JP2002523474A - Immunotherapy of staphylococci with donor selection and donor stimulation - Google Patents

Abstract

  (57) [Summary] Provided are methods and compositions for passive immunization of patients infected or susceptible to staphylococcal bacteria, such as Staphylococcus aureus and Staphylococcus epidermidis, the method and compositions comprising a carefully selected adhesion factor of staphylococci. That is, selection or production of a donor plasma pool having high antibody titers against MSCRAMMs or fragments or components thereof or sequences having substantial homology thereto. The donor plasma pool combines individual blood or blood component samples with antibody titers higher than normal antibody titers with one or more other proteins that bind to the selected adhesin or extracellular matrix protein of the cell. By combining, or by administering a carefully selected protein or peptide to the host to induce expression of the desired antibody, and subsequently recovering the enhanced high titer serum or plasma pool from the treated host Can be manufactured. In any case, it is preferred that the donor plasma pool be purified and concentrated prior to intravenous administration to the patient, and that the present invention allows the patient to be immunized against a wide range of potentially dangerous staphylococcal infections. Is advantageous. Kits for identifying high titer potential donors for selected adhesins are also provided. Thus, the present invention provides methods and compositions that can be highly effective against infections associated with staphylococcal bacteria.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

【Technical field】

 The present invention is in the field of biological products for the treatment, prevention and diagnosis of bacterial infections.

[0002]

BACKGROUND OF THE INVENTION

Staphylococci are gram-positive spherical cells that are usually arranged as grape-like irregular clusters. Some staphylococci are constituents of the normal bacterial flora of human skin and mucous membranes, while other staphylococci cause suppuration, abscess formation, various purulent infections, and even lethal sepsis. The pathogen Staphylococcus causes hemolysis of blood, coagulation of plasma,
It often produces a variety of extracellular enzymes and toxins. The most common type of food poisoning is caused by heat-stable staphylococcal enterotoxins.

There are at least 30 species of Staphylococcus. The three major species of clinical importance are Staphylococcus aureus and Staphylococcus aureus.
epidermidis) and Staphylococcus haemolyticus
haemolyticus). Staphylococcus aureus is coagulase-positive
And is distinguished from other species in this respect. Staphylococcus aureus is a major human pathogen. Almost every human suffers from some form of Staphylococcus aureus infection during its lifetime, ranging in severity from food poisoning or small skin infections to severe, life-threatening infections. Coagulase-negative staphylococci are human normal bacterial flora, which sometimes cause infections that often involve transplantation devices, especially in very young, elderly and immunocompromised patients. Approximately 75% of infections caused by clotting enzyme-negative staphylococci are due to parasitic Staphylococcus epidermidis. Staphylococcus haemolyticus, Staphylococcus hominis
Infections due to (Staphylococcus hominics) and other staphylococcal species are less common. Staphylococcus saprophyticus is a relatively common cause of urinary tract infections in young women.

[0004] Thus, staphylococcal bacteria such as Staphylococcus aureus can cause pneumonia, septic arthritis, sepsis, endocarditis and biomaterial-associated infections from wound infections, lesions of the skin such as impetigo and furuncel. It causes a wide range of infectious diseases, ranging from life-threatening diseases including: The colonization of Staphylococcus aureus of articular cartilage, where collagen in the joint space is the main component, appears to be a significant factor contributing to the development of septic arthritis. Bacterial arthritis acquired by hematogenesis remains a serious medical problem. This rapidly progressive, highly destructive joint disease is difficult to treat.
Typically, less than 50% of infected patients cannot recover without significant joint injury. S. aureus is a major pathogen isolated from adult patients with hematogenous and secondary osteomyelitis.

[0005] For hospitalized patients, Staphylococcus aureus is a major cause of infection. Initial local infection of a wound or indwelling medical device can cause more invasive infections such as sepsis, osteomyelitis, mastitis and endocarditis. In infections involving medical devices, plastic and metal surfaces are coated with host plasma immediately after implantation and with substrate proteins such as fibrinogen and fibronectin.
You. The ability of staphylococci such as S. aureus to attach to these proteins is essential for the initiation of infection. Vascular transplants, intravenous catheters, prosthetic heart valves and heart assist devices are thrombogenic and prone to bacterial metastatic growth. Staphylococcus aureus is the most damaging pathogen of such infections, and other staphylococcal bacteria, such as Staphylococcus epidermidis, cause a significant amount of dangerous infections, especially those associated with implanted devices. ing.

[0006] There is a strong and rapid need for therapeutics for treating infections from bacterial infections of staphylococci, such as Staphylococcus aureus and Staphylococcus epidermidis, which are effective against antibiotic resistant strains. It is expanding. The National Institutes of Health recently indicated that this goal is now an international priority.

[0007] Successful transplantation of MSCRAMMs hosts is one of the steps necessary for most microorganisms to cause infections in animals and humans. Microbial attachment is the first critical step in the chain of events that ultimately results in disease. The pathogen's microorganisms bind to the host's tissues or to serum-adjusted endograft biomaterials, such as catheters, artificial joints and vascular grafts, through specific adherens on the bacterial surface. To transplant. MSCRAMMs (adhesive substrate molecules that recognize surface components of microorganisms:
Microbial Surface Components Recoginizing Adhesive Matrix Molecules)
It is a member of cell surface adhesins that are recognized in the extracellular matrix of the host and specifically bind to distinct components. As bacteria successfully attach to and transplant into host tissues, their physiology changes dramatically and harmful components such as toxins and proteolytic enzymes are secreted. Furthermore, attached bacteria often form biofilms, which quickly become more resistant to the killing action of most antibiotics.

For example, Staphylococcus aureus is known to express a repertoire of various MSCRAMMs that can act individually or in concert to promote the attachment of microorganisms to specific host tissue components. ing. MSCRAMMs provide an excellent target for immunological offensive action by antibodies. The presence of a suitable anti-MSCRAMM high affinity antibody has a double-edged aggressive action, the first action is that the antibody prevents microbial attachment and the second action is increased MS.
The CRAMM antibody titer promotes rapid clearance of microorganisms from the body via bacterial disruption, opsonization, phagocytosis and complement activation.

Passive Immunization Against Bacterial Infection Immunoglobulins (A, D, E, G and M) have been used in the body as a primary defense against infection. Complements that are activated by the presence of microorganisms and globulin and that are available as precursor proteins also exhibit antimicrobial activity.
After prior exposure to the antigen, the immune system produces a series of globulin that attach to and coat the bacteria or neutralize the virus, so that the bacteria are easily recognized by neutrophils and macrophages, Received and destroyed. Foreign proteins that invade microorganisms also stimulate a humoral immune response that proliferates a large number of cells intended to recognize and destroy specific invaders over a period of three to six weeks.

In the last decade, intravenous immunoglobulin (IVIG) therapy has become the primary regimen for bacterial infections, especially for immunocompromised patients (Siber's New
Eng. J. Med. 327: 269-271. (1992)). IVIG therapy has shown efficacy against more than 35 diseases caused by immunopathological mechanisms. Passive immunization against infection
Immunoglobulins specific for tetanus, hepatitis B, rabies, varicella and cytomegalovirus have been particularly successful. The response to the use of immunoglobulins to prevent nosocomial infections, which may be due to the emergence of various bacterial strains and new serotypes found in hospitals, has been blind and frustrating. Passive immunization requires the presence of high and sustained antibody titers against the infecting pathogen.

[0011] Additional immunoglobulin therapy includes Hemophilus influenzae
And has been shown to provide some measure of protection against some surrounding bacteria, such as Streptococcus pneumoniae. Infants deficient in antibodies are susceptible to infection from these bacteria, and bacteremia and sepsis are common. When anti-streptococcal and anti-Hemophilus antibodies are present, they provide protection by promoting clearance of the respective bacteria from the blood. In the case of antibodies to staphylococci, the potential use of additional immunoglobulins to prevent or treat infection was less apparent.

[0012] Early attempts to treat staphylococcal infections have been directed to the potential use of additional immunoglobulins to amplify peritoneal defenses, such as opsonic activity, in patients undergoing continuous ambulatory peritoneal dialysis. I was Standard intravenous immunoglobulin
(IVIG) showed that there was lot-to-lot variability in opsonic activity against Staphylococcus epidermidis (L, A, Clark and C, S, F, J. Clin.
Pathol. 39: 856 (1986)). In this study, 1 / IV of the tested IVIG lots
3 had poor opsonization with complement, and only 2 out of 14 lots had opsonization without complement. That is, despite the fact that the IVIG lot was formed from a large number of plasma donor pools, there were not uniformly good opsonic antibodies to Staphylococcus epidermidis. Furthermore, this study did not test whether IVIG could be used to prevent or treat Staphylococcus epidermidis infection or bacterial sepsis.

[0013] Previous studies have implicated coagulase-negative staphylococcal bacteria, such as S. epidermidis, as the most common species that causes bacteremia in neonates undergoing lipid emulsion infusion. (Freeman, J. et al., N. Engl. J. Med. 323)
: 301, (1990)). These neonates had low opsonic antibodies to Staphylococcus epidermidis despite the fact that the serum had clearly detectable IgG antibodies to Staphylococcus epidermidis peptidoglycan (Fleer, A. J. Infect.
Dis. 2: 426, (1985)). This is surprising. This is because anti-peptidoglycan antibodies were considered to be the major opsonic antibodies.
Although these studies suggest that the neonate's susceptibility to Staphylococcus epidermidis may be related to impaired opsonic activity, these studies also suggest that a number of antibodies directed against Staphylococcus epidermidis have no Suggests that it cannot provide protection when given passively.

[0014] Furthermore, using an antigen binding assay, IgG antibodies to Staphylococcus epidermidis were analyzed in patients with uncomplicated bacteremia and patients with bacteremia and endocarditis.
(See F. Espersen et al., Arch. Intern. Med. 147: 689 (1987)). In this test,
An ultrasonic extract of Staphylococcus epidermidis was used to identify IgG specific to Staphylococcus epidermidis. None of the uncomplicated bacteremia patients had IgG antibodies to Staphylococcus epidermidis. These data suggest that IgG does not provide effective treatment of Staphylococcus epidermidis from the blood. Furthermore, bacteremia patients with endocarditis
89% expressed high IgG antibodies against Staphylococcus epidermidis. In these patients, the IgG was not protective. Because high IgG antibodies engage with significant bacteremia and endocarditis. Based on these studies, the protective role of IgG in Staphylococcus epidermidis sepsis and endocarditis is particularly important for immaturity, weakness,
Suspicious in the presence of intralipidal injection or immunosuppression.

[0015] In animal studies of the literature demonstrating immunoglobulin protection against staphylococcal infection,
Enzyme-linked immunosorbent assays (ELISA) showed the specificity of the strain, and normal adult mice were used for protection studies. Animal models typically used mature animals with normal immunity to overwhelming counterdoses of abnormally pathogenic strains or bacteria. Human patients are generally immunologically immature or frail. Human patients also get somewhat painless infections with co-morbid pathogens, such as Staphylococcus epidermidis, which can usually die due to secondary complications. Models with abnormal strains or overwhelming bacterial doses generally induce rapid and lightning death. These are important factors. This is because antibodies generally work in concert with the host's cellular immune system (neutrophils, monocytes, macrophages and the fixed reticuloendothelial system). Therefore, the effectiveness of antibody therapy can depend on the host's functional immune competence. Predictably, animal models must closely mimic the clinical conditions under which the infection occurs, and must capture the settings of the therapy. Furthermore,
Animal studies have yielded indeterminate results.

One model using an abnormally pathogenic strain of S. epidermidis has been reported. Infected mature mice developed 90-100% mortality within 24-48 hours (K. Yoshida
Et al., Japan. J. Microbiol, 20: 209 (1976)). Antibodies to S. epidermidis surface polysaccharide are protective in these mice. Protection was shown to occur in the IgM fraction but not in the IgG fraction (see K. Yoshida and Y. Ichiman, J. Med. Microbiol, 11: 371 (1977)). However, this model presents a very different pathology from that typically seen in infected patients. Intra-peritoneal countermeasures developed signs of sepsis within minutes of injection,
He died in 48 hours. This particular pathology is not seen in humans infected with staphylococci. A highly pathogenic strain of S. epidermidis can represent an atypical type of infection. Furthermore, isolates of S. epidermidis from infected humans did not kill mice in this model.

In 1987, these animal studies were expanded to include the evaluation of antibodies in human serum against selected pathogenic strains of Staphylococcus epidermidis (Y. Ichiman et al., J. Appl.
Bacterial. 63: 165 (1987)). In contrast to conventional data, the protective antibody
Found in gA, IgM and IgG immunoglobulin fractions. No definitive role could be established for any single type of immunoglobulin (IgG, IgM, IgA).

In this animal model, normal adult mice were used and mortality was measured.
Death was thought to be related to the action of specific bacterial toxins, not sepsis
(See K. Yoshida et al., Japan J. Microbiol, 20: 209 (1976)). Most clinical isolates did not cause lethal infection, and no quantitative blood culture was performed. Furthermore, this study provided little insight into whether antibodies could successfully prevent or treat S. epidermidis in immature or immunosuppressed patients.

In subsequent studies, serotype-specific antibodies directed against Staphylococcus epidermidis capsular polysaccharide were tested in animal models. The results show that serotype-specific antibodies are protective, but each antibody is directed against one serotype as determined by ELISA. Protection was equally serotype specific. No protection against heterologous strains occurred. Furthermore, it was concluded that protection was provided by the IgM antibody.

IVIG is effective in treating Staphylococcus epidermidis infection or sepsis, especially if the patient is immature or immunosuppressed or contains multiple staphylococcus epidermidis serotypes There was no strong evidence. Thus, for example, recent extensive reviews of the etiology, diagnosis and treatment of Staphylococcus epidermidis infection did not use immunoglobulins as potential prophylactic or therapeutic agents (CC Patrick
J. Pediatr. 116: 497 (1990)). Further, as noted above, no US patent describes the effective use of IVIG therapy with antibodies to MSCRAMMs.

US Pat. No. 5,505,945 discloses a passive immunization composition containing an entire repertoire of immunoglobulins, including IgA, IgM and IgG, for inhibiting infection from microorganisms and viruses at the site of a wound, surgery or burn. The thing is described. The composition is Staphylococcus aureus, Coagulase Negative
Staphylococci), Enterococci (Enterococci), Staphylococcus epidermidis, Pseudomonas aeruginosa (P. aeruginosa),
Contains enhanced antibody titers against some pathogens, including E. coli and Enterobacter spp. However,
These compositions are specifically intended to avoid the use of intravenous immunoglobulin or IVIG therapy, and instead are applied in the form of ointments, creams, sprays, etc., intended only for topical application.

US Pat. No. 4,717,766 describes a method for producing high titers of anti-respiratory syncytial virus intravenous immunoglobulin.

US Pat. No. 5,219,578 describes an immunostimulatory composition and method in mammals, and furthermore, contains I or I from goats free of foreign or artificially induced antigens.
It details the isolation of the gG fraction and the use of the isolated immunoglobulin fraction to elicit a stimulated immune response.

US Pat. No. 5,548,066 describes a method of drawing blood from a donor animal, coagulating the blood, separating fluid from cellular material, and then clarifying, concentrating, and sterilizing the product.

US Pat. No. 4,412,990 discloses an intravenous pharmaceutical composition containing an immunoglobulin (IgG) and fibronectin that exhibits synergistic opsonic activity resulting in enhanced phagocytosis of bacteria, immune complexes and viruses. The thing is described.

US Pat. No. 4,994,269 describes the topical use of monoclonal antibodies for the prevention and treatment of pulmonary experimental P. aeruginosa infections. Specifically, the antibody is administered to the lung via an aerosol spray. The results show a beneficial effect in the treatment of infected patients. U.S. Patent No. 4,714,612 describes the use of non-specific gamma globulin IgG in gargles to prevent gingivitis. Another gargle with monoclonal antibodies is
35 et al., Arch. Orai Bial. 35 Supp: 115S-122S (1990). Monoclonal antibodies are specific for Streptococcus mutans, and patients treated with gargles remain free of S. mutans for up to two years. Patients who have not used gargles will experience re-metastatic growth within 2 days.

US Pat. Nos. 5,718,889 and 5,505,945 disclose compositions having a full repertoire of immunoglobulins (IgG, IgM and IgA) for biomaterials, implants, tissues,
And direct and focused local delivery of passive immunity performed by application to wound and burn sites.

US Pat. No. 5,571,511 describes the use of immunoglobulins from individual samples or from serum, plasma, whole blood or a pool of tissues for the treatment of staphylococcal infections. Performing a first assay to identify immunoglobulins that are reactive with the specimen of the first staphylococcal organism and a second assay to identify immunoglobulins reactive with the second specimen of the staphylococcal organism And the second of the first staphylococcal organisms
By selecting immunoglobulins that are reactive with specimens from both staphylococcal organisms. Reactivity is measured by an immunological assay, which can be a binding assay, an opsonization assay or a clearance assay. Preferably, the specimens of the first and second staphylococcal organisms are derived from different serotypes or different genus species, such as Staphylococcus epidermidis and Staphylococcus aureus, and the first specimen is derived from Staphylococcus epidermidis. Is more preferred (Hay, ATCC 55/33).

[0029] US Patent No. 5,412,077 describes sieving plasma samples of effective antibody titer for the treatment or prevention of infections caused by respiratory viruses.

Thus, there remains a need to provide more effective products and methods using antibodies against MSCRAMMs, which products and methods are used to prevent and / or treat staphylococcal infections. Can be used for internal immunoglobulin therapy
Moreover, it can preferably exhibit a wide range of immunizations against various strains of staphylococcal bacteria.

Active Immunization Against Bacterial Infection Historically, studies in bacterial adhesion have been primarily directed to Gram-negative bacteria that express various adherent proteins on the bacterial cell surface (Falkow, S et al., Cell. , 6
5: 1099 to 1102 (1992)). These adhesins or adhesins recognize specific glycoforms exposed on the surface of host cells, especially in the epithelial layer. The use of cretin-like structures for attachment can effectively transfer microorganisms to epithelial surfaces. This provides the bacterium with an excellent replication site and also an opportunity to spread to neighboring host tissues. It has been demonstrated that immunization with a pilus adhesion factor can elicit protection against microbial challenges, for example in Haemophilus influenzae that induced otitis media in a chinchilla model (Sirakova et al., Infect, Immun 62 (5): 2002-2020 (1994)). In experimentally induced infectious bovine keratoconjunctivitis in Moraxella bovis (see Lepper et al., Vet. Microbial, 45 (2-3): 129-138 (1995)), and diarrhea in rabbits (See McQueen et al., Vaccine, 11: 201-206 (1993)). In most cases, immunization with the adhesion factor will prevent infection by inhibiting bacterial attachment and metastatic growth and by promoting bacterial opsonophagocytosis and antibody-dependent, complement-mediated killing. Produce immune antibodies that prevent.

