cys Glu Lys 165 170 175 Val Thr Asn Ser Val Ser Ser Lys Leu Gln Pro Tyr Phe Gln Thr Leu 180 185 190 Pro Val Met Thr Lys He Asp Ser Val Wing Gly He Asn Tyr Gly Leu 195 200 205 Val Wing Pro Pro Wing Thr Thr Wing Glu Thr Leu Asp Val Gln Met Lys 210 - 215 220 225 Gly Glu Phe Tyr Ser Glu Asn His His Asn Pro Pro Pro Phe Pro Wing 30 235 * 240 Pro Val Met Glu Phe Pro Ala Ala His Asp Arg Met Val Tyr Leu Gly 245 250 255 Leu Ser Asp Tyr Phe Phe Asn Thr Wing Gly Leu Val Tyr Gln Glu Wing 260 - 265 270 Gly Val Leu Lys Met Thr Leu Arg Asp Asp Met He Pro Lys Glu Ser 275 280 285 Lys Phe Arg Leu Thr Thr Lys Phe Phe Gly Thr Phe Leu Pro Glu Val 290 295 300 305 Wing Lys Lys Phe Pro Asn Met Lys He Gln He His Val Ser Wing Ser 310- 315 320 Thr Pro Pro His Leu Ser Val Gln Pro Thr Gly Leu Thr Phe Tyr Pro 325 330 335 Wing Val Asp Val Gln Wing Phe Wing Val Leu Pro Asn Ser Ser Leu Wing 340 345 350 Ser Leu Phe Leu He Gly Met His Thr Thr Gly Ser Met Glu Val Ser 355 360 365 Wing Glu Ser Asn Arg Leu Val Gly Glu Leu Lys Leu Aep Arg Leu Leu 370 375 380 385 Leu Glu Leu Lys His Ser Asn He Gly Pro Phe Pro Val Glu Leu Leu 390 395 400 Gln Asp He Met Asn Tyr He Val Pro He Leu Val Leu Pro Arg Val 405 410 415 Aen Glu Lys Leu Gln Lys Gly Phe Pro Leu Pro Thr Pro Wing Arg Val 420 425 430 Gln Leu Tyr Asn Val Val Leu Gln Pro His Gln Asn Phe Leu Leu Phe 435 440 445 Gly Wing Asp Val Val Tyr Lys 450 455

Claims (16)

R E I V I N D I C A C I O N E S:

1. The use of a therapeutically effective amount of a bactericidal / permeation enhancing protein (BP1) product to manufacture a medicament for treating chlamydial infections in a patient suffering from chlamydial infection.

2. The use of claim 1, wherein the BPl protein product is a fragment of the N-terminal of BP1 or a numerical form thereof.

3. The use of claim 2, wherein the N-terminal fragment has a molecular weight of about 21 kD to 25 kD.

4. The use of claim 1, wherein the BP1 protein product is holoprotein BP1, rBP133 or

5. The use of claim 1, wherein the BPl protein product is at a dose of between 1 microgram per kilogram to 100 milligrams per kilogram per day.

The use of claim 1, wherein the BPl protein product is a unit dose of between one microgram per milliliter to 1 milligram per milliliter of the drug.

7. The use of claim 1, wherein the infection is by a species of chlamydia which is selected from the group consisting of C. trachomatis, C. pneumoniae, C. psi ttaci and C. pecorum.

8. The use of claim 7, wherein the species of chlamydia is C. trachomatis.

The use of claim 1, comprising the step of adding additional to the medicament, an anti-chlamydial agent that does not contain BPl: 10.

The use of a bactericidal / permeant enhancing protein product (BPl) for producing a medicament for killing or inhibiting the duplication of chlamydia, the use being characterized by contacting chlamydia with a bactericidal / permeation enhancing protein product (BP1).

The use of claim 10, further comprising contacting chlamydia with an anti-chlamydial agent that does not contain BPl.

12. The use of claim 1, wherein the patient suffers from coronary artery disease.

13. The use of a bactericidal / permeability enhancing protein product to manufacture a medicament for killing or inhibiting the duplication of chlamydia in a patient suffering from coronary artery disease, the use being characterized by contacting chlamydia with a Bactericidal protein product / that increases permeability.

The use of claim 1, wherein the medicament is for concomitant administration with an anti-chlamydial agent that does not contain BPl.

The use of claim 14, wherein the medicament is for administration before or after the anti-chlamydial agent that does not contain BPl.

16. An in vitro method for killing or inhibiting chlamydial duplication, comprising contacting chlamydia with a BPl protein product.