The use of molecules that mediate the attachment of pathogenic microorganisms to host tissue components as vaccine components has emerged as an important step in future vaccine development. Bacterial attachment is an attractive target for the development of new vaccines, since bacterial attachment is a critical first step in the development of most infections. Increased elucidation of the interaction between MSCRAMMs and host tissue components at the molecular level, combined with new technologies in recombinant DNA technology, has laid the foundation for a new generation of subunit vaccines. The entire region or specific regions of MSCRAMMs can now be manufactured, either in their native form or in a location-specific modified form. Furthermore, the ability to mix and balance MSCRAMMs from various microorganisms is:
It has the potential to create a single vaccine that protects against multiple bacteria.

In addition to inactivated pertussis toxin, purified Bordetella pertussis
Recent clinical trials with a novel subunit vaccine against pertussis, consisting of MSCRAMMs filamentous hemagglutinin and pertactin, are the major agents that have been successful in this type of stage. Some modifications of the new acellular vaccine have shown that it is safer and more effective than older vaccines containing whole bacterial cells (Greco et al.
N Eng J Med, 334: 341-348 (1996): Gustattson et al., N Eng J Med, 334: 349-
355 (1996)).

[0034] Natural immunity to staphylococcal infections has not been fully elucidated. Typically,
Healthy humans and animals show a high degree of innate resistance to staphylococcal bacteria, such as Staphylococcus aureus. Protection is due to a complete epithelial and mucosal barrier and normal cellular and humoral responses. Antibody titers to Staphylococcus aureus components were elevated in severe infections (see Ruding et al., J. Med. Microbiol 43 (5): 328-334 (1995)).
To date, there is no serological evidence of an association between antibody titers and human immunity.

Over the past decades, live, heat-sterilized and formalin-fixed specimens of S. aureus cells have been tested as vaccines to prevent staphylococcal infection. To study the effects of a commercial vaccine consisting of staphylococcal toxoid and whole-killed staphylococci on the incidence of peritonitis exit site infection and nasal transport of Staphylococcus aureus in continuous peritoneal dialysis patients Intended to be a multicenter clinical trial
(See Poole-Warren, LA et al., Clin Nephrel, 35: 198-206 (1991)). Although immunization with the vaccine elicited increased levels of specific antibodies to S. aureus,
The incidence of peritonitis was not affected. Similarly, immunization of rabbits with whole cells of S. aureus does not prevent or alter any stage in the development of experimental endocarditis, does not reduce the incidence of renal abscesses, or It did not reduce bacterial loading in infected kidneys (see Greenberg, DP et al., Infect Immun, 55; 3030-3034 (1987)). Recently, no vaccine has been approved by the FDA for the prevention of S. aureus infection. However, a Staphylococcus aureus vaccine based on capsular polysaccharide (Staph VAX) is available from NABI (North
American Biologicals). This vaccine consists of a type 5 or type 8 capsular polysaccharide conjugated to Pseudomonas aeruginosa exotoxin (rEPA). The vaccine is intended to elicit type-specific opsonic antibodies and promote opsonic phagocytosis (see Karakawa, WW et al., Infect Immun, 56: 10910-95 (1988)).
See). Using a purified model of lethal challenge mouse (Fattom, A et al., Infect Immun, 61: 1023-1032 (1996)), type 5 specific IgG was injected intraperitoneally with Staphylococcus aureus. It has been shown to reduce mortality of mice. 17 patients with end-stage renal disease were also vaccinated with the capsular polysaccharide-rEPA vaccine of type 5 (Welch et al., J. Amer, Soc. Nephrol, 7 (2): 247-253 (1996)). reference)
. The geometric mean (GM) IgG antibody level for the Formula 5 formulation was 1 after the first immunization.
There was a 3- to 17-fold increase, but no additional increase was detected after the additional injection. Moreover, these vaccination therapies failed to treat various bacterial strains. Interestingly, vaccinated patients have significantly lower GM IgG levels than control individuals. As demonstrated by animal studies, the vaccine has recently completed Phase II trials in continuous ambulatory peritoneal dialysis patients. Clinical trials have shown that the vaccine is safe but not effective in preventing staphylococcal infections (NABI SEC FORM 10-K405 12/31/95). Inability of Staph VAX to prevent infections associated with peritoneal dialysis in vaccinated patients.
Possible explanations are that the immunogenicity of the vaccine is too low due to the suboptimal vaccine dose or that antibodies in the bloodstream cannot affect infection of certain anatomical areas such as the peritoneum .

The incidence of Gram-positive bacteria-associated sepsis is increasing. Virtually 1/3 to 1/2 of the sepsis in all cases is caused by Gram-positive bacteria, especially Staphylococcus aureus and Staphylococcus epidermidis. In the United States, it is estimated that more than 200,000 patients will develop gram-positive-related sepsis this year. Using a mouse model (see Bremell et al., Infect. Immun, 59 (8): 2615-2623 (1991)), active immunization of the M55 region of Col-conjugated MSCRAMM against sepsis-induced mortality It is clearly demonstrated in PCT WO 97/43314 that the mais can be protected. Mice is M55 or control antigen (
(Bovine serum albumin) and then administered intravenously with S. aureus. 83% (35/42) of mice immunized with M55 survived compared to only 27% (12/45) of mice immunized with BSA. It is the compilation of three separate studies.

According to Schnnings et al., Immunization with fibronectin binding protein from S. aureus protects against experimental endocarditis in rats
(See Micro Pathog, 15: 227-236 (1993)). Rats were immunized with a fusion protein (gal-FnBP) that included β-galactonidase and a region of the fibronectin binding protein from S. aureus that was responsible for binding to fibronectin. gal-
Antibodies to FnBP have been shown to block binding of S. aureus to immobilized fibronectin in vivo. Endocarditis in immunized and non-immunized control rats is induced by catheterization via the right carotid artery, resulting in impaired aortic heart valves coated with fibrinogen and fibronectin. The catheterized rats were then infected intravenously with 1 × 10 ⁵ cells of S. aureus. One and a half days after the challenge infection, the number of bacteria associated with the aortic valve was measured,
Significant differences in bacterial counts between the immunized and non-immunized rat groups were then observed.

To study the effects of vaccination with fibrinogen-binding proteins (especially FnBP-A) and collagen-binding proteins from S. aureus against challenging infection with S. aureus, a mouse mastitis model was developed using Mamo. Used in 1994 by
(Vaccine, 12: 988-992), mice vaccinated with fibrinogen binding protein showed reduced rates of mastitis compared to controls. Meis' challenged mammary gland examination showed that maize glands immunized with fibrinogen binding protein developed mild intramammary infection or pathological changes compared to mammary glands from control mice. Did not have. A significantly reduced number of bacteria could be recovered in mammary glands from mice immunized with fibrinogen binding protein as compared to controls. Mamo then found that vaccination with FnBP-A in combination with staphylococcal α-toxoid did not improve protection (Mamo et al., Vaccine,
12: 988-992 (1994)). Mamo et al. Then immunized mice with collagen-binding protein only, which did not elicit protection against challenge with S. aureus.

Whole-killed staphylococci were used in vaccine studies in people undergoing peritoneal dialysis (see Poale-Werren et al., Clinical Nephrology 35: 198-306 (1991)). In this clinical trial, a commercially available vaccine of α-hemolysin toxoid in combination with a suspension of whole killed bacteria was administered intramuscularly 10 times over a 12 month period, while control patients received saline. Have received an injection. Vaccination elicited a significant increase in antibody levels against S. aureus cells in peritoneal fluid and against α-hemolysin in serum. However, immunization did not reduce the incidence of peritonitis, catheter-related infections, or nasal metastatic growth among vaccine recipients. The lack of protective effect in this study was due to a suboptimal vaccine formulation.

The secreted protein was used as a vaccine component at the subcellular level. Alpha (α) toxin is among the most potent staphylococcal exotoxins, which has cytolytic activity, induces tissue necrosis, and kills laboratory animals. Immunization with formaldehyde-detoxified alpha-toxin may reduce the clinical severity of infection, but does not protect animals from systemic or local infections (Ekstedt, RD
Staphylococci, 385-418 (see 1972).

One study evaluated the protective efficacy of antibodies against S. aureus microcapsules in an experimental model of staphylococcal infection (Nemeth J. and Lee JC Infect.
Immun, 61; 1023-1092 (1993)). Rats were either actively immunized with killed microencapsulated bacteria or passively immunized with a high titer of rabbit antiserum specific for capsular polysaccharide. Control animals were injected with saline or passively immunized with normal rabbit serum. The protection against catheter-induced endocarditis resulting from intravenous administration with the same strain was then evaluated. Immunized animals are susceptible to staphylococcal endocarditis despite increased levels of anti-capsular antibodies, and immunized and control animals have similar numbers of bacteria in the blood. did.

As described in the detailed description below, a number of patents and published patent applications cover fibronectin, fibrinogen, collagen, elasticity and MHC.
Describes the gene sequence for a II serotype binding protein. These patents and patent applications are incorporated by reference in their entirety. These documents teach that proteins, fragments, or antibodies immunoreactive with these proteins or fragments can be used for vaccination to treat S. aureus infections. PCT / US97 / 087210 is a collagen binding protein (M55 fragment),
Vaccination of mice with a combination of a fibronectin binding peptide (FnBPA (D1-D3) treated with formalin) and a fibrinogen binding peptide (ClfA) is described.

Despite the technical advantages of compositions for treating infections from staphylococcal bacteria, such as Staphylococcus aureus, there is still a need to provide more effective products and, preferably, various staphylococcal strains. There is a need to provide products that exhibit widespread immunization against bacterial strains. As described in the Detailed Description of the Invention, one approach to generating prophylactic immunotherapy against bacteria is to stimulate a donor with a vaccine containing a combination of MSCRAMMs. This technique of producing hyperimmune globulin
Plasma with a high concentration of a particular type of disease-antibody may be constantly supplied. Newborn,
To provide passive immunity to infection in trauma patients, immunocompromised patients, or those who are immediately at risk and have no time to deploy their own antibody response
MSCRAMM hyperimmune globulin may be used. Hyperimmune globulin has a high benefit-to-cost ratio, can be manufactured from non-human or human sources, and has a high level of physician acceptance based on past use.

It is therefore an object of the present invention to provide new therapeutic compositions for active and passive immunization against staphylococcal infections.

For mastitis, arthritis, endocarditis, sepsis, osteomyelitis, cholecystitis, cellulitis, pulmonary disease, pneumonia, pyoderma, suppuration of wounds, food poisoning, bladder infections and other infectious diseases It is another object of the present invention to provide for active and passive immunization.

Immunizing against staphylococcal bacteria such as Staphylococcus aureus and Staphylococcus epidermidis, increasing the rate of opsonization and phagocytosis of various staphylococcal infections,
Yet another object of the present invention is to provide a therapeutic composition that promotes the intracellular killing of staphylococcal bacteria.

It is another object of the present invention to provide an immunological serum for staphylococci.

It is another object of the present invention to provide such a serum that produces humoral and cellular immunity against staphylococci.

It is another object of the present invention to provide such a serum that confers short-term immunity to staphylococci.

It is another object of the present invention to provide a method for detecting, diagnosing, treating, preventing or monitoring the progress of a staphylococcal infection.

[0051]

DISCLOSURE OF THE INVENTION

Provided are methods and compositions for passive immunization of patients infected or susceptible to Staphylococcus bacteria such as S. aureus and S. epidermidis infections. And a composition comprising a carefully selected donor plasma pool (a) having high antibody titers against a carefully selected staphylococcal adhesin, ie, MSCRAMMs or a fragment (fraction) or component thereof, or a sequence having substantial homology thereto. selection or manufacture of a donor plasma pool; purification, concentration and processing of the donor plasma pool as necessary to obtain a purified immunoglobulin having a higher than normal antibody titer to the selected adhesin; and then immunization And administering an effective amount of purified immunoglobulin to a patient in need of the globulin. The donated plasma pool is, for example, a protein that binds an individual blood or blood component sample with a higher than normal antibody titer to a selected adhesin or extracellular matrix protein or a fragment having substantial homology thereto. Alternatively, it can be produced by producing a desired complex in combination with one or more of the sequences. Kits for the identification of high titer donating plasma pools of selected adhesins are also provided. In another embodiment, there is provided a method of obtaining a donor plasma pool that is highly effective against staphylococcal bacterial infection, comprising administering a carefully selected protein or peptide to a host to produce a desired antibody. Inducing expression of, regenerating an enhanced high titer serum or plasma pool from the host, optionally purifying and concentrating the immunoglobulin, and providing the immunoglobulin to a patient in need thereof. Is included. As used herein, a "high titer" of an antibody is an antibody that is immunoreactive with a selected adhesin or a fragment thereof, in a normal population of 100 random samples of blood or blood components. Two times higher or higher than found
It means the presence of the antibody is higher than 10 to 20 times.

In one embodiment of the present invention, at least a fibrinogen binding protein such as Clumping factor A (“ClfA”) or Clumping factor B (“ClfB”) or a fragment or component thereof or a combination thereof. Donor plasma compositions having high titers of antibodies to proteins or fragments with sufficiently high homology are selected or produced.

In another embodiment of the present invention, at least a collagen binding protein or peptide (or a suitable site-directed mutant sequence thereof), a fragment or component thereof, such as collagen binding domain protein M55 or sufficiently high homology thereto. A donor plasma composition having a high titer antibody against a protein or fragment having the same potential is selected or produced.

In another embodiment of the present invention, at least a fibronectin binding protein or peptide (or an appropriate site-directed mutant sequence thereof) or a protein or fragment having sufficiently high homology thereto, and a fibrinogen binding protein For A and B (ClfA or ClfB) or a useful fragment or component thereof or a protein or fragment with sufficiently high homology thereto, a donor plasma composition having a high titer antibody is selected or produced.

In another embodiment, at least fibrinogen binding protein A (ClfA)
And selecting or producing a donor pool with high titer antibodies against fibrinogen binding protein B (ClfB) or a useful fragment thereof or a protein or fragment having sufficiently high homology thereto. I do. In yet another embodiment, a donor having a high titer antibody against at least a fibronectin binding protein or peptide (or an appropriate site-directed mutant sequence thereof) or a protein or fragment having sufficiently high homology thereto. Pool, (i) a donor pool in combination with a high titer antibody to fibrinogen binding proteins A and B (ClfA and ClfB) or useful fragments thereof or proteins or fragments having sufficiently high homology thereto; and ii) Select or produce a donor pool that combines high titer antibodies to collagen binding protein or useful fragments thereof.

In another embodiment, the invention relates to the above-described embodiments, wherein said protein or fragment has a high degree of homology with an elastic element (elastin) binding protein or peptide in combination with a high titer antibody. A donor pool with high titer antibodies, such as any, is selected or produced.

In another embodiment, a high titer antibody, as described in the preceding embodiments, is combined with a high titer antibody against an MHCII-like protein or peptide or a protein or fragment with sufficiently high homology thereto. A donor pool with titer antibodies is selected or produced.

In an additional embodiment, high titer antibodies against one or more fibrinogen binding proteins SdrC, SdrD or SdrE or useful fragments thereof or proteins or fragments having sufficiently high homology thereto And selecting or producing a donor pool having the high titer antibody of any of the above embodiments.

In yet another embodiment, at least fibrinogen binding protein SdrC,
Fibrinogen binding protein SdrD and fibrinogen binding protein
For SdrE or a useful fragment thereof or a protein or fragment with sufficiently high homology thereto, a donor pool with high titer antibodies is selected or produced.

A kit for identifying a plasma pool having a high titer of a desired antibody is also provided.
In one embodiment, an appropriate amount of antibody to antibody of a protein or peptide combination as described herein can be immobilized on a solid support and is preferably labeled with a detection agent. Antibodies can be immobilized on various solid substrates by known methods. Suitable solid support substrates include materials having a membrane or coating layer supported on or bonded to a stick, bead, cup, flat pack or another solid support.
Other solid substrates include cell culture plates, ELISA plates, tubes and polymeric membranes. Antibodies can be labeled with a detecting agent such as a fluorescent dye, a radiolabel, biotin or another enzyme such as horseradish peroxidase, alkaline phosphatase and 2-galactosidase. If the detection agent is an enzyme, the kit can be provided with a means for detecting the detection agent. A preferred means for detecting the detection agent uses an enzyme as the detection agent and an enzyme substrate that changes color upon contact with the enzyme. The kit can also include a means for evaluating the assay product, such as a color table or a numerical control table.

Preferably, the isolated immunoglobulin is of the IgG fraction or isotype, but the isolated immunoglobulin is not limited to any particular fraction or isotype and may be of the IgG, IgM, IgA, IgD, IgE or It can be a combination of these. Also preferably, the isolated immunoglobulin is purely or antigenically human immunoglobulin, which can be obtained from human sources or fused to human antibody producing cells against human antibody producing cells. Alternatively, it can be formed directly by substituting a human DNA sequence for some of the non-human DNA sequence encoding the antibody, while retaining the antigen-binding ability of the original antibody molecule.

[0062]

DETAILED DESCRIPTION OF THE INVENTION

Methods and compositions for the passive immunization of patients infected or susceptible to staphylococcal cell infections, such as those caused by Staphylococcus aureus or Staphylococcus epidermidis, are carefully selected for staphylococcal adhesion factors or fragments thereof. Or selection or preparation of a donor plasma pool having a high antibody titer to a sequence having substantial homology thereto; a purified immunoglobulin having a higher than normal antibody titer to the selected staphylococcal adhesin Provided, comprising purifying, enriching and processing the donor plasma pool as necessary to obtain a purified immunoglobulin; and then administering to a patient in need thereof an effective amount of purified immunoglobulin. The donated plasma pool can be obtained, for example, by combining individual blood samples with higher than normal antibody titers against one or more selected adhesin or fragments or sequences having substantial homology thereto. Can be manufactured. Kits for identifying a donor plasma pool having a high titer of a selected adhesin are also provided. In another embodiment,
Methods for obtaining a donor plasma pool that is highly effective against staphylococcal infections include:
A carefully selected protein or peptide is administered to the host to elicit the expression of the desired antibody, recovering the enhanced high-titer plasma pool from the host, optionally purifying and concentrating the immunoglobulin, Provided to include providing to the patient in need.

The donor plasma pool is selected or produced or purified, processed and then administered to a patient in need thereof in an effective amount, while the donor plasma pool contains high titer antibodies to the following proteins: Ie, at least (i) a fibrinogen binding protein such as clamping factor A ("ClfA")
Or clamping factor B ("ClfB") or a fragment or component thereof or a protein or fragment having sufficiently high homology therewith; (ii) a collagen binding protein or peptide (or an appropriate site-directed mutant sequence thereof). A fragment or component thereof, such as collagen binding domain protein M55 or a protein or fragment with sufficiently high homology thereto; (iii) fibrinogen binding proteins A and B (ClfA and ClfB) or a useful fragment thereof or A fibronectin binding protein or peptide (or an appropriate site-directed mutant sequence thereof) or a protein or fragment having sufficiently high homology thereto, in combination with a protein or fragment having high homology to (iv) ) Fibrinogen binding protein A (ClfA) and fibrinogen binding protein B (ClfB) or useful fragments thereof or proteins or fragments having sufficiently high homology thereto; (v) (I) fibrinogen binding proteins A and B (ClfA and (ClfB) or a useful fragment thereof or a protein or fragment having sufficiently high homology thereto; and (II) a fibronectin binding protein or peptide (or thereof) in combination with a collagen binding protein or a useful fragment thereof. (Appropriate site-directed mutant sequence) or a protein or fragment having sufficiently high homology thereto; (vi) an elastin binding protein or peptide or a protein or fragment having sufficiently high homology thereto (Vii) an MHCII type binding protein or peptide or a component with any of the above specific examples in combination with a protein or fragment having sufficiently high homology thereto; (viii) fibrinogen A binding protein SdrC, SdrD or SdrE or a useful fragment thereof or a component having any of the foregoing embodiments in combination with a protein or fragment having sufficiently high homology thereto; (ix) fibrinogen binding protein SdrC, fibrinogen binding protein
SdrD and fibrinogen binding protein SdrE or useful fragments thereof or proteins or fragments having sufficiently high homology thereto; or (x) proteins SdrF, SdrG and SdrH or proteins from coagulase negative bacteria such as S. epidermidis Contains high titer antibodies to useful fragments or proteins or fragments having sufficiently high homology therewith.

Using isolated peptide fragments from wild-type or naturally occurring variants and synthetic mutants corresponding to wild-type naturally occurring variants or introduced mutants that do not correspond to the naturally occurring binding region of the binding protein. The donor plasma pool can be selected or manufactured.

I Definitions Terms FnBP-A protein, FnBP-B protein, ClfA protein, ClfB protein
Quality, SdrC protein, SdrE protein, CNA protein, EbpS protein and
MHC II protein is herein referred to as FnBP-A, FnBP-B, ClfA, ClfB, SdrC, respectively.
SdrD, SdrE, CNA, EbpS and MHC II subdomains, and each
FnBP-A, FnBP-B, ClfA, ClfB, SdrC, SdrD, SdrE, CNA, EbpS and MHC II protein activity or antigenic fragments or components, or containing a protein or fragment with sufficiently high homology with it Defined. FnBP-A,
Active fragments or components of the FnBP-B, ClfA, ClfB, SdrC, SdrD, SdrE, CNA, EbpS and MHC II proteins can inhibit binding of staphylococcal bacteria to host extracellular matrix proteins herein. Defined as a peptide or polypeptide
You. FnBP-A, FnBP-B, ClfA, ClfB, SdrC, SdrD, SdrE, CNA, EbpS and MHC II
An antigenic fragment of a protein is defined herein as a peptide or polypeptide that can generate an immune response.

The term “adhesin” as used herein refers to naturally occurring and synthetic or recombinant proteins and peptides that can bind to extracellular matrix proteins and / or mediate adhesion to host cells. Is included.

The term “amino acid,” as used herein, includes, but is not limited to, naturally occurring and synthetic amino acids, alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan, methionine, glycine,
Includes serine, threonine, cysteine, tyrosine, asparagine, glutamate, aspartic acid, glutamic acid, lysine, arginine and histidine. An "antibody" is any immunoglobulin, including immunoglobulin antibodies and fragments, that binds a specific epitope. Antibodies of the term as used herein include monoclonal antibodies, polyclonal, chimeric, single chain, bispecific, simiauized and humanized antibodies and Fab immunoglobulin expression libraries. Contains Fab fragment including product.

The term “antibody molecule” in its various grammatical forms as used herein includes both intact immunoglobulin molecules and immunologically active portions of immunoglobulin molecules.

As used herein, “pg” means picogram, “ng” means nanogram, “ug” or “μg” means microgram, and “mg” means milligram. , “Ul” or “μl” means microliter, “ml” means milliliter, and “l” means liter.

A “cell line” is a clone of a primary cell that can grow stably for many generations in vitro.

A “clone” is a population of cells derived from a single cell or a common ancestor by mitosis.

A “coding sequence” of DNA is a double-stranded DNA sequence that is transcribed and translated into a polypeptide in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5 '(amino) terminus and a Hong translation stop codon at the 3' (carboxyl) terminus. A coding sequence can include, but is not limited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genetic DNA sequences from eukaryotic (eg, mammalian) DNA, and even synthetic DNA sequences. The polyabnylation signal and transcription termination sequence are usually placed 3 'to the coding sequence.

“DNA molecule” describes a polymeric form of a deoxyribonucleotide (adenine, guanine, thymine or cytosine) in its single-stranded or double-stranded helix. The term describes only the primary and secondary structure of the DNA molecule and is not limited to any particular tertiary form. That is, the term includes, inter alia, linear DNA molecules (eg, restriction fragments),
Includes double-stranded DNA found in viruses, plasmids and chromosomes. In discussing the structure of a particular double-stranded DNA molecule, the sequence is referred to herein as D
It can be described by the usual convention of providing only the sequence in the 5 'to 3' direction along the non-transcribed strand of NA (ie, the strand having a sequence homologous to the mRNA).

As used herein, the term “extracellular matrix protein” or ECM refers to a general family of four macromolecules that provide support and regulate cell behavior: collagen, structural glycoproteins, proteoglycans and elastin Is described.

An “immune effective amount” is an amount capable of stimulating a B cell and / or T cell response. The term "in vivo vaccine" as used herein, refers to immunizing an animal with a protein to elicit a humoral and cellular response that protects against subsequent exposure to a pathogen. Describe.

The term “ligand” is used to include molecules, including those in host tissues, to which pathogenic bacteria bind.

The term “MHC II-like protein” as used herein describes a cell-surface molecule that causes rapid transplant rejection and is required for presentation of antigen to T-cells.

The term “monoclonal antibody” in its various grammatical forms describes an antibody having only one genus of antibodies that combines sites capable of immunoreacting with a particular antigen.

As used herein, the term “pharmaceutically acceptable” refers to a molecule that when administered to a human is physiologically tolerable and does not typically produce an unacceptable allergic or similar stiff reaction. The parts and compositions are described.

A “protective antibody,” as used herein, is an antibody that, when used to passively immunize a naive animal, provides protection against infectious diseases caused by infection with staphylococci. is there.

A “protective epitope” as used herein is an epitope recognized by a protective antibody and / or any of the diseases that can result from infection with staphylococci when used to immunize an animal. Epitopes that elicit an immune response sufficient to prevent or reduce one severity for a period of time.

The term “wound” is used herein to mean a wound that normally covers the epidermal cell layer, wherein another surface structure is injured by mechanical, chemical or other effects.

“Immunologically effective amount” means the amount of a peptide composition that is capable of producing an immune response in a recipient animal. This includes both the generation of an antibody response (B cell response) and / or the stimulation of a cytotoxic immune response (T cell response). The generation of such an immune response finds use in both the production of bioreactive agents useful in diagnostic embodiments, such as CTLs and more particularly reactive antibodies, and also in various prophylactic or therapeutic embodiments. There is.

II Fibronectin-Binding MSCRAMMs Fibronectin (Fn) is a 440-kDa glycoprotein found in ECN and animal body fluids. The major biological function of fibronectin appears to be related to its ability to serve as an adhesion substrate for cells expressing the appropriate integrins. Several bacterial species have been shown to specifically bind fibronectin and adhere to fibronectin-containing substrates. Most Staphylococcus aureus isolates bind Fn,
It binds to different extents, which reflects changes in the number of MSCRAMM molecules expressed on the bacterial cell surface. Fn interaction with S. aureus is highly specific
(See Kuusela, P., Nature 276: 718-20 (1978)). Fn binding is determined by FnBP-A and
It is mediated by two surface-exposed proteins of 110 kDa molecular weight called FnBP-B. The major Fn binding site consists of a 35-40 amino acid motif repeated 3-5 times. These genes have been cloned and sequenced (Jonsson K et al., Eur.
Biochem, 202: 1041-1048 (1991)). Potential uses for vaccination with anti-FnBP antibodies include, but are not limited to, mastitis, endocarditis and wound infection in cattle.

WD-A-85 / 05553 discloses bacterial cell surface proteins having fibronectin, fibrinogen, collagen and / or laminin binding capacity.

US Patent Nos. 5,320,951 and 5,571,514 to Hook et al. Disclose the gene sequence of fibronectin binding protein A (fnbA), and biological products and methods based on this sequence.

US Patent No. 5,175,096 to Hook et al. Discloses the gene sequence of fnbB, a hybrid DNA
Disclosed are molecules (fnbB) and biological products and methods based on this sequence.

US Pat. No. 5,652,217 discloses a specific sequence of hybrid D from Staphylococcus aureus.
Disclosed are isolated and purified associatively active proteins encoded by NA molecules.

US Pat. No. 5,440,014 describes mastitis caused by staphylococcal infection for treating wounds, blocking protein receptors, immunizing another animal, or for use in a diagnostic assay. Disclosed are fibronectin binding peptides within the D3 homology unit of the S. aureus fibronectin binding protein, which can be used for vaccination of ruminants.

US Pat. No. 5,189,015 discloses a method for the prophylactic treatment of colonization of a bacterial strain of Staphylococcus aureus capable of binding fibronectin in a mammal, the method requiring treatment. Administering to the mammal a prophylactically effective amount of a protein having fibronectin binding properties and a molecular weight of 87 kDa to 165 kDa to prevent the occurrence of infection caused by a Staphylococcus aureus bacterial strain capable of binding fibronectin.

US Pat. No. 5,416,021 discloses a DNA encoding a fibronectin binding protein from Streptococcus dysgalactiae and a DNA encoding a fibronectin binding protein from Streptococcus dysgalactiae contained in E. coli. Disclosed is a fibronectin-binding protein-encoding DNA from Streptococcus dysgalactiae, as well as a plasmid containing Streptococcus dysgalactiae transformed by E. coli and DNA encoding a fibronectin-binding protein from an E. coli microorganism.

It was observed that antibodies against wild-type fibronectin binding protein did not substantially inhibit the ability of S. aureus to bind to fibronectin and thus did not show a significant therapeutic effect in vivo. PCT / US98 / 01222 discloses an antibody that blocks fibronectin from binding to a fibronectin binding protein. The antibodies are raised against site-directed mutant sequences of the fibronectin binding protein that do not bind to fibronectin. It was identified that rapid complexation of fibronectin with fibronectin binding proteins and fragments occurs in vivo. Even based on the fibronectin binding region of the fibronectin binding protein, peptide epitopes that do not bind to fibronectin do not form a complex with fibronectin in vivo. This allows antibodies to be formed against unconjugated peptide epitopes, which inhibit or block fibronectin from binding to the fibronectin binding protein.
III Collagen Binding MSCRAMMs Collagen is a major component of cartilage. Collagen (Cn) binding proteins are commonly expressed by staphylococcal strains. Staphylococcus aureus Cn-binding MSCRAMM adheres to cartilage in a process that constitutes an important part of the pathogen mechanism in staphylococcal infections
(See Switalski et al., Mol. Micro, 7 (1), 99-107 (1993)). Cn binding by staphylococcal bacteria such as Staphylococcus aureus has been found to play a role, at least, but not exclusively, in arthritis and sepsis. CNA proteins with molecular weights of 133, 110 and 87 kDa have also been identified (Patti J. et al., J. Biol. Chem. 267: 4766-4772 (1992). Strains expressing CNAs with different molecular weights have their Cn binding ability. (Switalski LM et al., Mol. Microbial, 7: 99-107 (1993
)reference).

[0093] Staphylococcal strains recovered from the joints of patients diagnosed with septic arthritis or osteomyelitis almost always express CNA, but significantly fewer isolates obtained from wound infections Express adhesin (Switalski LM et al., Mol. Microbial, 7:
99-107 (1993)). Similarly, a strain of Staphylococcus aureus isolated from bone of a patient with osteomyelitis is a bone-specific protein, MSCRAMM that recognizes bone sialoprotein (BSP).
(See Ryden, C. et al. Lancet, 11: 515-518 (1987)). Metastatic growth of Staphylococcus aureus in articular cartilage in the joint space appears to be a significant factor contributing to the development of septic arthritis.

The cloning, sequencing, and expression of the gene encoding the CNA protein of S. aureus has also been reported (Patti. J et al., J. Biol. Chem. 267;
4766-4772, (1992)). The CNA gene encodes a 133-kDa adhesin that contains structural features that characterize surface proteins isolated from Gram-positive bacteria.

Recently, the ligand binding site was localized within the N-terminus of half of the CNA (Patti
.J et al., Blochemistry, 32: 11428-11435, (1993)). Analysis of the Col binding activity of the recombinant proteins corresponding to the different segments of MSCRAMM identified a 168 amino acid long protein fragment (corresponding to amino acid residues 151-318) with recognizable Col binding activity. . Short truncation of the protein at the N- or C-terminus resulted in loss of ligand binding activity, but also resulted in a conformational change of the protein.

PCT WO 92/07002 discloses a hybrid DNA molecule containing a nucleotide sequence from Staphylococcus aureus encoding a protein or polypeptide having collagen binding activity and a plasmid or phage containing the nucleotide sequence. ing. Also disclosed are strains of Escherichia coli expressing collagen-binding proteins, microorganisms transformed with recombinant DNA, methods for producing collagen-binding proteins or polypeptides, and protein sequences of collagen-binding proteins or polypeptides.

[0097] Patti et al. (See J of Biol, Chem. 270, 12005-12011 (1995)) disclose a collagen binding epitope in the S. aureus adhesin that is encoded by the CNA gene. In this study, researchers synthesized peptides derived from the protein sequence and used them to produce antibodies. Some of these antibodies block proteins from binding to collagen.

PCT / US97 / 08210 discloses that certain identified epitopes of collagen binding proteins (M55, M33 and M17) can be used to generate protective antibodies. This patent application also discloses a crystal structure of CNA that provides the critical information needed to identify compositions that prevent or completely prevent Col from binding to CNAs. The ligand binding site in the CNA and 25-amino acid peptide of S. aureus has been characterized to directly block the binding of S. aureus to 125 I-labeled type II Col.

IV Fibrinogen-Bound MSCRAMMs Fibrin is a major component of blood clots and fibrinogen / fibrin is one of the major host proteins attached to implanted biomaterial. There is considerable evidence to suggest that bacterial adhesion to fibrinogen / fibrin is important for the initiation of device-associated infection. For example, as shown by Vaudaux et al., Staphylococcus aureus adheres to fibrinogen-coated in vitro plastics in a dose-dependent manner (see J. Infect, Dis. 160: 865-875 (1989)). Furthermore, in a model that mimics damage to blood clots or heart valves, Herrmeun et al. Have demonstrated that Staphylococcus aureus binds greedily to platelets that adhere to surfaces via fibrinogen cross-links (J. Infect. Dis. 167: 312-322 (1993)). Staphylococcus aureus can adhere directly to fibrinogen in clots formed in vitro and to cultured endothelial cells via fibrinogen deposited from plasma which acts as a crosslink (Moreillon et al., Infect. 63: 4738-4743 (1995); Cheung et al., J. Clin.
Invest. 87: 2236-2245 (1991)). As shown by Vaudaux et al. And Moreillon et al., Mutants deficient in fibrinogen binding protein clumping factor (ClfA) can adhere to fibrinogen in vitro, adhere to explanted catheters, clots. Adhesion to injured heart valves in a rat model of endocarditis (Vaudaux et al., Infect. Immun. 63: 585-590 (1995); Moreillon et al., Infect. Immun. 63: 4738-). 4743 (1995)).

The fibrinogen adhesin, often described as a “clumping factor,” is located on the surface of S. aureus cells. Interaction between bacteria and fibrinogen in solution results in immediate clamping of bacterial cells. The binding site on fibrinogen is located at the C-terminus of the gamma chain of dimeric fibrinogen glycoprotein. The affinity is very high and clamping occurs even at low concentrations of fibrinogen. Scientists have recently shown that clamping factors also promote adhesion to solid-phase fibrinogen, to blood clots and to damaged heart valves (McDevitt et al., Mol.Microbial. 11: 237). ~ 248 (1994): Vaudaux et al. Infect.
Immun. 63: 585-590 (1995); Moreillon et al., Infect. Immun. 63: 4738-4743.
(1995)).

Two genes in Staphylococcus aureus encoding two Fg binding proteins, ClfA and ClfB, were found. The gene, ClfA, was cloned, sequenced, and
Was found to encode the polypeptide of ClfA binds the gamma chain of fibronectin and ClfB binds the α and β chains (see Eidhin et al., Mol Micro, awaiting publication (1998)). ClfB binds to both soluble and immobilized fibrinogen and acts as a clamping factor, with a predicted molecular weight of 88 kDa and a
It is a cell wall engaging protein with an apparent molecular weight of 4 kDa.

The gene for the clamping factor protein, designated ClfA, has recently been cloned, sequenced, and analyzed in detail at the molecular level (MeDevitt et al., Mol.
Microbial. 11: 237-248 (1994); MeDevitt et al., Mol Microbiol. 16: 895-907.
(1995)). The predicted protein consists of 933 amino acids. A 39 residue signal sequence occurs at the N-terminus followed by a 520 residue region (region A), which contains the fibrinogen binding region. It is followed by a 308 residue region (region R) consisting of 154 repeats of the dipeptide serine-aspartate. The sequence of the R region is
It is encoded by a repeat of 18 base pairs, GAYTCNGAYTCNGAYAGY, where Y equals pyrimidine and N equals any base. The C-terminus of ClfA is responsible for anchoring the protein to the cell wall, to membrane anchors and to positively charged residues at the extreme C-terminus, a number of gram-positive surface proteins such as the LPDTG motif. It has features that exist in

Platelet integrin αIIb β3 recognizes the C-terminus of the γ chain of fibrinogen. This is a significant event in the initiation of a clot during coagulation. ClfA and αIIb β3
Appears to accurately recognize the same site on the fibrinogen gamma chain. This is because ClfA can block platelet aggregation and the peptide corresponding to the C-terminus of the gamma chain (198-411) can block both integrins and ClfA, which interacts with fibrinogen (McDevitt et al. Eur. J. Biochem, 247: 416-424 (1997)). αII
b The fibrinogen binding site of β3 is close to or overlaps the Ca ²⁺ binding determinant described as the “EF hand”. ClfA region A carries several EF-hand-like motifs. Concentrations of Ca ²⁺ in the range of 3-5 mM block these ClfA-fibrinogen interactions and alter the secondary structure of ClfA protein. Mutations affecting the ClfA EF hand reduce or prevent interaction with fibrinogen. Ca ²⁺ and the fibrinogen γ chain appear to bind to the same or overlapping sites in ClfA region A.

The α-chain of leukocyte integrin, αMB2, has an insertion of 200 amino acids (A or I region) due to ligand binding activity. The I region requires a novel metal ion-dependent adhesion factor site (MIDAS) motif for ligand binding. Among the recognized ligands are fibrinogen. The binding site on fibrinogen is in the gamma chain (residues 190-202). Candida albicans (Candibla albicans) has recently been reported to have a surface protein, αIntlp, which has the property of storing eukaryotic integrins. The backside protein has amino acid sequence homology to the I region of Mβ2, including the MIDAS motif. Intlp also binds to fibrinogen.

The ClfA region A also shows some sequence homology to αIntlp. Examination of the ClfA region A sequence revealed a potential MIDAS motif. Of the MIDAS motif in ClfA
Mutations in putative cation coordination residues in the DxSxS moiety significantly reduce fibrinogen binding. Peptides corresponding to the γ-chain binding site for αMβ2 (190-202) can block ClfA-fibrinogen interaction
Indicated by O'connel et al. (In O'connel et al., Biol. Chem. Printing). That is, ClfA is 2
It is likely that they can bind to the γ-chain of fibrinogen at two distinct sites. The ligand binding site on ClfA is similar to the site used by eukaryotic integrins and includes a divalent cation binding EF-hand and a MIDAS motif. Despite the low degree of identity between ClfA and ClfB, both proteins do not share an apparent metal-binding motif, but are susceptible to inhibition by divalent cations to fibrinogen (different strands). Above).

Other fibrinogen binding proteins are disclosed in US patent application Ser. No. 09 / 220,650, filed by the present applicant, which is incorporated herein by reference. This U.S. application discloses isolated fibrinogen binding proteins ClfB, SdrC, SdrD and SdrE.
Also disclosed are antibodies against the protein and diagnostic kits containing the protein or antibody. Also comprising administering to the patient an effective amount of ClfB, SdrC, SdrD, SdrE or a binding fragment thereof, a method for preventing Staphylococcus aureus infection and
Also described is a method of eliciting an immune response comprising administering to a patient a pharmaceutical composition containing ClfB, SdrC, SdrD, SdrE or an active fragment thereof.

ClfB has a predicted molecular weight of approximately 88 kDa and an apparent molecular weight of approximately 124 kDa.
ClfB is a cell wall engaging protein that binds both soluble and immobilized fibrinogen. In addition, ClfB binds both the α and β chains of fibrinogen and acts as a clamping factor. ClfB protein has proven to be a virulence factor in experimental endocarditis.

The SdrC, SdrD and SdrE proteins are found in major sequence and structural organizations
It is related to ClfA and ClfB proteins and is located on the cell surface. SdrC,
The SdrD and SdrE proteins are cell wall engaging proteins with a signal sequence at the N-terminus, an LPXTG motif, a hydrophobic region, and a positively charged residue at the -terminus. Each also has an SD of sufficient length to allow efficient expression of the ligand binding region A on the cell surface.
It has a repeating body containing region R. With the A region of the SdrC, SdrD and SdrE proteins placed on the cell surface, the proteins can interact with proteins in plasma, with extracellular matrix or with molecules on the surface of host cells. The protein is ClfA and Cl
It shares some limited amino acid sequence similarity to fA. Furthermore, SdrC, SdrD and SdrE also show cation-dependent ligand binding to extracellular matrix proteins.
For example, SdrC binds hydronectin and SdrE binds bone sialoprotein (BSP).

It has been found that in the A region of SrdC, SrdD, SrdE, ClfA and ClfB, there is a highly conserved amino acid sequence that can be used to induce a common TYTFTDYVD motif. The motifs can be used in multi-component vaccines to confer a wide range of immunity to bacterial infections, and can be used to produce monoclonal or polyclonal antibodies that confer a wide range of passive immunity. In another embodiment, the Sdr and Clf protein families, (T / I) (Y / F) (T / V) (F) (T) (D / N) (Y) (V) (D / N) Any combination of variable sequence motifs derived from can be used to confer immunity or to produce protected antibodies.

Thus, ClfB, SdrC, SdrD and SdrE share a common TYTFTDYVD motif that overlaps the ligand binding / Ca ²⁺ binding region of ClfA. Thus, the protein interacts with fibrinogen and other host components. ClfB, SdrC, depending on the protein
The SdrD and SdrE subdomains include subregions A and B1-B5. Additional information regarding extracellular matrix binding proteins is disclosed in US Patent Application Ser. No. 09 / 200,650, which is incorporated herein by reference.

V. Elastin-binding MSCRAMMs The major role of elastin is to impart reversible elastic properties to tissues and organs (see Rosenbloom, J et al., FASEB J. 7: 1208-1218 (1993)). Although elastin expression is highest in lung, skin and blood vessels, the protein is widely expressed in mammalian hosts for S. aureus. Binding of Staphylococcus aureus to elastin was found to be rapid, reversible, with high affinity and ligand specificity. In addition, a 25 kDa cell surface elastin binding protein (EbpS) was isolated and proposed to mediate S. aureus binding to the elastin-enriched host ECN. EbpS binds to a region in the N-terminal 30 kDa fragment of elastin.

[0112] PCT / US97 / 03106 discloses a gene sequence for an elastin binding protein. The disclosed DNA sequence data indicates that the ebps open reading frame consists of 606 bp and encodes a novel polypeptide of 202 amino acids. The EbpS protein has a predicted molecular weight of 23,345 daltons and has a pi of 4.9. EbpS is N-
It is expressed in E. coli as a fusion protein with a polyhistidine residue attached to the end. The polyclonal antibody raised against recombinant EbpS is 25 kDa EbpS on the cell surface
And specifically inhibited staphylococcal elastin binding. Furthermore, the recombinant EbpS specifically bound to immobilized elastin and prevented Staphylococcus aureus from binding to elastin. Degradation products of the recombinant EbpS, which lacks the first 59 amino acids of the molecule, and the C-terminal fragment of the CNBr-cleaved recombinant EbpS, however, did not interact with elastin. These results strongly suggest that EbpS is a cell surface molecule that mediates S. aureus binding to elastin. The finding that some constructs of recombinant EbpS do not interact with elastin
It suggests that the elastin binding site in EbpS is contained in the first 59 amino acids of the molecule.

[0113] Several separate criteria indicate that EbpS is a surface protein that mediates elastin binding in cells. First, rEbpS specifically binds to immobilized elastin and prevents Staphylococcus aureus cells from binding to elastin in a dose-dependent manner. These results establish that EbpS is a functionally active elastin binding protein in a soluble form. Next, the antibody raised against rEbpS recognizes the 25 kDa protein expressed on the cell surface of S. aureus cells. In addition to dimensional similarity and antibody reactivity, another evidence that this 25 kDa protein is cell surface EbpS is that binding of the 25 kDa protein to immobilized anti-rEbpS IgG in the presence of excess unlabeled rEbpS. Provided by experiments that show that Finally,
Fab fragments produced from the anti-rEbpS antibody but not from the pre-immune control block the binding of S. aureus elastin. The results suggest that the surface EbpS topology is such that the elastin binding site is accessible to interact with the ligand (ie, elastin and the anti-rEbpS Feb fragment) and is not buried in the cell wall or membrane region. It is something. Composite data demonstrate that EbpS is a cell surface protein that is responsible for binding S. aureus to elastin.

The findings of the present invention and previous findings suggest the existence of a functionally active 40 kDa intracellular precursor form of EbpS that requires processing at the C-terminus before surface expression. This concept is based on the following observations: i) Intracellular cells that are never detected during cell surface labeling experiments
The 40 kDa elastin binding protein is present, ii) the 25 kDa EbpS and the 40 kDa elastin binding protein have identical N-terminal sequences, and iii) a single gene is present in EbpS. Initially, we ignored this hypothesis because the dimensions of the ebps open reading frame were not sufficient to encode a 40 kDa protein. However, rEbpS
The inventor's work demonstrated that, while the actual size of the recombinant protein was 26 kDa, it migrated abnormally as a 45 kDa protein in SDS-30PAGE. The present invention concludes that full-length native EbpS with a predicted size of 23 kDa may be migrating into SDS-PAGE as a 40 kDa intracellular precursor, and that the 25 kDa surface form of EbpS is actually more than that of molecules processed at the C-terminus. Suggests a small form. EbpS is N
-Although lacking terminal signal peptides and other known sorting and anchoring signals, the proposed intracellular processing results can explain some questions as to how EbpS was directed to the cell surface. In practice, the C-terminal signal peptide has been identified in several bacterial proteins (Fath, MJ and kalter, R. Microbiol. Rev. 57:
995-1017 (1993)) Yet another means of anchoring proteins to the cell surface has been reported in Gram-positive bacteria (Yother, JJ and White, JM Bacteriol,
176: 2976-2984 (1994)).

Using the overlapping EbpS fragment and the recombinant construct, the elastin binding site in EbpS was mapped to the amino-terminal region of the molecule (PCT / US97 / 03106). Overlapping synthetic peptides spanning amino acids 14-34 were then used to better define the binding region. Of these, the peptides corresponding to residues 14-23 and 18-34 specifically blocked elastin binding by more than 95%. Common to all active synthetic peptides and proteolytic fragments and recombinant fragments of EbpS is the hexameric sequence 18Thr-Asn-Ser-His- Gln-Asp 23. Further evidence that this sequence is important for elastin binding the Asp ^{2 3} when substituted by Asn in the synthetic peptide corresponding to residues 18 to 34, the activity is lost. However, the synthetic hexamer TNSHQD itself did not prevent elastin from binding to staphylococci. These discoveries are TNSHQD
Although the presence of the sequence is essential for EbpS activity, carboxyl side chains flanking amino acids and Asp ²³ in the N- or C- terminal direction indicates that it is necessary to recognize elastin.

VI. NHC II Similarity Protein (MAP) In addition to fibrinogen, fibronectin, collagen and elastin
Thus, Staphylococcus aureus associates with another adhesive eukaryotic protein, many of which belong to the family of adhesive matrix proteins such as pitronectin (Chatnul, GS et al.
Infect. Immun. 55: 1878-1883 (1987)). U.S. Pat. No. 5,648,240, incorporated herein by reference, discloses a DNA segment comprising a gene that encodes a broad-sequence adhesin for S. aureus having a molecular weight of about 70 kDa. The adhesin is capable of binding fibronectin or hydronectin and includes an MHCII mimetic unit of about 30 amino acids. Further analysis of the binding specificity of this protein indicates that it is functionally similar to the MHCII antigen in binding synthetic peptides. That is, in addition to mediating bacterial adhesion to the ECN protein, it may play a role in staphylococcal infection by suppressing the host's immune system. The U.S. patent discloses a recombinant vector containing a specific DNA sequence, a recombinant host cell transformed with the vector, and DNA hybridized with the DNA of the specific sequence. Also disclosed is a composition comprising a protein or polypeptide encoded by a particular DNA sequence and a method of eliciting an immune response in an animal comprising administering an immune composition comprising the encoded protein or polypeptide. Have been. The next step, ie, inserting the specific DNA sequence into an appropriate expression vector and culturing host cells transformed with the vector under conditions that produce the MHC II antigen protein analog, forms an MHC antigen protein analog. A method of doing so is further disclosed in the US patent.

VII SDR Protein from Staphylococcus epidermidis Staphylococcus epidermidis, a clotting enzyme-negative bacterium, is commonly resident in human skin,
Often causes heterogeneous infections. Pathogenesis is facilitated by the ability of microorganisms to first adhere to indwelling medical devices, such as prosthetic valves, orthopedic devices and venous and peritoneal dialysis catheters, and then form a biofilm thereon. Device-related infections jeopardize the success of medical procedures and significantly increase patient mortality. Thus, the ability to develop vaccines that can regulate or prevent the outburst of Staphylococcus epidermidis infection is an important tool for preventing or treating infection from a wide range of bacteria, including both coagulase positive and coagulase negative bacteria. Like development, it is of great importance.

The three Sdr (serine-aspartate (SD) repeat region) proteins expressed by S. epidermidis are named SdrF, SdrG and SdrH, and the amino acid sequences of these proteins and their nucleic acid sequences are Foster U.S. patent application Ser. Nos. 60 / 098,443 and 60 /
Based on 117,119. All of these applications are incorporated herein by reference. According to the present invention, the methods of donor selection and donor stimulation described herein can be performed on SdrF, SdrG or SdrH proteins. In these methods, individuals with higher than normal antibody titers to SdrF, SdrG or SdrH proteins are identified and selected and antibody titers higher than normal to one or more of these proteins are identified. Can be produced. Thus, donor plasma can be produced according to the present invention which is useful in a method for preventing or treating infections from clotting enzyme-negative staphylococcal infections, such as those associated with Staphylococcus epidermidis.

VII Proteins and Peptides Having Substantial Homology or Equivalent Function as Described in the Present Invention Donor plasma pools can be sieved or stimulated as desired, including proteins, peptides, proteins of the full sequence. Or a peptide fragment, an isolated epitope,
It has a fusion protein or any other protein that binds to the target ECN either in the form of a wild-type, site-directed mutant or a sequence that is substantially homologous thereto.

The term “substantially similar” when used in the context of an amino acid sequence means an amino acid sequence that is not identical to the published sequence but results in a protein or peptide having the same functionality and activity. I do. Because one amino acid is replaced by another similar amino acid or the change (either a substitution, deletion or insertion) does not substantially affect the active site of the protein.
When at least about 70% (preferably at least about 80%, and most preferably at least about 90 or 95%) of the amino acids are matched over a sequence of limited length, the two amino acid sequences are "substantial" Homology ".

It is to be understood that each of the NMSCRAM polypeptides of the invention can be part of a larger protein. For example, a ClfA polypeptide of the invention can be fused at its N-terminus or C-terminus to a ClfB polypeptide or to a non-fibrinogen binding polypeptide or a combination thereof. The following is a discussion of changing the amino acids of a protein to produce an equivalent or even improved second generation molecule. Amino acid changes can be achieved by changing the codons of the DNA sequence according to Table 1. The amino acid residue abbreviations are shown in Table 1, retaining the standard polypeptide nomenclature (see J. Bial, chem, 243; 3552-3559 (1969)). It will be understood by those skilled in the art that the codons specified in Table 1 are for the RNA sequence. The corresponding codon for DNA has T substituted for U.

For example, certain amino acids may be present in a protein structure without appreciable loss of interactive binding capacity for structures such as the antigen binding region of an antibody or a binding site on a substrate molecule. Can be substituted for another amino acid. Because the ability to interact with a protein limits the biological functional activity of the protein, certain amino acid sequence substitutions can be made in the protein sequence and, of course, in its underlying DNA coding sequence. And yet nonetheless obtain proteins of similar properties that can stimulate the production of substantially similar antibodies. The peptide sequence of the disclosed composition, the corresponding DNA sequence encoding the peptide or an antibody against the peptide without appreciable impairment of the biological use or activity of the donor plasma pool immunoglobulin thus recovered. It is envisaged by the inventors that various changes can be made.

[0123]

It is understood in the art that similar amino acid substitutions can be made effectively on the basis of hydrophilicity. U.S. Pat.No. 4,554,10, incorporated herein by reference.
No. 1 states that the maximum local average hydrophilicity of a protein, as governed by the hydrophilicity of its neighboring amino acids, correlates with the biological properties of the protein.

As detailed in US Pat. No. 4,554,101, the following hydrophilicity values are assigned to amino acid residues: arginine (+3.0); lysine (+1.0); aspartic acid (+ 3.0 ± 1). )
Glutamic acid (+ 3.0 ± 1) serine (+3.0); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4); proline (−0.5 ± 1)
Acnin (-0.5); Histidine (-0.5); Cysteine (-1.0); Methionine (
Valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4). It is understood that an amino acid can be substituted for another amino acid of similar hydrophilicity value and still obtain a biologically equivalent protein, especially an immunologically equivalent protein. In such changes, substitution of amino acids having a hydrophilicity value within ± 2 is preferable, amino acid substitution having a hydrophilicity value within ± 1 is particularly preferable, and hydrophilicity value of ± 0.5 is preferable.
Substitution of amino acids within is more particularly preferred.

Therefore, as described above, amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. Representative substituents taking into account the various properties described above are well known to those skilled in the art and include the following substituents; arginine and lysine; glutamic and aspartic acid; serine and threonine; glutamine and asparagine; and valine; Leucine and isoleucine.

The following non-classical amino acid sequences can be incorporated into peptides to introduce specific conformational motifs: 1,2,3,4-tetrahydroisoquinoline-3-carboxylate
(See Kazmierski et al., J. Am. Chem. Soc. 113; 225-2283 (1991)); (2S, 3S)-
2-amino Tetrahydronaphthalene-2-carboxylic acid (see Landis, Ph, D, Thesis, University of Arizona (1989));
Hydroxy-1,2,3,4-tetrahydroinknoline-3-carboxylate (see Miyake et al., J. Takeda Res, Labs 45; 53-76 (1989)); β-carboline (D and L) ( Kazmierski. Ph.D. Thesis. University of Arizona (1988)); HIC
(Histidine innoline carboxylic acid) (Zechel et al., Int. J. Rep. Protein Res.
43 (1991)); and HIC (histidine cyclic urea (Dharaniprada)).

The following amino acid analogs and peptiomimetics can be incorporated into peptides to induce or favor specific secondary structures;
LL-Acp (LL-3-amino-2-propenidone-6-carboxylic acid), a β-turn induced dipeptide analog (Kemp et al., J. Org. Chem, 50; 5834-5838, (1985)
)); Β-sheet induced analogs (see Kemp et al., Tetrahedron Lett, 29; 5081-5082 (1988)); β-turn induced analogs (Kemp et al., Tetrahedron Lett, 29; 505).
7-5060, (1988); α-helix-induced analogs (Kemp et al., Tetrahedron Lett, 29;
4935-4938)); β-turn induced analogs (Kemp et al., J. Org. Chem, 54; 109-
115 (1989)); and the following references: Nagai and Sato, Tetrahedron Lett,
26; 647-650 (1985); DiMaio et al., J. Chem. Soc. Perkin Trans p. 1687 (1989).
And Gly-Ala turn analogs (Kahm et al., Tetrahedron Le
tt, 30; 2317 (1989)); amide bond isostere (Jomes et al., Tetrah
edron Lett, 29; 3853-3856 (1988); tetrazole (Zabrocki et al., J. Am. C.
hem. Soc. 110; 5875-5880 (1988)); DTC (Samanen et al., Int. J. Prote.
in Pep Res. 35: 501-509 (1990)); and Dlsam et al., J. Am. Chem. Sci.
2; 323-333 (1990); Analogs taught by Org. Chem. 56; 436 (1990). Conformationally restricted mimetics of β-turns and β-bulges and peptides containing them are described in Kahn, US Pat. No. 5,440,013.

IX Production of Purified Immunoglobulins In one embodiment, purified immunoglobulins (A, D, E or G) having high titer antibodies to a selected adhesin are produced. As used herein, the term "high titer" refers to an amount that is twice or more, for example, 10 to 20 times or more, higher than the titer found in a normal population of 100 disordered samples of blood. Indicates the presence of the antibody.

Blood products can be produced by the following procedures: (i) selection and purification of donor immunoglobulins with natural high titer antibodies to the selected adhesin, (ii) desired composite distribution a combination of donor immunoglobulins from several individuals with high titer antibodies to one or more of the adhesins selected to produce the (profile); or (iii) a donor against the selected antigen One or more donors to form the desired composite antibody distribution by obtaining a blood sample of the exposed donor after a time sufficient to produce and collect the resulting immunoreactive antibodies. Stimulation of the desired antibody in The first two embodiments are described as a "donor selection" program, and the third embodiment is described as a "donor stimulation" program.

Stimulation of Donor Using the peptide antigens described herein, the present invention also provides an immunologically effective amount of MSCRAM
Provided is a method of stimulating high antibody levels in a donor, comprising administering a pharmaceutically acceptable composition comprising a peptide composition derived from M to an animal, eg, a human. The pharmaceutical composition can contain partially or significantly purified MSCRAMM-derived peptide epitopes obtained from natural or recombinant sources, wherein the protein or peptide is naturally available or chemically purified. It can be synthesized or alternatively produced in vitro from a recombinant host cell expressing a DNA segment encoding such an epitope. Smaller peptides encompassing a reactive epitope are often preferred, such as peptides of between about 30 and about 100 amino acids in length. If desired, the antigenic protein or peptide can be combined with another reactant, such as another staphylococcal or streptococcal peptide or nucleic acid composition. Pharmaceutical compositions can also contain staphylococcal-produced bacterial components as described above, obtained from natural or recombinant sources, wherein the protein can be obtained naturally or chemically synthesized or otherwise. As such, it can be produced in vitro from recombinant host cells expressing a DNA segment encoding such a peptide.

Another approach conceived by the inventors to generate an immune response in animals is
Is a pharmaceutically acceptable composition comprising an immunologically effective amount of a nucleic acid composition encoding a peptide epitope or an immunologically effective amount of an attenuated living microorganism containing and expressing such a nucleic acid composition. The substance may be administered to an animal or a human subject. Antigenic functional equivalents of the proteins and peptides described herein are also included within the scope of the invention. Antigenic functional equivalents or epitope sequences can be first designed or predicted and then tested, or simply tested directly for cross-reactivity.

In the case of preventing bacterial adhesion, it is particularly desirable to produce an epitope that produces an antibody that blocks the interaction of a specific gene product or the interaction of a proteobrican that is structurally similar to the specific gene product.

The identification or design of suitable MSCRAMM epitopes and / or functional equivalents of epitopes suitable for use in vaccines or simply as antigens (eg, for use in detection protocols) is a relatively straightforward matter. . For example, the method of Hopp can be used as allowed in U.S. Pat.No. 4,554,101, incorporated herein by reference, which teaches the identification and production of epitopes from amino acid sequences based on hydrophilicity. I have. The amino acid sequence of these "epitope core sequences"
It can then be easily incorporated into the peptide either by application of peptide synthesis or by recombinant techniques.

[0135] The term "plasmapheresis" refers to the technology of removing blood from an animal, separating it into its cellular and plasma components, and then returning the cells to the animal to retain the plasma. Describe. Large volumes of plasmapheresis require that the removed plasma be replaced by a suitable fluid, and when doing so, the technique is often known as plasmapheresis. Any components found in plasma can be removed by plasma exchange. Plasmapheresis is a method still used in most blood banks and public blood donation centers in the United States. Such extracted plasma for commercial use can be used in a preferred embodiment of the present invention.

During plasma donation, it is necessary to replenish the collected blood fluid to prevent circulatory collapse. Under most circumstances, the osmotic action of plasma requires replenishment. A 5% solution of human albumin obtained from donor blood is a safe and effective supplement.
It is standard practice in the medical community to add 2 ml of KCl solution and 2 ml of 10% calcium gluconate solution to albumin. Most plasmapheresis units replenish 1.5 liters of human albumin solution and 0.5 liters of normal saline for every 2 liters of plasma removed.

The currently used methods for plasma separation are centrifugation and filtration. If this is not commercially available, the donor plasma pool can be prepared using the techniques of US Patent No. 5,548,066, which is incorporated herein by reference. First, a plurality of blood donors are identified. These donors are mature mammals, typically mammals of the same species using serum. When trying to treat or prevent a particular disease, such as mastitis or another disease caused by staphylococcal bacteria, such as Staphylococcus aureus, in a mammal, the microorganism that naturally exposes or causes the donor Or exposure to immunization against an antigenic portion thereof. Furthermore,
To achieve a constant serum product, the donor population is preferably relatively large. Preferably, the donor plasma pool is produced using a human host. Once the donor has been identified, blood is drawn from the donor. Since serum is purified directly from blood, it is thus desirable to obtain the maximum amount of blood to obtain the maximum amount of serum. For humans,
An established limited amount of blood is drawn periodically over a long period of time.

It is preferred to identify and maintain a group of donors by repeated withdrawal of blood in small volumes, for example, once a month from humans. The frequency of withdrawal will of course affect the amount that can be safely withdrawn. In general, it is desirable to draw a maximum amount of blood over a time setting that is not detrimental to the donor's health. This may indicate, depending on the particular species, whether to extract a small amount at a high frequency or to extract the maximum amount possible at a reduced frequency. Donor blood volume can be estimated by standard formulas available from the Centers for Disease Control.

If long-term voting is desired, of course, the health of the donor is taken into account in this process. Prior to donation, the donor can check for general good health and, if the donor is unhealthy, postpone drawing blood until the next scheduled date. Beyond this period, it is preferable to maintain a long-term health record, preferably including more detailed information. In this regard, the production of this serum is considered to be a good indicator of donor health.

Typically, serum is separated from donor blood, which consists of material from the immune system. In one method, a detailed record is kept of the amount of serum generated from the blood as a percentage yield, for example, 7 l serum from 14 l blood gives a 50% yield. In a preferred method, a record of percent yield is kept for each donor for each blood draw. These percentages can then be used to determine whether the donor should draw blood at the next scheduled time. In particular, if the action to be taken is expressed as a function of yield, percentage, the guideline can be expressed as: yield percentage <1 = 30% rest; 31-35% caution; 36-59% normal; ~ 64% caution;> 1 = 65% rest. Clearly, if the serum yield is above or below the normal range, the donor will not draw blood. Such yield rates can indicate underlying disease. Depending on the donor's medical history and / or further tests, the subject may draw blood at the cautionary and possibly reduced volume. In this regard, it has been found that a small percentage of individuals constantly yield about 60-62% yield.

[0141] Methods of blood and plasma collection are generally standard and well known to those skilled in the art. Any method that achieves the desired result can be used. Once the blood has been collected, it is subjected to a technique for extracting the desired components. The first critical step in this process is to allow each container of blood collected, usually for one hour, to sit at room temperature, at least until substantial clotting has occurred. During this period, blood transitions from body temperature to room temperature and is exposed to air. This exposure to the atmosphere changes fibrinogen into fibrin, causing blood clotting.

This clotting period is an important aspect of serum recovery. Coagulation provides a coarse separation of cellular material from liquid. In addition, although the exact mechanism is not known, the clotting period kills leukocytes and, for a certain percentage of such cells, ruptures or destroys the scientific material therein, including antibodies, so that they are released from the leukocytes. It seems to collapse. It is believed that this material remains in the serum and acts to provide "information" to the immune system of the serum recipient. This "information" can be used to identify the recipient in such a way that the recipient's leukocytes respond quickly and in a manner similar to a vaccinated or immunized subject, as well as to provide the leukocytes with microbial memory. Can help to "program" leukocytes against different microorganisms.

This non-refrigerated period also results in a coarse filtration of the collected blood. In particular, clotted blood with relatively substantial red blood cells descends toward the bottom of the container, while liquid plasma, immunoglobulins and chemicals are pumped toward the top. To assist in this method, and to assist in the method described below, the collection vessel is preferably tall and narrow with a similar balance as a standard test tube.

The liquid portion obtained at this stage is a coarse serum, which can be immunized after filtration and sterilization. Further steps are optionally performed. However, in order to increase the yield, various alternative steps before filtration are preferred.

After having sufficient time for the collected blood to clot, the first of these steps is roughly
Cooling to 20-60 ° C. This cooling reduces the temperature of the blood from room temperature to the cooling temperature. Such cooling, of course, prevents the growth of bacteria, mold and the like. Furthermore, during this cooling, the clotted blood further sediments and the clotted blood contracts. This shrinkage (and possibly cooling) can rupture a further percentage of leukocytes. In addition, the contraction of the clotted blood helps to express the immunoglobulins and chemical materials trapped therein from the clot. This cooling should last until the blood reaches the cooling temperature and preferably for at least about 14-18 hours or at least one part.

[0146] The second preferred step is physical pressing of the clotted blood. This pressing is likely to cause even more rupture of the leukocytes, thus producing more transfer factors. In addition, pressing helps to push immunoglobulins and transfer factors out of the clot, in a manner similar to cold shrinkage.

[0147] A preferred method of pressing is to insert a sterile bolus into a cooled container of collected blood. For example, a cylinder with a snug glass slide is mounted in a container and a roughly two pound stake is installed. As imagined, when the clotted blood settles to the bottom of the container, the liquid material flows around the cylinder until the cylinder rests. It is preferred that the pressing be maintained in place for about 6 to 24 hours.

It is recognized that pressing can also serve as the first effective filtration step. Although a precise fit of the limestone is not required in the container, a close fit of the limestone helps to separate the liquid coarse serum upwards and the solid material downwards. This can serve as a first coarse filtration step and can therefore be conveniently used to determine the amount of crude serum generated for the calculation of the percentage yield. In particular, looking at the column height of the crude serum and knowing the diameter of the container provides the volume of crude serum produced.

At this point, the appropriate filtration process begins. This further processing involves filtration to remove all cellular material. This filtration is accomplished in multiple steps. The first filtration is a total filtration. This can be easily achieved by forcing the contents of the container into a collection vat while holding the screen over the opening of the collection container. If a high yield step of cooling and pressing is used, the pressing cylinder still in the vessel can serve to filter with the screen, and the screen can mainly filter out the cylinder itself. If these high yield steps were not employed, finer filter screens may be desirable. The coagulated cells remaining in the container are properly discarded and the container is sterilized for subsequent use.

This is a preferred point to combine serum from various donors.

However, it is recognized that samples from multiple donors can be combined at any point after the first overall filtration step.

Crude serum still contains large amounts of cells and cell debris. For the next filtration step, the reconstituted liquid is then placed in a continuous flow centrifuge. For example, the liquid is placed in a Sharples AS16NF continuous flow centrifuge operating at approximately 13,000-15,000 rpm. Fluid is drawn off during this process, thus removing even more cells and cellular contaminants.

Following isolation of the plasma, the antibodies are purified from other cellular products. This can be accomplished by various protein isolation methods known to those skilled in the art of immunoglobulin purification, such as ion exchange, affinity purification, and the like. Means for producing and characterizing antibodies are well known in the art. For example, a serum sample can be passed over a Protein A or Protein G Sepharose column that binds IgG (depending on the isotype). The bound antibody is then eluted, for example, with a citrate buffer at pH 5.0. The eluted fraction containing Abs is dialyzed against an isotonic buffer. Alternatively, the eluate is passed over an anti-immunoglobulin-Sepharose column. The Ab is then eluted with 3.5M magnesium chloride. Abs purified in this way is then subjected, for example, to an isotype-specific ELISA.
The binding activity can be tested by the method and immunofluorescence assay of the target cells.

[0154] In another embodiment, the liquid is instead subjected to another filtration step. This separate step actually consists of several sub-steps, in which the liquid is passed progressively through several filters of fine gauge. In particular, the liquid passes through at least a 0.65 micron filter, then a 0.2 micron nominal filter and then a 0.2 micron absolute filter. By first passing the liquid through a 0.2 micron nominal filter, most of the bacteria, mold and fibrin are removed before passing through the 0.2 micron absolute filter.

At this point, the liquid has removed essentially all of the solid cellular material. However, chemical material and immunoglobulin remain in the liquid, which liquid is described as clarified serum.

The clarified serum may be used as the final serum (after sterilization described below), however, it is preferred to concentrate the clarified serum. This concentration reduces the volume and thus the amount of material that must be transported. In addition, some receptors, such as infant mammals, cannot accept large amounts of intravenous medication due to lack of capacity. Until then, the enrichment may allow the entire dose of serum to be administered. Concentration is preferably performed by repeated ultrafiltration to remove water molecules, as is known in the art. Such filtrations have molecular weight cut-off filters of 10,000-100,000. During this process, a sample of the clarified serum can be used to determine whether the serum has been sufficiently concentrated. Preferably, the final serum is concentrated about 2-6 times the clarified serum, most preferably 2-4 times.

The measurement of the concentration concentration (level) is performed by examining the amount of IgG (or another immunoglobulin) in the serum. Initial tests can be performed on clarified sera, and the results can be compared to tests performed on sera during the ultrafiltration process. For example, if the first test yields a clarified serum having an IgG concentration of 1 g / 100 ml, then a subsequent test will require about 2-6 g / 100 ml, preferably about 3 g / 100 ml.
The concentration process can be stopped when reporting the IgG concentration. The measurement of the amount of IgG can be performed by a radial immunodiffusion test. However, it is preferred that serum protein electrophoresis be performed on the whole serum to obtain an overall gamma-globulin fight. This appears to be more accurate and gives a clear indication of IgG levels. Once the enrichment process is complete, the concentrated sterile serum is bottled or packaged by standard methods.

When the concentration and packaging process is completed, the result is a non-sterile serum. The next step is to sterilize the serum. It is important that the sterilization is performed, but that the sterile serum is not denatured. For sterilization without denaturation, the sterile serum is frozen to severe freezing conditions. For sterile serum, this is approximately -29 ° C (-21 ° F).
While still frozen, the serum material is then subjected to sufficient gamma irradiation to kill it but not denature it. This illumination level can vary among various material species, but can be measured without undue experimentation. It is important that the serum material be sufficiently cooled (severely frozen) so that the serum material remains frozen during the irradiation step, otherwise denaturation will occur. It is for this reason that the serum material is frozen at a relatively low temperature. If the irradiation process is short enough or produces cooling during irradiation, then it has been found that higher temperatures (albeit still below freezing) are acceptable.

At this point, the final serum, although frozen, was obtained. Thus, if the serum packages are placed refrigerated and they are stored until use, they can be thawed to refrigerated temperatures.

After administration, the serum is found to produce a cellular immunity similar to the vaccine and can be used with or without the introduction of toxicants. In general, the sera of the present invention should provide protection against bacteria against which the donor population is immune. A variety of vaccination applications are possible in humans, including general vaccination of individuals with impaired immunity, such as that caused by diabetes, and vaccination of individuals who are ready to undergo surgery by in-hospital trunk. In addition to humans, the sera of the present invention have uses for a large number of mammals, for example, farm mammals and domestic mammals and humans. For livestock, there is one particular use in avoiding mastitis in cattle, a common disease that costs the dairy industry millions of dollars per year.

XMSCRAMM and Uses for Antibody Compositions The immunization products of the present invention are purified and concentrated extracts of plasma or serum from a purified donor pool. Serum contains antibodies released from leukocytes in the extracted blood and, optionally, other chemicals present in the extracted blood. This serum would provide information that is "read" by the recipient's immune system to confer an extended period of immunity, typically on the order of six to eight weeks. The purified donor plasma pool may be used for treating wounds, blocking protein receptors or for immunization (vaccination).

The plasma pool interferes with the initial physical interaction between the pathogen and the mammalian host underlying the infection, eg, against mammalian extracellular matrix proteins on an underlying device. Or an antibody useful for preventing the adhesion of bacteria, particularly Gram-positive bacteria, to extracellular matrix proteins in the wound, wherein the antibody inhibits protein-mediated mammalian cell entry; To prevent bacterial adhesion between the extra-stromal protein and the bacterial protein that mediates tissue damage; and to prevent the normal progression of the onset of infection that is initiated other than by implantation of an indwelling device or surgical technique , Contains.

In general, both polyclonal and monoclonal antibodies to MSCRAMM peptides can be used in various embodiments. For example, the antibodies can be used in antibody cloning protocols to obtain cDNAs or genes encoding the peptides or related proteins discussed herein. The antibodies can also be used in inhibition studies to analyze the effects of MSCRAMM-derived peptides in cells or animals. Anti-MSCRAMM epitope antibodies also analyze the distribution of MSCRAMMs in various cellular events. For example, it is useful for immunolocalization studies to determine the cell-specific or tissue-specific distribution of MSCRAMM peptides under various physiological conditions. A particularly useful use of such antibodies is in purifying natural or recombinant MSCRAMMS, for example, using an antibody affinity column. The operation of all such immunization techniques is known to those of skill in the art in light of the present disclosure.

[0164] Immune compositions, including vaccines, and other pharmaceutical compositions containing plasma concentrates of selected donor pools are included within the scope of the invention. The combination of immunoglobulins against the binding protein or active or antigenic fragment thereof or fusion protein thereof can be disposed of and packaged, alone or in combination with other antibodies, using methods and materials known to those skilled in the art of vaccines. it can. The immune response can be used therapeutically or prophylactically and can provide passive immunity.

XI Preparation of Proteins and Antibodies One of skill in the art of preparing the protein, peptide and antibody compositions described herein can employ conventional molecular biology, microbiology and recombinant DNA techniques. Such techniques are explained fully in the literature. For example, Sanbrook et al.
"Molecular cloning; A Laboratory Manual"(1989);"Current Protocols in
Molecular Biology, Volumes I-III (Ausubel, RQ-1 ed., 1994); Cell Biology;
"A Laboratory Handbook", Volumes I-III (edited by JE celis, 1994); "Current Protocols in Tommunslogy" Volumes I-III (edited by coligan, JE, 1994); "Oligonucleotide Synthesis" (edited by MJ Gait, 1984); "Nucleic Acid Hybridization" ( BDHames & SJHiggins eds. 1985); “Transcription
And Translation '' (B, D, Hames & S, J, Higgins ed., 1984); `` Animal Cell Cul
ture "(RI Freshney ed. 1986);" Immobilized Cells And Enzymes "(IRL
Press, 1986); B. Perbal, "A Practical Guide To Molecular Cloning" (1984
)reference.

Antibodies obtained according to the present invention can be directly labeled with a detectable label for the identification and quantification of staphylococcal bacteria, such as Staphylococcus aureus, Staphylococcus epidermidis and the like. Labels for use in immunoassays are generally known to those of skill in the art and include oxygen, radioisotopes,
And chromogenic substances, including fluorescent substances, luminescent substances, and colored particles such as colloidal gold and latex beads. Enzyme-linked immunosorbent assay for appropriate immunoassay
(ELISA) method.

Alternatively, an antibody can be labeled indirectly by reaction with a labeled substance having affinity for immunoglobulins, such as protein A or G or another antibody. The antibody can be conjugated to the second substance, and can be detected with a labeled third substance having affinity for the second substance conjugated to the antibody. For example, antibodies can be conjugated to biotin and antibody-biotin conjugates can be detected using labeled avidin or streptavidin. Similarly, antibodies can be conjugated to haplan and antibody-hapten complexes can be detected using labeled anti-hapten antibodies. These and other methods of labeling antibodies and assay complexes are well known to those of skill in the art. Antibodies to the binding protein may also be used in production plants or laboratories to isolate additional amounts of the protein, such as by affinity chromatography.

In another identification embodiment, one of the target cells is bound to each well using a microliter plate pre-treated with poly-L-lysine, and the cells are then treated with, for example, 1% glutar. The antibodies are fixed using aldehydes and the antibodies are tested for their ability to bind whole cells. In addition, FACS, immunofluorescent staining, idiotype-specific antibodies, antigen binding competition assays, and other methods commonly used in antibody characterization techniques can be used in conjunction with the present invention to identify preferred donors.

A humanized antibody is an antibody of animal origin that has been modified using genetic engineering techniques to replace certain regions and / or variable framework sequences with human sequences while retaining the original antigen specificity. It is.

Such antibodies are commonly derived from rodent antibodies having specificity for a human antigen. Such antibodies are generally useful for in vivo therapeutic applications. This strategy can reduce the host's response to foreign antibodies and allow for the selection of human effector functions.

Techniques for producing humanized immunoglobulins are well known to those skilled in the art. For example,
US Patent No. 5,693,762 discloses one or more complementary determining regions (CDRs).
A method for producing a humanized immunoglobulin having the formula: and the composition of the humanized immunoglobulin are disclosed. When combined with a whole antibody, the humanized immunoglobulin is substantially non-immunogenic in humans, and is substantially similar to the donor immunoglobulin for antigens, such as proteins or other compounds containing epitopes. Retain the same affinity. Another U.S. patent, each of which is incorporated herein by reference, which teaches the production of antibodies useful in the present invention, includes U.S. Pat.No. 5,565,332, which describes the production of chimeric antibodies using combinatorial techniques. There are US Patent No. 4,816,567 describing recombinant immunoglobulin preparations and US Patent No. 4,867,973 describing antibody-therapeutic agent conjugates.

US Pat. No. 5,565,332 describes a method for producing an antibody or antibody fragment that has the same binding specificity as a parent antibody, but has enhanced human characteristics. Humanized antibodies can be obtained by chain mixing, possibly using phage display techniques, and as long as such methods are useful in the present invention, U.S. Patent No. 5,565,332 is hereby incorporated by reference in its entirety. .

XII Production of High-Titer MSCRAMM-Specific IgG from Biological Fluids via Affinity Purification In accordance with the present invention, affinity for producing high-titer MSCRAMM-specific immunoglobulins from biological fluids such as blood or plasma Modes of isolation and purification can also be utilized. In a preferred mode of this aspect of the invention, a recombinant or wild type / native MS
CRAMMs can be covalently linked to a substrate or resin such as Sepharose® or agarose to form an affinity matrix (affinity substrate). Antibodies can be selectively isolated from serum, plasma or other biological fluids using MSCRAMM affinity substrates. In a preferred embodiment, the biological fluid is passed over an MSCRAMM affinity substrate, which is then washed to remove non-specifically bound antibodies. The washed substrate is then subjected to conditions such as a low pH value or a high salt concentration such that the MSCRAMM specific antibody remaining on the substrate is eluted. The anti-MSCRAMM titer of the eluted material is much higher than that of the original biological fluid, and the eluted material can then be used in the same manner as other donor-selected or donor-stimulated compositions of the present invention.

XIII Pharmaceutical Compositions MSCRAMM Pharmaceutical compositions for immunizing a donor containing a protein, nucleic acid molecule, antibody or fragment thereof include a pharmaceutical carrier such as saline, dextrose,
It can be formulated in combination with water, glycerol, ethanol, other therapeutic compositions and combinations thereof. The formulation should suit the mode of administration. Suitable administration methods include, but are not limited to, oral, anal, intracavitary, intravenous, intraperitoneal, intramuscular,
There are subcutaneous, intranasal and intradermal administrations. The preferred mode is by intravenous administration.

A pharmaceutical composition for treating any of the diseases described herein should contain, in a pharmaceutically acceptable excipient, an effective amount of an immunoglobulin to treat or prevent the target disease. is there.

Compositions containing immunoglobulins as active ingredients are well understood in the art.
Typically, such compositions are prepared as injectables, either as liquid solutions or suspensions, but may also be prepared in solid forms suitable for solution or suspension in liquid prior to injection. The formulation can also be emulsified. The therapeutically active ingredient is often mixed with excipients that are pharmaceutically acceptable and compatible with the active ingredient. Therapeutic donor immunoglobulin pool compositions are usually administered intravenously, for example, by injection of a unit dose. The term "unit dosage" when used in reference to the therapeutic compositions of the invention describes physically discrete units, suitably as unit dosages for humans, each unit being combined with the required diluent or carrier or vehicle. Contain the predetermined amount of active ingredient calculated to produce the desired therapeutic effect.

The compositions are administered in a manner compatible with the dosage formulation, and in a therapeutically effective amount. The amount to be administered depends on the ability of the subject's immune system to utilize the subject active ingredient to be treated and the degree of suppression or neutralization of the desired MSCRAMM binding ability. Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner and are peculiar to each individual. However, a suitable dosage is about 0.1-20 mg / kg of the individual's body weight per day,
Preferably about 0.5 to about 10 mg, more preferably 1 to several mg of active ingredient,
Moreover, it depends on the mode of administration. First dose and booster shots
The regimen suitable for is also variable, but is typified by the first administration at one or more intervals by subsequent injections or another administration and subsequent repeated dosing. Alternatively, continuous intravenous infusion sufficient to maintain a concentration of 10 nanomolar to 10 micromolar in the blood is envisioned.

Immune and other pharmaceutical compositions such as vaccines can be used alone or protect against human and animal infections caused by staphylococcal and other bacteria, including Staphylococcus aureus. May be used in combination with another inhibitor. In particular, the compositions can be used to protect humans against endocarditis or humans or ruminants against mastitis caused by staphylococcal infection. Vaccines can also be used to protect dogs and horses against similar staphylococcal infections. To enhance immunogenicity, the plasma pool concentrate protein of the donor can be conjugated to a carrier molecule. Suitable immunogenic carriers include proteins, polypeptides or peptides such as albumin, hemocyanin, thyroglobulin and derivatives thereof, especially bovine serum albumin (BSA) and keyhole limpet hemocyanin (KLH), polysaccharides, carbohydrates, polymers and solids There is a phase. Other protein inducers or non-protein inducers are known to those skilled in the art. The immunogenic carrier typically has a molecular weight of at least 1,000 daltons, preferably
It has a molecular weight of 00 Dalton or more. Carrier molecules often contain reactive groups that promote covalent attachment to the hapten. The carboxylic acid or amine groups of amino acids or the sugar groups of glycoproteins are used in this manner. Carriers lacking such groups are often capable of reacting with a suitable agent to produce such groups. Preferably, the immune response is an immunogen, wherein the immunogen is identified as a product such as mice, rabbits, rats, goats, sheep, guinea pigs,
Occurs when injected into chickens and other animals, most preferably mice and rabbits. Alternatively, multiple antigenic peptides containing multiple copies of the protein or polypeptide or polypeptides on antigen or immunologically equivalent are sufficiently antigenic to improve immunogenicity without the use of a carrier. possible.

In a preferred embodiment, the donor-stimulated vaccine is packaged for immunization by parenteral (ie, intramuscular, intradermal or subcutaneous) administration or by nasopharyngeal (ie, intranasal) administration. Most preferably, the vaccine is injected intramuscularly into the deltoid muscle. Preferably, the vaccine is combined with a pharmaceutically acceptable carrier to facilitate administration. The preferred carrier is usually water or buffered saline, with or without preservatives. The vaccine may be lyophilized for resuspension or dissolution upon administration.

The carrier to which the protein can be conjugated can be a polymeric delayed release system. Synthetic polymers are particularly useful in vaccine formulations to modulate the release of antigen. For example, polymerizing methyl methacrylate into spheres less than 1 micron in diameter is known as Krente
r, J, `` Microcapsules and Nanoparticles in Medicine and Pharmacology '' M.
Donbrow, CRC Press, p125-148.

The amount of the immunogen composition used to produce the polyclonal antibody will be distributed depending on the nature of the immunogen and the animal used for immunization. Preferred dosage for human administration is 0.01 mg
/ Kg to 10 mg / kg, preferably approximately 1 mg / kg. Based on this range, for heavier weights, an equivalent dosage can be determined. The dosage should be adjusted to be compatible with the individual receiving the immunogenic composition, and will vary with the age, weight and metabolism of the individual. The vaccine may further contain a stabilizer such as thimerosal [ethyl (2-methylcaptobenzoate-S) mercury sodium salt] (Sigma Chemical Co., St. Louis) or a physiologically acceptable preservative.

The production of polyclonal can be monitored by sampling the blood of the immunized animal at various time points following immunization. A second booster injection may also be given. The augmenting and titrating home is repeated until a suitable titer is obtained. The process can be sustained when the desired level of immunogenicity is obtained.

[0183] The immunogenic composition preferably further comprises auxiliaries. Numerous auxiliaries are known for use in vaccination of animals and are readily adapted to this composition. at this point,
The only auxiliaries widely used in humans were alum (aluminum phosphate or aluminum hydroxide). Saponin and its purified component Quil A. Freund (Freun
The complete auxiliaries of d) and other auxiliaries used for research and veterinary use have toxicities that limit their potential use in human vaccines.

The isolated peptide can be linked to a selected amino acid sequence to form a fusion protein. By way of example, and not limitation, the fusion protein may comprise at least a first peptide of a fibronectin binding region of a fibronectin binding protein that is substantially bound to a selected amino acid sequence, provided that the first peptide specifically binds to fibronectin. (Not bonded). In a preferred embodiment, the first peptide binds to a selected carrier molecule or amino acid sequence, including but not limited to keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA).

One of the key features provided by the donor stimulation embodiments of the present invention is a polyclonal serum that is relatively homogeneous with respect to the specificity of the antibodies in the serum. Typically,
Polygonal antisera is derived from a variety of different "clones", i.e., different lineages of B-cells. In contrast, monoclonal antibodies are defined as coming from antibody-producing cells that have a common B-cell ancestry and therefore have their "mono" clonality.

When using peptides as antigens to stimulate the production of polyclonal sera,
A much smaller change in the clonal properties of the serum than with the whole antigen is expected. Unfortunately, if they display incomplete fragments of the epitope, the peptide is well inferred to have a multiple (and possibly non-natural) conformation. As a result, even short peptides produce polyclonal antisera with relatively multiple specificities, and unfortunately produce antisera that either do not react with natural molecules or react poorly.

A polyclonal antiserum according to the present invention is produced against a peptide predicted to contain the entire intact epitope. It is therefore likely that these epitopes are more stable in an immunological sense and thus express a more constant immunological target to the immune system. Under this model, the number of potential B-cell clones responding to this peptide is fairly small and therefore the homogeneity of the resulting serum is higher. In various embodiments, the present invention provides polyclonal antisera in which the clonality, ie, the percentage of clones that react with the same molecular determinant, is at least 80%. Higher clonality, ie, 90%, 95% or more, is envisioned.

XIV Kits (KITS) The present invention also includes kits for identifying blood or plasma having high titers of a desired antibody. Preferred kits contain enough antigen to bind substantially all of the antibody in the sample in about 10 minutes or less, or contain enough antibody to target antibodies in the sample to be detected. . The antigens or antibodies in the kit, e.g., MSCRAMMs or any of their binding regions as described above, are preferably immobilized on a solid support, and are labeled with a detection agent as described above or commonly known in the art. obtain.
The kit optionally contains means for detecting the detection agent. If the antigen or antibody in the kit is labeled with a fluorescent dye or radiolabel, the means for detecting the detection agent is typically because the user is expected to have a suitable spectrophotometer, scintillation counter or microscope. It is not provided. If the detection agent is an enzyme, the kit can be provided with a means for detecting the detection agent, typically an antigen-
It contains a substrate for the enzyme in an amount sufficient to detect all of the antibody complexes.
One preferred means of detecting the detection agent is a substrate that is converted to a colored product by an enzyme. A common example is the use of the enzyme horseradish peroxidase with 2,2'-azino-di [3-ethyl-benzothiazoline sulfonate] (ABTS).

The invention encompasses a method of detecting a biological sample with an increased titer of antibodies against selected staphylococcal MSCRAMMs. A biological sample of the term as used herein,
Tissue or body fluid samples isolated from a host, typically a human, including but not limited to plasma or serum, are described. It is a simple matter to confirm that factors in the donor plasma are immunologically cross-reactive with one or more epitopes of the disclosed peptides. This is a sort that can be easily measured using, for example, a specific assay for a single proposed epitope sequence, or using, for example, a more general screen of a pool of randomly generated synthetic peptides or protein fragments. Screening assays can be used to identify either equivalent antigens or cross-reactive antibodies. In each case, the principle is the same, ie, based on binding site competition between the antibody and the antigen.

Any test that measures the binding of an antigen to an antibody can be used to assess antigen or antibody levels in a biological sample of a host according to the invention. Many such alternative methods are known and commonly used in industry.

Immunocytochemistry and immunohistochemistry are techniques that use antibodies to identify antigens on the surface of lysed cells or on tissue parts, respectively. Individual cell populations are quantified by surface markers using immunocytochemistry. Specific cell populations or antigens are localized using immunohistochemistry. These techniques are also used to identify autoantibodies, using tissues or cells containing the putative autoantigen as substrates. Antibodies are usually identified using an enzyme-conjugated antibody to the original antibody, followed by a chromogen that deposits an insoluble colored end product on cells or tissues.

Another common method of evaluation is a radioimmunoassay in which an antigen or antigen is detected using a radiolabeled reactant. Antibodies can be detected using plates sensitized with the antigen. The test antibody is applied, and detection is performed by adding a radiolabeled ligand specific to the antibody. The amount of ligand bound to the plate is proportional to the amount of test antibody. This test can be reversed to a test for antigen. Variants of the radioimmunoassay are competitive RIA, direct binding RIA, capture RIA, sandwich RIA and immunoradiometric assay (RAM).

Enzyme-linked immunosorbent assay (ELSA) is a widely used panel of techniques for detecting antigens and antibodies. The principle is similar to that of a radioimmunoassay, except that the enzyme is more complex to the detection system than the radioactive molecule. Representative enzymes used are peroxidase, alkaline phosphatase and 2-galactosidase. These can be used to produce a colored reaction product from a colorless substrate. The color density is proportional to the amount of reactant under study. These assays are more convenient than RIA, but are less sensitizing.

The unknown proteins are characterized using Western blotting (immunoblot). The components of the biological sample are separated by gel electrophoresis. SDS gels separate by molecular weight, and IEF gels separate samples by charge characteristics. Separated proteins were transferred to membranes (blotted) and identified by immunocytochemistry.

[0195] Appropriate evaluation methods, which are often used less often, include the following methods: Farn
Assay (radiolabeled ligand binds to and detects dissolved specific antibody, ligand is precipitated and quantified); Precipitin reaction (antibody and antigen are cross-linked in a large grid Forms an insoluble immune complex; only works if antigens and antibodies are present in sufficient quantities near the equivalents and only when there are sufficient epitopes available to form the lattice); (The immune complex formed in the solution is measured by the ability of the immune complex to scatter light.); Immunodiffusion (detects antigens and antibodies in agar gel); Countercurrent electrophoresis (immunodiffusion As above, but using current to drive antibodies and antigens away from each other; useful for low concentrations of antigens and antibodies); Single Radial Immunodiffusion (SRID) (antigen containing antibodies outward from pits) Diffused in the gel Quantifying the antigen by Rukoto; The technique can be reversed by diffusing unknown antibody solutions into recess containing antigen); rocket electrophoresis (SR
Similar to ID, except that the test antigen is transferred into the gel by an electric field); and immunofluorescence (similar to immunochemistry, but using fluorescence rather than enzyme conjugate). The antibody used to contact the sample of the body fluid is preferably immobilized on a solid substrate. Antibodies can be immobilized using various means, as described in "Antibodies: A Laboratory Manual" (described above). Suitable solid substrates include sticks, synthetic glass, agarose beads, cups, flat packs or substrates having a membrane or coating layer carried or bonded to another solid support. Other solid substrates include cell culture plates, ELISA plates, tubes and polymer membranes.

Means for labeling antibodies with a detection agent are also described in “Antibodies; A Laboratory Mannal”. The amount of antigen in a biological sample of a host can be determined by any means that involves the assay of choice. For example, a selected immunoassay can be performed on a known increasing amount of antigen to generate a standard curve or color diagram, and then the amount of test antigen can be determined using the standard curve or the amount of antigen-antibody complex. It can be determined by comparing the results of the test on the color diagram with known amounts of antigen. The amount of antigen determined to be present in a biological sample of a host can be used to assess a patient's health in a number of ways. First, the level of antigen is compared to a population criterion based on statistical data. Second, the level of antigen may be considered from the patient's own history of antigen levels.

The kit may optionally include a lysing agent (lys) to lyse cells present in the retired sample.
ing agents). Suitable lysing agents include surfactants such as Tween.
) -80, Nonidet P40 and Triton x-100. Preferably, the lysing agent is immobilized on the solid support together with the antibody.

The kit may optionally contain various concentrations of the ready-made antigen to calibrate the assay. The kit can further contain a visual or numerical presentation of the amount of antigen in a standard assay calibrated for control purposes. For example, if an assay that produces a colored product is used, a sheet may be included that provides a depiction of increasing concentrations that engage different amounts of antigen.

The kit can optionally contain two antibodies in the detection system. The first antibody, present in small amounts, is specific for the antigen being assayed. The first antibody is detected using the second antibody provided in a large amount. For example, rabbit antibodies can be used to detect LOOH / amine antigens, and then anti-rabbit IgG antibodies can be used to detect bound rabbit antibodies. Goat antibodies and anti-antibodies are also commonly used.

As one non-limiting example, a rabbit antibody specific for the desired antibody, an anti-rabbit IgG antibody in an amount sufficient to detect the bound first antibody, a second antibody,
A kit for detecting a lipid peroxidation state of a patient, comprising an enzyme conjugated to the above antibody and a substrate for the enzyme that changes color when exposed to the enzyme.

[0201]

【Example】

The following examples are included to illustrate preferred embodiments of the present invention. It should be understood by those skilled in the art that the technology disclosed in the following examples represents the technology found by the present inventor to function sufficiently for the practice of the present invention, and thus forms a preferred mode of the practice of the present invention. it is conceivable that. However, it will be apparent to one skilled in the art, in light of the present disclosure, that numerous changes may be made in the particular embodiment disclosed and still achieve similar results without departing from the scope of the invention.

EXAMPLE 1 Preparation of a Prototype Fourth Generation MSCRAMM Vaccine A series of recombinant proteins representing regions from collagen as previously described,
Fn and Fbg-linked MSCRAMMs (see Figure 1) were overexpressed in E. cali and affinity purified by metal chelation chromatography (e.g.,
Joh et al., Biochemistny, 33 (20); 6086-6092, 1994; Patt et al., J. Biol. Chem, 270, 1200.
5-12011, 1995; McDevitt et al., Mol, Micro, 11 (2); 237-248, 1994; Ni Eidhin et al.
Infect, Immun, Submitted, 1998), the following amino acids were used; amino acids contained in recombinant collagen-bound MSCRAMM expressed from CNA (M55, for example, according to Applicants' application incorporated herein by reference). Amino acids contained in recombinant fibrinogen-conjugated MSCRAMM expressed from clfA (region A, e.g., US patent application Ser. No. 08 / 856,253, incorporated herein by reference). 293
728); recombinant fibrinogen-conjugated MSCRAMM expressed from clfB
(Region A, for example, disclosed in US patent application Ser. No. 09 / 200,650, incorporated herein by reference); and recombinant fibronectin-conjugated MS.
Amino acids contained in CRAMM (disclosed in DUD4, eg, Ser. No. 09 / 010,317, filed by the present applicant and incorporated by reference herein). Recombinant FN
The bound MSCRAMM protein DUD4 converts this to the region A and cl from M55, clfA.
Treated with formalin (5% formalin overnight at 4 ° C.) before combining with region B from fB.

Example 2 Growth of an Escherichia coli Strain for Production of Recombinant Protein Herboring a Recombinant Plasmid An overnight culture of E. coli JM101 or TOP3 cells (Stratagene) contains 50 mg / mL ampicillin. Luria gravy
1:50 dilution with 1 L of (Lntia Broth) (Gibco BRL). Cells of E. coli were grown until the culture reached oD ₆₀₀ of 0.5 to 0.8. Expression of the recombinant protein was induced by adding IPTG to a final concentration of 0.2 mM. After a 3 hour induction period, cells were harvested by centrifugation, resuspended in 15 mL of buffer A (5 mM imidazole, 0.5 M Nacl, 20 mM Tris-HCl, pH 7.9) and 20,000 lb / in ² 2 in 2
Disintegrated by passing through a French press twice. Cell debris at 50,000 xg 10
The mixture was removed by centrifugation for one minute, and the supernatant was passed through a 0.45 μM filter.

Example 3 pQE-30 (Qiagen (registered trademark): Qiagen) or PV-4 substrate
Purification of HIS _6- containing recombinant protein expressed from the recombinant plasmid was immobilized using an iminodiacetic acid / Sepharose (registered trademark) 6B Fast Flow column (Sigma) loaded with Ni ^2+. (Porath et al. 1975; Hochuli et al. (1988). Proteins labeled with HIS ₆ were purified by immobilized metal chelate affinity chromatography. More specifically, EPLCC® A column containing Iminodiacetate / Sepharose 6B FF, which had been disinfected in a system (Pharmacia), was packed with 150 mM Ni ⁺⁺ and buffered with buffer A (5 mM imidazole, 0.5 M NaCl, 20 mM Tris, pH 7.9). After equilibration, the supernatant of the bacteria was applied to the column, and the column was filled with 10 beds (
Washed with bed) volume of buffer A. Subsequently, the column was eluted with buffer B (200 mM imidazole, 0.5 M NaCl, 20 mM Tris, pH 7.9). The eluate was monitored for protein by absorbance at 280 nm and peak horn was analyzed by SDS-PAGE. Endotoxin was removed from purified recombinant protein by detergent extraction with 1% Triton x-114, followed by metal chelate affinity chromatography and by passage over a polymyxin B-Sepharose column. Endotoxin levels are determined by the chromogen Limulus Amebocyte Lysate
) (Bio Whittaker) assay.

Example 4 Quaternary MSCRAMM Vaccine-Animal immunized with MSCRAMM IV Akagegal; 100 μg of M55 (1 EU / mg), ClfA (2.5 EU / mg), ClfB (<1.0 EU / mg) and DUD4 (<10 EU / mg)
EU / mg) were mixed together to form the MSCRAMM IV vaccine. The compound was mixed with TiterMax® Gold (CytRX, Norcross) in a 1: 1 ratio. Two female rhesus monkeys, ID No. 495Z & 664U (-9.4 kg) were vaccinated intramuscularly (IM) in the posterior quadriceps with 200 μl of the vaccine. Twenty-eight days later, two rhesus monkeys were injected with 200 μl of the same vaccine formulation 1M to enhance the efficacy of the vaccine. Two additional female monkeys, ID No. 215W & 203U (~ 8.00 kg), were immunized with MSCRAMM IV formulated with aluminum hydroxide (2% Alhydrogel: Superfos) in a 1: 1 ratio. did.
Twenty-eight days later, two rhesus monkeys were boosted by IM injection of 200 μl of the same vaccine formulation.

The clinical regimen performed is described below; 15 ml of pre-immunized plasma samples per day, complete blood chemistry IM vaccination of the posterior quadriceps with 0.2 ml of MSCRAMM IV (100 μg) per day Injection site examination, temperature recording 7 days Liver panel, body temperature recording, injection site examination 14 days 15 ml plasma sample 21 days 15 ml plasma sample 28 days Complete blood chemistry, body temperature recording, 15 ml plasma Increased effect by IM injection of 0.2 ml sample of MSCRAMM IV (100 μg) 30 days Liver panel, recording body temperature, examination of injection site 35 days Liver panel, recording body temperature, injection site, 15 ml plasma sample 42 days 15 ml plasma sample 49 Day 15 ml plasma sample Day 106 ml 15 ml plasma sample All four animals were seroconverted following the first immunization.

[0207] At 106 days after the first vaccination, antibody concentrations more than 3 times higher than background could be detected by ELISA. Four animals received another enhanced immunization on the 21st week of the study. Each animal received booster with four subcutaneous injections of 125 μl of vaccine, for a total of 600 μl of booster of vaccine. 189 days after the first vaccination, antibody concentrations at least 3 times higher than background and 15 times higher than background could be detected by ELISA. See FIG. No adverse reactions at the injection site were detected by direct observation by a veterinarian. In addition, liver enzyme distribution, CBC, and blood distribution were within normal ranges for rhesus monkeys.

Example 5 Plasma samples from vaccinated monkeys were analyzed by ELISA using an Immulon-2 microtiter plate (manufactured by Dynex Technologies) with collagen-binding MSCRAMM (M55) and fibrinogen-binding MSCRAMM (clfA). ; pCF44),
Fibrinogen-binding MSCRAMM (clfB; region A) and fibrinectin-binding MSCR
50 μl of diluted plasma samples coated overnight at 4 ° C. with 10 μg / ml (50 μl) of AMM (DUD4) were added to the MSCRAMM-coated wells and incubated for 1 hour at room temperature. Wash buffer consisting of PSB containing 0.05% (v / v) Tween-20, 1% in PSB
Blocking solution of (vol / vol) BSA with 0.05% Tween-20, and 0.1%
An antibody dilution buffer consisting of PBS containing 0.1% BSA and 0.05% Tween-20 is used. Incubation (incubation) with primary and secondary antibodies is at 25 ° C. for 60 minutes. The secondary antibody was alkaline phosphatase-conjugated goat anti-monkey immunoglobulin G (Rockland) diluted 3500-fold with antibody dilution buffer. ELISA plates were developed with 1 mg / ml p-nitrophenyl phosphate (Sigma) (pH 9.8) (pH 9.8) in 1 M Jetanolamine and 0.5 mM MgCl ₂ for 30 minutes at 37 ° C. -Quantified at 405 nm with an Assay reader. Each plasma sample was prepared in phosphate buffered saline (p.p.) containing 0.05% Tween-20 and 0.1% BSA.
H7.4). ELISA data is shown in FIG.

Example 6 Inhibition Assay Methicillin-resistant Staphylococcus aureus strain 601 (Gene of Smeltzer, M, S, 196: 249-15)
9 (1997)) in BHI broth at 37 ° C. for 15 hours under constant rotation. A 1: 100 dilution of the overnight culture was made in BHI, and the bacteria were grown at 37 ° C to a medium exponential growth phase. Bacteria were harvested by centrifugation, washed three times in sterile PBS (pH 7.4), and then resuspended in carbonate buffer (50 mM NaCO ₃ , pH 8.5). Bacteria are 50 mM N ₂ HCO ₃ (pH 8
.5) and mixed with (mg / ml FITC (Sigma; F-7250)) and incubated at 25 ° C. for 1 hour in the dark in an end-over-end manner. Cells were centrifuged and stopped by removing the supernatant containing unreacted FITC Labeled bacteria were washed three times in PBS to remove unincorporated FITC and resuspended in PBS. Adjusted to 1 × 10 ⁸ cfu / ml and stored at −20 ° C. in PBS (pH 7.4).

Example 7 Purification of IgG from immunized monkeys IgG was purified from monkey plasma by affinity chromatography on PROSEP® (high capacity resin) (Bioprocessing). Briefly, plasma was thawed and passed through a 0.45μ filter. Plasma was applied to a bench top column containing Procept high volume resin. Unbound material was removed by extensively washing the column with PBS. IgG was eluted from the column with 0.1 M sodium citrate (pH 3.0). The pH of the eluted IgG was adjusted by adding 1 M Tris (pH 9.0).
Neutralized immediately to pH 6.8-7.4. The IgG was then dialyzed into PBS (pH 7.4), concentrated and filter sterilized. The concentration of purified IgG was measured by absorbance at 280 nm.

Example 8 Competition Inhibition ELISA A 96-dent black plate of Costar (Costar) contains 10 μg / ml of a substrate component consisting of bovine collagen, human fibrinogen and bovine fibronectin in PBS (pH 7.4). The solution was coated overnight at 4 ° C or 2 hours at room temperature. The plate coated with the substrate protein is washed three times with PBS, 0.05% Tween 20, then PBS,
Blocked with 1% BSA. Blocked plate in PBS, 0.05% Tween
Washed three times with 20. 500 μl of FITC-labeled Staphylococcus aureus cells were added to PBS,
Mixed with increasing amounts of purified monkey IgG in 0.05% Tween 20, 0.1% BSA. The labeled cells and IgG were mixed on a double-ended shaker for 1 hour at 25 ° C. 50 μl of the labeled cell / IgG mixture was added to each well on the microtiter plate and incubated at 25 ° C. on a rocker platform. The dents were washed three times with PBS, 0.05% Tween20. The amount of bacteria bound to the immobilized substrate protein was determined on a Perkin Elmer HIS7000 bioassay reader with an excitation filter set at 485 nm and an emission filter set at 535 nm. The data is shown in FIGS.

Example 9 Animal Model of Sepsis Using a mouse model of sepsis (Bremell, TA et al., Infect, Immun, 62 (7): 297).
6 to 2985 (1992)), purified Ig from rhesus monkeys immunized with MSCRAMM IV.
Passive immunization with G has demonstrated that mice can be protected against sepsis-induced death. In one day, naive male NMRI mice (5-8 weeks old) were purified IgG from rhesus monkeys (n = 12) immunized with MSCRAMM IV or non-immunized rhesus monkeys (n
= 13) was passively immunized ip with 20 mg of any of the IgGs from On day 0, mice were administered iv with 2.4 × 10 ⁷ CFU / mouse S. aureus strain LS-1. Mortality and weight changes were monitored over the next three days. Three days after inoculation, 3/13 mice (13%) died in the control group compared to 0/12 mice (0%) in the passive immunization group. On day 13, the mortality of the control group was 53.8% (7/13), indicating that MSCRAMM IV
Compared with only 16.2% (2/12) of the passive immunization group. Control mice are MSCRAMM IV
Significant weight loss was observed when compared to IgG passively immunized mice (28.0 ± 2.5% vs.
21.3 ± 3.1%: p <0.01).

Example 10 ELISAs ELISAs consist of a wash buffer consisting of PBS containing 0.1% (vol / vol) of Tween 80 and a block of 1% (polymerized / vol) BSA and 0.1% Tween 80 in PBS. Solution and an antibody dilution buffer consisting of PBS containing 0.05% Tween-80, Imron II 96 convex microtiter plate (Dynex Technologie
s company). Incubation with primary and secondary antibodies was at 25 ° C for 60 minutes. The secondary antibody is alkaline phosphatase-conjugated goat anti-human immunoglobulin G (Chemicon) diluted 3000-fold in antibody dilution buffer. The ELISA plate was developed with 1 mg / ml p-nitrophenyl phosphate (Sigma) in 1 M diethanolamine, 0.5 mM MgCl ₂ (pH 9.8) for 30 minutes at 20 ° C. and equipped with a 405 nm filter. Quantification was performed on a Molecular Dynamics ELISA plate reader. Each human serum sample was diluted 100-fold with phosphate buffered saline containing 0.05% Tween 20 and 0.1% BSA (pH 7.4). The ELISA plate is a collagen-bound MSCRAMM (M55: see Patti, JM et al., J. Biol. Chem, 270, 2005-12011 (1995)).
And fibrinogen-binding MSCRAMM (clfA: pCF44; see MeDevitt, D. et al., Mol, Micro, 11 (2): 237-248 (1994)) and fibrinogen-binding MSCRAMM (clfB; region A; Ni Edh
in, D. et al. (see Infect. Immun. Submitted (1998)) and fibronectin-bound MSCRAM.
M (DUD4; see Joh, HJ et al., Biochemistry 33 (20): 6086-6092 (1994)) at 1 μg /
ml (100 μl) at 4 ° C. overnight. 100 μl of the diluted serum material was
Added to AMM-coated dents and incubated for 1 hour at room temperature.

[0214] One hundred human plasma donor samples were analyzed using the ELISA protocol described above. Eight donors were selected as having elevated MSCRAMM antibody titers ("selected MSCRAMM"). 700-850 ml of plasma units from 8 donors were pooled and IgG was purified by affinity chromatography on Procept high volume resin (Bioprocessing). Briefly, human plasma was thawed at 4 ° C. for 24 hours and pooled as single plasma units. Pour the plasma pool through a cotton cloth, then 0.45
Filtered through a μ filter. Plasma was applied to a column of Procept high volume resin connected to a preparative scale HPLC (Waters). Unbound material was removed by thoroughly washing the column with PBS. IgG is 0.1 M sodium citrate (pH 3.0
) To elute from the column. The pH of the eluted IgG was immediately neutralized to pH 6.8-7.4 by the addition of 1M Tris (pH 9.0). The IgG was then dialyzed into PBS, concentrated and filter sterilized. The concentration of the purified IgG was measured by the absorbance at 280 nm.

Example 11 Animal Model from Staphylococcus aureus Infection Rabbit Model of Infectious Endocarditis (Perlman, BB and LS Freedman, Yale J
Biol. Med, 42: 394-410 (1971)).
The protective potential of gG was evaluated. This model has been used over the past decade to study the etiology of endocarditis and to test various new antibiotics and vaccines. In this model, a 2.5 kg rabbit was placed in the transcarotid artery with an indwelling polyethylene catheter.
He underwent transaortic valve catheterization. The eight rabbits then received 18ml of the "Selected MSCR"
AMM "was treated intraperitoneally with human IgG (28 mg / ml; 504 mg total). Eight control rabbits received 18 ml of sterile PBS and 10 rabbits received 500 mg of normal human IVIG.
(Alpha therapeutics Veniglobulin S) was received intraperitoneally. An intraperitoneal injection of 10 ⁹ cfu of Staphylococcus aureus strain (Reynolds) resulted in infective endocarditis 18 hours after IgG administration. Animals were followed for 72 hours and blood samples were obtained at 12 hour intervals. After 72 hours,
The animals were euthanized and the kidneys and valvulus removed aseptically. Tissue samples were processed for quantitative culture. A rabbit is considered positive for endocarditis if bacteria are recovered from the nodule, regardless of bacterial density. The lowest concentration of bacteria detected in this model is found to be = 2 log ₁₀ cfu / tissue. The number of microorganisms recovered from tissue sites (kidney and varices) was statistically compared using a two-tailed Student's t-test. A p value of 0.05 or less for individual comparisons is considered significant. Table 2 shows the results.

[0216] Example 12 Studies on ClfA and CNA Selected Human IVIG Prophylactic administration of ClfA and CNA donor selection SA-IVIG IVIG The purpose of the study described here was to increase the ability of microbial surface component-recognizing adhesion substrate molecules (MSCRAMM) proteins to be expressed by S. aureus. Immunization with Donor-Selected IVIG Products Prepared from Human Donor Plasma Containing Monovalent Antibodies Prevents Mortality Induced by Antibiotic-Resistant Staphylococcus aureus Clinical Isolates in a Murine Sepsis Model It measures whether it can be done.

SA-IVIG MS502, an Intravenous Immunoglobulin of Staphylococcus aureus Increased IgG in ELISA assay specific for the A region of ClfA MSCRAMM expressed by Staphylococcus aureus (5-fold higher than normal IVIG) Above titer increase)
Human IgG was purified using 15 units of plasma collected from seven donors that were determined to have Plasma was obtained from Serologicals and IgG was purified by Cangene as a sterile filtered solution in 10% maltose and 0.03% polysorbate 80. The sample was reported by Cangene to contain 47.55 mg / ml IgG by radioimmunodiffusion. This material was stored at 4 ° C. as directed by the manufacturer. On the day of dosing, MS502 was diluted to 40 mg / ml with 1 × D-PBS as a 0.5 ml IP injection formulation.

SA-IVIG MS503, Staphylococcus aureus intravenous immunoglobulin Specific concentrations for the A region of CNA MSCRAMM expressed by Staphylococcus aureus, increased concentrations of IgG in ELISA assays (5-fold higher than normal IVIG) Above titer increase)
Human IgG was purified using 12 units of plasma collected from five donors that were determined to have Plasma was obtained from Serologicals, and IgG was purified by Cangene as a sterile filtered solution in 10% maltose and 0.03% polysorbate 80. The material was reported by Cangene to contain 45.41 mg / ml IgG by radioimmunodiffusion. This material was stored at 4 ° C. as directed by the manufacturer. On the day of dosing, MS503 was administered as a 0.5 ml IP formulation for injection.
It was diluted to 40 mg / ml with × D-PBS.

Controls, human intravenous immunoglobulin, Polygum® (Baxter
IVIG) A sterile, lyophilized preparation of IgG was prepared from a large pool of human plasma by Baxter Healthcare. The material was reconstituted in sterile water for injection (Baxter Healthcare) for injection. This 5% solution contains 50 mg / ml total protein, 45 mg / ml IgG, 8.5 mg / ml NaCl, 3 mg / ml human albumin, 22.5 mg / ml glycine, and 20 mg / ml glucose. 2 mg / ml polyethylene glycol and 1
μg / ml tris (n-butyl) phosphate and 1 μg / ml octoxynol 9
And 100 μg / ml polysorbate 80. Prior to injection, the stock was diluted with sterile distilled water to a final concentration of 40 mg / ml IgG as a formulation for 0.5 ml IP injection.

S. aureus S. aureus strain 601 (Smeltzer et al., 1996) was obtained from Dr. MSSmeltzer (University of Arkansas). This Staphylococcus aureus strain was isolated from an intensive care unit. Isolates are cephalothin, ciprofloxacin, clindamycin, erythromycin, oxanline (methicillin), penicillin G and trimethopurine-sulfamethoxazole resistant. S. aureus cells of strain 601 from the frozen glycerose stock were inoculated into 10 ml of BHI culture broth and 37
Grow overnight at ° C. Dilute cells from overnight culture 1: 100 in BHI broth,
Grow at 37 ° C. for approximately 3 hours until the absorbance at 600 nm reaches 1.8-2.0 OD units. Bacteria are pelleted by centrifugation and 1/5 volume of a freezing medium (1 × D-PBS, pH7.
4, 10% DMSO, 5% BSA). Small aliquots were placed on blood agar plates at 10 ^-2 , 10 ^-4, and 10 ^-6, and incubated overnight to determine the production CFU concentration. Bacterial preparations were stored at -86 C until use. On the day of injection, the frozen bacterial stock was thawed and pelleted by centrifugation. Bacteria were washed once with D-PBS and resuspended to the appropriate concentration in D-PBS for IV injection. Aliquots of the bacterial suspension were placed on blood agar plates as 10 ⁻² , 10 ⁻⁴ and 10 ⁻⁶ dilutions and cultured overnight to determine the CFU concentration of the final injection formulation.

Sex, Species, Number, Age and Source of Animals 56 female mice (5-6 weeks old) with 56 draws were subjected to Taconic Quality Laboratory Animals a.
Purchased from nd Services for Research. Animals were acclimated for at least 14 days before the start of treatment. Upon arrival, the mice were inspected and bred (5 / cage) in polycarbonate shoebox cages lined with adsorbent. All mice were subjected to a 12 hour light-dark cycle under the required housing standards found in the NIH Guide for Care and Use of Laboratory Animals.

Identification and Randomization All animals were uniquely identified using tailing for dosing. Prior to the start of treatment, animals were weighed individually and their health was assessed. Mice were randomized and assigned to treatment groups using stratified body weight.

Experimental Mode On day 1, animals were treated with a single 0.5 ml IP injection of MS502, MS503 or Baxter IVIG. On day 0, 2.2 × 10 ⁸ CFU S. aureus was administered via a single IV injection (0.1 ml) to all animals via the tail vein.

Data mice were primed by intraperitoneal (IP) injection with either 20 mg of Baxter's normal IVIG product or 20 mg of SA-IVIG MS502 and 20 mg of SA-IVIG MS503. MS
502 is an immunoglobulin G (IgG) preparation purified from donor plasma containing an antibody of increased titer recognizing the A region of the clamping factor (ClfA), a Staphylococcus aureus fibrinogen binding MSCRAMM protein. Similarly, MS503 is the high titer formulation of choice for recognizing the A region of CNA, collagen-bound S. aureus MSCRAMM. The total IgG concentrations for the two MSCRAMM formulations and the normal product of the standard Baxter are given in Table 3 below. As shown below, the high titer MS502 sample has a total ClfA content of 2.29 units / mg as opposed to only 0.2 units / mg in the normal sample. The high titer MS503 sample has a total CNA content of 1.06 units / mg as opposed to only 0.2 units / mg in the normal sample. Twenty-four hours after IgG administration, mice were challenged with a single intravenous (IV) injection of a methicillin-resistant strain of S. aureus (strain 601). The mice were followed for 5 days, at which point all remaining mice were killed. Significant differences in relative survival times between treatment groups were detected. 63% of mice that received MS502 (12/19
) Survived the bacterial challenge (p = 0.003 vs. control: Mantel-Cox survival analysis). MS50
68% (13/19) of the mice that received S.A. survived the bacterial challenge (p = 0.008 vs. control: Mantel-Cox survival analysis). 22% of mice treated with IVIG of normal humans (4/18
Only) survived the entire study period. These results clearly show that ClfA
Prophylactic administration of SA-IVIG and selected CNA donors provides a significant degree of protection against lethal infection compared to the commercially available human normal IVIG product.

[0225] B. Therapeutic application of human SA-IVIG selected from ClfA SA-IVIG Staphylococcus aureus immunoglobulin intravenously Increased IgG (normal IVIG) by ELISA assay specific to the A region of ClfA MSCRAMM protein expressed by Staphylococcus aureus Using 15 units of plasma collected from seven donors that were determined to have a titer increase of
Human IgG was purified. Plasma was obtained from Serologicals and IgG was purified by Cangene as a sterile filtered solution in 10% maltose and 0.03% polysorbate 80. The sample was reported by Cangene to contain 47.55 mg / ml IgG by radioimmunodiffusion. This material was stored at 4 ° C. as directed by the manufacturer. On the day of dosing, MS502 was diluted to 40 mg / ml with 1 × D-PBS as a 0.5 ml IP injection formulation.

S. aureus S. aureus strain 601 (Smeltzer et al., 1996) was obtained from Dr. MSSmeltzer (University of Arkansas). This S. aureus strain was isolated from an intensive care unit. Isolates are cephalothin, ciprofuxacin, clindamycin, erythromasiin, oxacillin (methicillin), penicillin G and trimethopurine-sulfamethoxazole resistant. S. aureus cells of strain 601 from the frozen glycerose stock were inoculated into 10 ml of BHI broth and grown overnight at 37 ° C. The cells from the overnight culture were diluted 1: 100 in BHI broth and
Grow at 37 ° C. for approximately 3 hours until the absorbance at 1.8 m reaches 1.8-2.0 OD units. Bacteria are pelleted by centrifugation, and 1/5 volume of freezing medium (1 x D-PBS, pH 7.4, 10%
DMSO, 5% BSA). Small aliquots were placed on blood agar plates at 10 ⁻² , 10 ⁻⁴ , and 10 ⁻⁶ dilutions and incubated overnight to determine the CFU concentration of the formulation. Bacterial preparations were stored at -86 C until use. On the day of injection, the frozen bacterial stock was thawed and pelleted by centrifugation. Bacteria are washed once with D-PBS and used for IV injection.
Resuspended in the appropriate concentration. Portions of the bacterial suspension were placed on blood agar dishes as 10 ⁻² , 10 ⁻⁴ and 10 ⁻⁶ dilutions and cultured overnight to determine the CFU concentration of the final injection formulation.

Animal Organisms, Species, Numbers, Age and Source Female mice (5-6 weeks old) were collected from Tacomic Quality Laboratory Animals and Servi
Purchased from ces for Research. Animals were acclimated for at least 5 days before the start of treatment. Upon arrival, the mice were inspected and housed in sorbent-lined polycarbonate shoebox cages (5 / cage). All mice were subjected to a 12 hour light-dark cycle under the required housing standards found in the NIH Guide for Care and Use of Laboratory Animals.

Identification and Randomization All animals were uniquely identified using tail retraction before dosing. Prior to the start of treatment, animals were weighed individually and their health was assessed. Mice were randomized and assigned to treatment groups using stratified body weight.

Experimental Mode On day 1, animals were treated with a single 0.5 ml IP injection of MS502 IVIG. On day 0, 5.6 × 10 ⁷ CFU S. aureus was administered via a single IV injection (0.1 ml) to all animals via the tail vein. In addition, one group of mice was treated with MS 3 hours after the challenge of IV bacteria.
Received 502 IVIG. The control mice remained untreated.

Data Mais showed that 20 mg of 18 mg before or 3 hours after IV challenge with S. aureus
Treated with SA-IVIG MS502 by intraperitoneal (IP) injection. MS502 is a member of the clumping factor (ClfA), a Staphylococcus aureus fibrinogen-binding MSCRAMM protein.
FIG. 4 is an immunoglobulin G (IgG) preparation purified from donor plasma containing elevated titers of antibodies recognizing regions. The mice were followed for 5 days, at which point all residual mice were sacrificed. Nineteen percent of the mice that received MS502 SA-IVIG18 18 hours before the S. aureus challenge survived. Similarly, three hours after bacterial challenge, 93% of mice that received MS502 SA-IVIG3 survived. In contrast, only 76% of the control mice survived the bacterial challenge. These results clearly show that the therapeutic administration of SA-IVIG in ClfA donor-selected humans has a significant and effective effect on staphylococcal infection compared to the commercially available human normal IVIG product. Give a proper treatment.

[Brief description of the drawings]

FIG. 1 is a diagrammatic representation of the peptides used in an exemplary vaccine, MSCRAMM IV. This FIG. 1 illustrates the essential characteristics of collagen-bound MSCRAMM CNA, fibrinogen-bound MSCRAMM ClfA, fibrinogen-bound MSCRAMM ClfB and fibronectin-bound MSCRAMM FnBPA proteins.

FIG. 2 is a time diagram of three immune responses in rhesus monkeys vaccinated with MSCRAMMs as indicated by changes in antibody titers against MSCRAMMs CNA, ClfA, ClfB and FnBPA, respectively. Antibody titers were analyzed by the Eliga assay and measured as changes in absorbance (quantified at 405 nm) during each week over the course of the 6 month treatment period following initial immunization with antigen.

──────────────────────────────────────────────────続 き Continuation of front page (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE ), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR , BY, CA, CH, CN, CR, CU, CZ, DE, DK, DM, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL , IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, UZ, VN, YU, ZA, ZW (71) Applicant The Plavast Fellows and Scholars Of the Caret of the Harry and the Andeidedos de Trinity of Queen Elizabeth Nyah Dublin Ireland Dublin 2, Trinite i Carrez (no address) (72) Inventor Patty, Joseph, M. United States of America Georgia 30040, Cummings, Stradoford Place 6680 Dublin 16, Ireland, Temploag, Coolumbar Park 70 (72) Inventor Hook, Magnus United States Texas 77005, Howston, Oberlin 4235 F-term (reference) 4C085 AA13 AA14 AA16 BA13 BB33 BB34 BB35 BB36 BB37 CC14 CC21 DD33 EE01 EE05 FF01 FF02 GG02 GG03 GG04 GG05 GG06 GG08

Claims (32)

[Claims] 1. The following steps: a. Producing a source of donor plasma having higher than normal antibody titers against staphylococcal adhesins; b. Treating the donated plasma to obtain a purified immunoglobulin having an unusually high antibody titer against staphylococcal adhesin; and c. A method of immunizing a patient to treat or prevent staphylococcal infection, comprising internally administering to a patient an immunologically effective amount of purified immunoglobulin-containing donor plasma to treat or prevent staphylococcal infection. 2. The method of claim 1, wherein the donor plasma source is obtained from a single donor having an unusually high antibody titer against staphylococcal adhesins.
Law. 3. The donor plasma source is obtained by identifying donor plasma samples from individuals having an unusually high antibody titer against staphylococcal adhesion factors and combining plasma obtained from these individuals. The method of claim 1 obtained. 4. The donor plasma source comprises administering a staphylococcal adhesin to a host,
A method according to claim 1, wherein the method is obtained by inducing a higher than normal antibody titer in the administered staphylococcal adhesin and recovering plasma from the inducing host. 5. The staphylococcal adhesin comprises a fibronectin binding protein,
The method according to claim 1, wherein the method is selected from the group consisting of a collagen binding protein, a fibrinogen binding protein, an elastin binding protein, an MHC II-like protein, and another protein that binds to an extracellular matrix protein. 6. The staphylococcal adhesion factor is a protein, Fn-BP-A,
The method according to claim 1, wherein the method is selected from the group consisting of Fn-BP-B, ClfA, ClfB, SdrC, SdrD, SdrE, SdrF, SdrG, SdrH, CNA, EdpS and MHC II. 7. The method of claim 1, wherein the donor plasma source has a higher than normal antibody titer against the A region of the staphylococcal adhesin. 8. The donor plasma source is the protein ClfA, ClfB, SrdC,
8. The method according to claim 7, wherein the method has a higher than normal antibody titer against the A region of a staphylococcal adhesin selected from the group consisting of SrdD, SrdE, SdrF, SdrG, SdrH and CNA. 9. The method of claim 1, wherein the donor plasma source has a higher antibody titer than normal to fibrinogen binding protein and fibronectin binding protein. 10. The method according to claim 9, wherein the fibronectin binding protein is selected from the group consisting of FnBP-A and FnBP-B. 11. The method of claim 1, wherein the donor plasma source has a higher than normal antibody titer for fibrinogen binding protein and collagen binding protein. 12. The method of claim 1, wherein the donor plasma source has a higher than normal antibody titer against fibrinogen binding protein, fibrinogen binding protein and collagen binding protein. 13. The method of claim 1, wherein the donor plasma source has a higher than normal antibody titer against ClfA and ClfB. 14. The method of claim 1, wherein the donor plasma source further comprises a capsular polysaccharide. 15. The method of claim 1, wherein the donor plasma source has a higher than normal antibody titer against the M55 region of the collagen binding protein. 16. The staphylococcal adhesion factor is Staphylococcus aureus.
Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus hominis and Staphylococcus saprophyticus from Staphylococcus saprophytic bacterium selected from Staphylococcus saprophytic bacteria from Staphylococcus saprophyticus. The method of claim 1 obtained. 17. Donor plasma, comprising a therapeutically effective amount of purified donor plasma having a higher than normal antibody titer against staphylococcal adhesin, and a pharmaceutically acceptable carrier or excipient. Composition. 18. The staphylococcal adhesin is selected from the group consisting of fibronectin binding protein, collagen binding protein, fibrinogen binding protein, elastin binding protein, MHC II-like protein and another protein that binds to extracellular matrix protein. The method of claim 17. 19. The staphylococcal adhesion factor is a protein, FnBP-A,
FnBP-B, ClfA, ClfB, SdrC, SdrD, SdrE, SdrF, SdrG, SdrH, cAN, EbpS and
18. The composition according to claim 17, wherein the composition is selected from the group consisting of MHC II. 20. The composition of claim 17, wherein the donor plasma has an unusually higher antibody titer against the A region of staphylococcal adhesin. 21. The donated plasma contains proteins ClfA, ClfB, SrdC, SrdD,
21. The composition of claim 20, which has an unusually higher antibody titer to the A region of a staphylococcal adhesin selected from the group consisting of SrdE, SdrF, SdrG, SdrH and CNA. 22. The composition of claim 18, wherein the donor plasma has an unusually high antibody titer for fibrinogen binding protein and fibronectin binding protein. 23. The composition according to claim 22, wherein the fibronectin binding protein is selected from the group consisting of FnBP-A and FnBP-B. 24. The method according to claim 1, wherein the donor plasma has a higher than normal antibody titer against fibrinogen binding protein and collagen binding protein.
A composition according to claim 7. 25. The composition of claim 17, wherein the donated plasma has a higher than normal antibody titer for fibrinogen binding protein, fibrinogen binding protein and collagen binding protein. 26. The composition of claim 17, wherein the donor plasma has an unusually high antibody titer against ClfA and ClfB. 27. The composition according to claim 17, further comprising a capsular polysaccharide. 28. The composition according to claim 17, wherein the capsular polysaccharide is selected from the group consisting of type 5 or type 8 capsular polysaccharides. 29. The composition of claim 17, wherein the donor plasma source has a higher than normal antibody titer against the M55 region of the collagen binding protein. 30. A blood sample is obtained from a donor, a blood sample having an antibody titer higher than normal against staphylococcal adhesion factors is identified, and plasma is recovered from the identified high-titer blood donor. Treatment of the donor plasma to obtain purified immunoglobulins with higher than normal antibody titers against staphylococcal adhesion factors.
A method for obtaining a donor plasma composition having a higher than normal antibody titer against an adhesion factor of staphylococci. 31. A donated plasma composition obtained by the method of claim 30. 32. An antigen against a staphylococcal antibody, a support that binds the antigen, and a detectable label that can be attached to the bound antibody in an amount sufficient to bind the antibody, usually higher in titer. A kit for identifying blood or plasma having a higher than normal antibody titer against a staphylococcal adhesin, comprising: