HK1176077B - Monoclonal antibodies against the pbp2-a protein and homologous sequences for the treatment of infections by and immunodiagnostics of bacteria of the firmicutes phylum - Google Patents

Description

Monoclonal antibodies and homologous sequences of the PBP2-a protein for use in immunodiagnosis and treatment of infections caused by bacteria of the phylum firmicutes

Technical Field

The present invention relates to monoclonal antibodies capable of recognizing and binding to the PBP2a protein and other proteins homologous to PBP2a, including methicillin-resistant staphylococcus aureus-MRSA, coagulase-negative staphylococci and staphylococcus squirrel (r) ((m))Staphylococcus sciuri) Enterococcus bacteria (A)Enterococcus sppAnd any other bacterial pathogen having PBP2a or having sequences homologous to the protein. The invention also relates to the use of monoclonal antibodies capable of recognizing and binding to the PBP2a protein and other proteins expressed from sequences homologous to PBP2a in complementary immunodiagnostics for the detection of beta lactam resistance.

Background

Infections caused by methicillin-resistant Staphylococcus aureus-MRSA are of major concern to clinicians, and exhibit mortality and morbidity rates that are higher than those caused by methicillin-sensitive Staphylococcus (1). Moreover, these infections result in longer hospitalizations and higher antibiotic costs, resulting in higher treatment costs for patients infected with this pathogen (1).

Vancomycin is the first antibiotic of choice for the treatment of infections caused by MRSA. However, the isolation of the growth of MRSA strains (2, 3, 4) in the population in the united states and australia, from the identification of vancomycin-resistant MRSA strains as mediators in japan, the united states (5) and brazil (6), has led to the current situation becoming more severe. The description in 2004(7) of completely vancomycin-resistant MRSA strains is of high interest to the pharmaceutical sciences community. MRSA now represents a strong candidate for becoming a feared "superbugs or superbugs" -a pathogenic bacterium that is currently resistant to all available drugs.

Generally, the prevalence rate of MRSA (the rate of infection caused by staphylococcus aureus caused by MRSA) in hospital infectious diseases has gradually increased in recent decades. In a study conducted by Jarvis et al, the number of infections caused by MRSA in ICU rose from 660 to 2184 and prevalence rose from 35% to 64.4% (8) in 1268 ICUs (intensive Care Unit) included in 337 hospitals in the United states. In japan, the prevalence of nosocomial infections (HIs) caused by MRSA appears to be a disturbing value, rising from 60% to 90% (9). In the studies conducted in the united states, the percentage rose from 2% in 1974 to 50% (10, 11) in 1997, with over 80% of His resulting from MRSA in some hospitals in the united states. In the uk, the percentage rose from 1.5% to 15.2% between 1989 and 1995, now (2004) estimated to be 41.5% (13).

In addition to a high percentage, MRSA is considered to be the main causative bacterium of epidemics outbreaks in brazilian hospitals especially in teaching hospitals and major hospitals (14). More than 50% of Staphylococcus aureus from hospital sources isolated from patients in the university Hospital of St. Paul in 1986 were resistant to methicillin, and in 1993, the incidence of MRSA in the pediatric hospital of the medical college of the department of health, the Paulista Medicine School was 70% (15). Resend et al (16) indicated a prevalence of 71% of MRSA in a study conducted in the Belo Origante hospital (Belo Horizonte).

MRSA strains express penicillin binding proteins of the beta lactamase family with very low affinity for antibiotics, such as PBP2a (17). In the presence of this enzyme, encoded by the mecA gene, bacteria successfully synthesized peptidopolyases, even in the presence of beta lactamases. The enzyme is also usefulStaphylococcus aureus and Staphylococcus squirrel which are normally coagulase negative (Staphylococcus sciuri) Is found in staphylococcus squirrel, a bacterium found in the normal microflora of dogs. In addition to being resistant to beta lactamases, nosocomial MRSA strains exhibit resistance to most other available antibiotic families, and the use of glycopeptides (vancomycin and teicoplanin) remains the treatment of choice.

Two studies using DNA vaccines against PBP2a showed that the protein was immunogenic as detected in a murine model and that the resulting immune response could confer protection against MRSA (18, 19). However, it is well known that in nosocomial infections, a large proportion of patients are immunosuppressed (20). In this case, the vaccine cannot generate protective antibodies in a timely manner to control bacterial infection.

Immunogenicity of PBP2a

PBP2a is a class II multicomponent enzyme according to the classification by Goffin and Ghuysen (40). This 76-kilodalton enzyme consists of a membrane-bound region, a non-transpeptidase region and a transpeptidase region, including the 4-amino acid active center (STQK), responsible for bacterial transpeptidation (20 bis Ryfell, 1990).

The current state of the art in DNA vaccine studies against PBP2a shows that the outcome of bacterial reduction (kidney quantification) in vaccinated animals is susceptible to systemic infection with a 3-4 fold challenge in Ohwada et al and 1000 fold in Senna et al. The authors of these studies used the complete sequence of the mecA gene (except for the membrane-immobilized region) and internal fragments of the transpeptidase region, respectively.

However, according to the foregoing, vaccines cannot timely generate protective antibodies to control bacterial infections. Thus, in the case of MRSA infections, administration of monoclonal antibodies against PBP2a is the most appropriate treatment to treat these infections.

Disclosure of Invention

The invention mainly aims to provideMonoclonal antibodies capable of recognizing and binding to the PBP2a protein (SEQ ID NO:1) and other proteins characteristic of homology to PBP2a, including methicillin-resistant Staphylococcus aureus-MRSA, coagulase-negative staphylococci, Staphylococcus squirrel (S.matsutakeStaphylococcus sciuri) Enterococcus bacteria (A), (B)Enterococcus spp.) And any other pathogen that possesses PBP2a or a bacterium with a sequence homologous to the protein.

Another object of the invention is to use monoclonal antibodies capable of recognizing and binding to the PBP2a protein and characterizing other proteins homologous to the PBP2a sequence in complementary immunodiagnostics for the detection of beta lactamase resistance.

The monoclonal antibody of the invention is characterized by the sequences SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 11, SEQ ID NO 12, SEQ ID NO 13, SEQ ID NO 14, SEQ ID NO 15, SEQ ID NO 16 and SEQ ID NO 17.

Drawings

FIG. 1 is an enzyme-linked immunosorbent assay (ELISA) of sera from animals vaccinated with antibodies against PBP2 a;

FIG. 2 is the result of polyacrylamide gel of crude and purified samples;

FIG. 3 is an immunoblot assay of supernatant MRSA and MSSA lysates containing anti-PBP 2a monoclonal antibody;

FIG. 4 shows the results of flow cytometry assays of MRSA (CEB) and MSSA bacteria co-cultured with monoclonal antibodies against PBP2a and Phycoerythrin (PE);

FIG. 5 shows the test of the in vitro protective effect of inoculants of different MRSA strains given anti-PBP 2a purified monoclonal antibody and vancomycin (MIC-minimum inhibitory concentration);

FIG. 6A shows the results of renal quantification of animals treated and non-treated with anti-PBP 2a monoclonal antibody for systemic infection with MRSA CEB strain given a sub-lethal dose;

FIG. 6B shows the results of renal quantification of animals treated and non-treated with anti-PBP 2a monoclonal antibody, administered a sub-lethal dose of a systemic infection with MRSA ibiian (Iberian) strain (European epidemic clone);

FIG. 6C shows the results of renal quantification of animals treated and non-treated with anti-PBP 2a monoclonal antibody, given systemic infection with a sub-lethal dose of WB79CA-MRSA strain (clone in Brazil population);

figure 7A shows survival curves for treated (protected) and non-treated (controlled) animals following infection with lethal bacterial dose (MRSA CEB);

fig. 7B shows survival curves for treated (protected) and non-treated (controlled) animals following infection with lethal bacterial dose (ibiya MRSA);

figure 7C shows the survival curves of treated (protected) and non-treated (controlled) animals following infection with lethal bacterial dose (WB79 CA-MRSA);

FIG. 8A shows bacterial counts in treated and untreated kidneys of animals infected with bacteria (MRSA CEB) with anti-PBP 2a monoclonal antibody, vancomycin, and the antibody + vancomycin combination;

FIG. 8B shows the renal bacterial count of animals treated with anti-PBP 2a monoclonal antibody;

FIG. 9 shows the interaction of PBP2a (antigen) with the reassortment of monoclonal antibody clone 38 (FIG. 9A) and clone 10 (FIG. 9B);

FIG. 10 is a graph of secondary analysis by flow cytometry of MRSA samples in the presence of FITC-labeled anti-PBP 2a antibody;

FIG. 11 shows the results of in vitro protection of antibodies administered against an enterococcus VRE strain;

FIG. 12 refers to LD ₅₀ And lethal dose of abdomenDetermination of intramembranous infection;

FIG. 13 shows the potency results of the in vivo protection of the anti-PBP 2a and PBP5 monoclonal antibodies of the enterococcin origin against systemic infection.

Detailed Description

Infections by bacteria resistant to multiple antibiotics are presenting increasing concerns. The prevalence of nosocomial infections caused by MRSA is increasing worldwide with high morbidity and resulting in high costs of use due to the exacerbation of antibiotics, and longer patient hospitalization (24). Chemotherapy treatments are showing signs of exhaustion due to the advent of truly novel and effective drugs on the market.

In this context, passive immunotherapy (again) appears to be a promising option with certain advantageous features compared to traditional chemotherapy: among these, lower toxicity, higher blood half-life (which favours reduced dose treatment, possibly resulting in lower final treatment costs), and especially in the products presented herein, optional toxicity, is recommended by Paul Ehrlich (where the drug may be selected to eliminate the mentioned pathogenic bacteria) (25 a). This last feature is extremely useful for preventing the so-called selective pressure-acting through broad spectrum antibiotics-which provides resistance against the emergence of multidrug resistant bacteria.

To develop this product, the inventors selected PBP2a as a target, based on preliminary studies of DNA vaccines in murine models (18, 19, 21) and molecular analysis of immune structures. PBP2a, whose structure is elucidated in 2002(29), is deposited in PDB (protein database) under the code of lwmr.

Unlike other ways, the inventors worked with the internal region of a 76 amino acid molecule, flanked by an enzymatic activity region (SxxK), in the transpeptidase region. Epitope recognition analysis was performed at the molecular level, where they appeared in different class II CHM alleles (Tepitope project), and site recognition later on the molecular level for them (SPDB reader project). This approach allows us to assess the likelihood of antibodies approaching these targets of the native molecule.

This computerized analysis showed the presence of an epitope near the center of enzyme activity with excellent recognition by the class II CHM allele, located on the surface of PBP2a (data not shown).

These in silico are expected to have been confirmed by carrying out target recognition tests (immunoblotting and flow cytometry), and the binding of antibodies to the PBP2a region was shown to be able to confer higher protection, as evidenced by the results obtained from tests carried out in vitro and in vivo. The antibody generated from the 76 amino acid fragment was shown to confer greater protection than that conferred by immunization with whole PBP2a, as demonstrated by the results obtained by the inventors, as shown in the aforementioned work by Ohwada and Senna.

Epidemiologically prominent clonal types of infection caused by MRSA are responsible for outbreaks and epidemics in hospitals all over the world. These clones have higher replication and virulence compared to non-circulating MRSA strains (30).

As a way of confirming the results obtained, tests were performed with different MRSA clones (epidemic clones), representing strains in most infections caused by MRSA, in particular hospital infections.

The CEB strain (brazilian epidemic strain) is responsible for most of the infections of MRSA in brazilian hospitals (31) and is also recognized in other countries in south america (32, 33), portugal and czochralski (34). The European epidemic strain (IBIOA-MRSA) is found in European countries and in the United states.

One of these strains (WB79 CA-MRSA) was isolated by Brazil and included in the current assay due to an increase in population infections caused by MRSA. These tests exhibit characteristics different from those found in hospitals, with higher virulence (presence of the Panton-Valentine leukocidin gene) and profile (amino acid composition) of antibiotic resistance different from that of hospital MRSA strains (36). Recently, CA-MRSA infection was found to outbreak in people who intercourse men in the United states with other men (37). From this data, we can show that MRSA-induced infections are in the role of STD (sexually transmitted disease).

The protection results obtained from the administration of antibodies against PBP2a showed effectiveness against all the different tested clones, which lets us believe their suitability for infections (human population or hospital) caused by any MRSA type.

Some notable features of the products of the invention need to be mentioned

(i) The results of in vitro protection led us to believe that the mechanism involved in the action, the blocking of the active region of the molecule, was sufficient to inhibit bacterial growth and reproduction. Like one of the beta lactam antibiotics, this mechanism is time-dependent, requiring the bacteria to expose the target site to the drug during the reproductive phase.

(ii) This feature is given by the pharmacokinetic properties of the antibody, which represents a long-term blood half-life (38). This means that lower doses of the drug are administered, the treatment of the surrogate patient is facilitated and may therefore cost less ultimately than antibiotic therapy. These ideas were demonstrated in practice in comparison tests for vancomycin protection.

(iii) Inhibition of bacterial growth in vitro also means that the antibody does not require traditional auxiliary antigen-antibody reaction mechanisms, such as opsonization and complementary activity. This product feature is extremely important considering that most nosocomial infections occur in patients with immunosuppressive data.

(iv) Staphylococcus aureus is known to have protein a on its surface. This molecule characteristically binds itself to the Fc region of immunoglobulins, preventing opsonization of the immune system (38). Due to the results obtained in our assays, we believe that the antibody concentration administered is capable of saturating the protein a present on the bacterial surface, and that the antibody administered does not prevent the blocking of PBP2 a.

Systemic infections caused by MRSA are treated with glycopeptides, particularly vancomycin. However, this drug presents certain side effects, immunotoxicity (42), ototoxicity, nephrotoxicity and transient neutropenia, and requires long-term administration, resulting in ultimately high treatment costs.

By using a murine infection model, it is possible to compare the treatment of vancomycin against monoclonal antibodies and the combination of the two drugs. The results obtained indicate that the antibody dose has activity similar to or higher than the 5-fold vancomycin dose, and that the simultaneous administration of both drugs is absolutely more effective in reducing the bacterial count than the single administration. It is extremely important to have important data for administration using vancomycin and an antibody so that treatment costs are lower and recovery of patients becomes more effective in consideration of the use of the antibody.

In addition to the use of anti-PBP 2a monoclonal antibodies for the treatment of infections caused by MRSA, the present invention also contemplates the following applications:

(i) identification of molecular weights close to enterococci by anti-PBP 2a monoclonal antibody (II)Enterococcus sp.) The results for the protein of one of the strains of PBP5 show that it is possible to obtain a protective response by administration of antibodies against the pathogenic bacteria. PBP5 is homologous to PBP2a, and is classified as class B of multimolecular PBPs with low affinity for β -lactams (40). Enterococci are bacteria that cause serious nosocomial infections, exhibiting an intrinsic and acquired high degree of antibiotic resistance. Vancomycin-resistant (VRE) -resistant strains exhibit the presence of glycopeptide resistance genes and are capable of conversion to other pathogenic bacteria (41).

(ii) These antibodies have the ability to recognize PBP2a through immunodiagnostic testing. For example, immunoassays based on agglutination reactions of latex particles bound with antibodies can provide predictions of resistance to all β -lactam antibiotics within hours. In conventional antibiotic sensitivity tests (e.g., antibiotic sensitivity tests), these results are only released after 12 to 24 hours.

(iii) Once monoclonal antibodies have been successfully used for topical administration against chronic pain (infliximab, see Streit et al and WO1999041285a1), these antibodies can be administered topically to promote decolonization of nasal infections caused by MRSA, as well as for treatment of skin lesions caused by this pathogen.

Based on the knowledge and subject found in the art, the present inventors carried out immunization in animals with a DNA vaccine having a structure corresponding to the gene of mecA (SEQ ID NO:2) without a membrane immobilization region, immunization including the 76-amino acid internal region of the transpeptidase region (SEQ ID NO:3) of the enzyme activity center.

Immunization was performed under the same conditions as in Ohwada et al and Senna et al, who developed a DNA vaccine against PBP2a using the complete sequence of the mecA gene (except for the membrane-fixed region) and an internal fragment of the transpeptidase region. Immunization was performed at 4 doses, vaccinated animals and non-vaccinated control groups were challenged by systemic infection with MRSA, and the number of bacteria present in the kidneys of the animals was determined at two different time periods. The results obtained show that the immunization with the transpeptidase fragment gives a more marked reduction in the number of bacteria present in the kidney of the animals compared to the number of bacteria in the kidney of the animals immunized with the mecA gene.

Thus, we see that under the conditions used (DNA vaccine in murine models) the antibodies generated against the transpeptidase fragment provide better protection than the antibodies generated against PBP2 a. The transpeptidase fragment included the active center STQK (SEQ ID NO: 4).

Therefore, based on the results found, the inventors have indicated that the present invention produces a monoclonal antibody capable of recognizing and binding to PBP2a protein and other proteins having a sequence homologous to PBP2a protein, using a transpeptidase fragment including an enzyme active center.

The invention will now be described in connection with the following examples, but is not to be construed as being limited thereto.

Materials and methods:

1. bacteria

The following methicillin-resistant staphylococcus aureus strains were used: Ibiya-MRSA (Iberian-MRSA), COL-MRSA (defined by Unit Agents antibodies-Institut Pasteur [ Unit of Antimicrobial Agents-Institut Pasteur Institute ], Dr. Patrice Corvalin), WB79CA-MRSA, and CEB-MRSA (defined by Dr. Agents Figuered, Instituto to de Microbiology [ Microbiology Institute ] of RJ); and methicillin-resistant staphylococcus aureus strains (MSSA). Coli strains BL21 DE3 (Novagen) and TOP10 (Invitrogen) were used as controls.

2. Animal(s) production

Female Balb/C mice obtained from CECAL-FIOCRUZ and colonized in LAEAN-BioManguinhos, 4 to 8 weeks old, were used for in vivo protection experiments.

3. Immunization

Mice (4) received an initial dose of 100 micrograms of pCI-Neo plasmid: the fragment of the mecA gene of MRSA (18), 14 days later was then emulsified in Freund's complete reagent using 10. mu.g of purified recombinant protein, corresponding to the internal region of MRSA PBP2a (21). After 14 days, the animals with the best immune response (measured by enzyme-linked immuno-ELISA) received a dose of purified protein diluted intravenously in PBS (phosphate buffered saline) 10. mu.g. Three days after IV injection, animals were in CO₂(CEUA L0009-07 Protocol-FI ℃ RUZ) were subjected to euthanization by asphyxiation, and spleens were aseptically excised and subjected to cell fusion. The blood of the animal with the best immune response was used for positive control of other immunological tests.

4. Preparation of monoclonal antibodies

Lymphocytes from the spleen were recorded for monoclonal antibody production by Current Protocols in Immunology (22) and SP2/0-Ag14 myelomaCells (ATCC 1581) were fused using polyethylene glycol at 37 ℃ with 10% CO₂The hypoxanthine-aminopterin-thymus medium. The synthesized hybridomas were evaluated after 14 days by ELISA detection using purified recombinant protein as antigen, as described below. The best hybridoma cells were used for cloning, the best clones were selected by ELISA and these clones were then stored in liquid nitrogen.

5. Enzyme immunoassay-ELISA

Maxisorb96 well plastic plates were coated with 500 nanograms/well of recombinant protein (PBP2a fragment) in carbonate/bicarbonate buffer and incubated overnight at 4 ℃. On the following day, the plates were washed three times with PBS containing 0.05% Tween 20(Tween 20) and blocked for two hours in PBS and 5% skim milk at 37 ℃. The samples (immune sera of vaccinated animals diluted 1:100 or cell culture suspension) were analyzed and incubated at 37 ℃ for 2 hours. The plate was washed three more times with PBS and (0.05%) Tween 20, and the anti-Ig conjugate (anti-murine HRP Ig SIGMA A0412) was added to a 1:5000 dilution followed by incubation at 37 ℃ for 90 minutes. After this time, the plate was washed three times with PBS and (0.05%) tween 20, developed by adding TMB peroxidase (BioRad), and incubated for 15 minutes in the dark. 0.5N H was added₂SO₄The reaction was stopped and read at 450 nm. A1: 200 dilution of hyperimmune polyclonal serum was used as a positive control.

5.1 affinity assays based on enzyme immunoassays

5.1.1 affinity assay with Urea (Niedeerhouser et al-5.1):

the method is similar to the aforementioned immunoassay method (5) with the following modifications: after two hours of sample incubation at 37 ℃ (100 ng of clone 10 of purified monoclonal antibody and 2.0 ng of clone 38 of purified monoclonal antibody), the samples were washed three times with 8M urea in PBS and tween 20(0.05%) and four times with PBS and tween 20(0.05%) with the same normal treated samples as control (without urea treatment). After the optical density value of the sample was read, the affinity ratio was calculated by dividing the reading of the urea treated sample by the reading of the non-urea treated sample, multiplied by 100 (percentage result).

5.1.2 affinity determination with ammonium thiocyanate

The method is similar to the aforementioned immunoassay method (5) with the following modifications: after two hours of sample incubation at 37 ℃ (100 ng of clone 10 of purified monoclonal antibody and 2.0 ng of clone 38 of purified monoclonal antibody), the samples were treated with ammonium thiocyanate at 37 ℃ for 30 minutes at the following concentrations: 3M, 1.5M, 1.0M, 0.75M, 0.50M, 0.25M and 0.125M. Samples not treated with ammonium thiocyanate were used for control of each clone.

After reading the optical density of the sample, the affinity ratio is calculated by the following formula: AR (affinity ratio) = [ (log50-logA) x (B-a)/log B-logA ] + a; where log50 = 1.70. A is the lowest ammonium thiocyanate concentration, which reduces the absorbance by less than 50%, and B is the highest ammonium thiocyanate concentration, which reduces the absorbance by more than 50%.

6. Preparation and purification of monoclonal antibody:

samples of preselected clones 10 and 38 were placed in 100mL vials of serum free media (GIBCO VP-SFM) supplemented with 1% BSA under 10% CO aeration₂Growing in the furnace of (1). The suspension was centrifuged, filtered through a 0.22 micron filter and purified by High Performance Liquid Chromatography (HPLC) using SelectSure protein a MAB resin (GE). The antibody was adjusted to pH10.0 with 1M Tris salt, pH7.0, and dialyzed against 0.5 Xdeionized water in PBS. The samples were processed by lyophilization, resuspended in deionized water, quantified by the Lowry method, and evaluated by electrophoresis on polyacrylamide gels.

7. Targeted recognition

7.1 in vitro PBP2a recognition-immunoblotting

MRSA (CEB) Strain, methicillin-sensitive Staphylococcus aureus (MSSA) Strain, vancomycin-resistant enterococci: (Enterococcus faecium) The strain, BL-21 DE3 E.coli strain, grew in the growth exponential phase. One ml of each sample was centrifuged and lysed by glass Bead agitation in a mini-Bead blender (Biospot Products), 3 times for 30 seconds each. A portion of each sample was electrophoresed in a 12% denaturing polyacrylamide gel (SDS-PAGE) and the proteins were later transferred to a nylon membrane (Hybond N-BioRad). The membranes were blocked after two hours of gentle stirring in phosphate buffer containing 10% skim milk and 1% BSA (calf serum). The membrane was washed 3 times in PBS and (0.05%) tween 20 and 3 times in PBS. The latter was then incubated for two hours with a suspension of monoclonal antibody against PBP2a diluted in PBS at a ratio of 1: 1. After incubation, the membranes were washed as before and alkaline phosphatase was bound at a ratio of 1:15,000 (murine anti-IgG antibody Sigma A3688) and incubated for 90 minutes. After this time, it was washed again in PBS and developed with Western Blue enzyme substrate of alkaline phosphatase (Promega).

7.2 recognition of PBP2a on the surface of bacteria-flow cytometry

The mrsa (ceb) strain grows either in stationary phase (ON) or in exponential growth phase. This sample was washed in 1 × PBS at 0.6 DO₆₀₀ (~10⁸bacteria/mL) were resuspended. These latter were used in an amount of 1mL (10)⁸Bacteria) and resuspended in 0.5% BSA and 1:10 in a dilution of normal serum (mouse/human). Then, the samples were incubated at 4 ℃ for 30 minutes, and then the concentrate (pellet) was washed twice in PBS, resuspended in 100. mu.l of PBS containing 0.5% BSA and a dilution of 1:10,000 monoclonal antibody against PBP2a, and incubated at 4 ℃ for 30 minutes. After this step, the samples were washed again as before, resuspended in a dilution of 100. mu.l PBS and 0.5% BSA and (1:1000) PE (phycoerythrin) murine anti-Ig conjugate, and then incubated for 30 minutes at 4 ℃ in the dark. Then, the sample was washed again and fixed in PBS containing 2% paraformaldehyde at 4 ℃ for 15 minutes. After this preparation step, the samples were analyzed in a flow cytometer (Becton and Dickinson-FACScalibur).

8. In vitro protection test-determination of minimum inhibitory concentration

The MRSA strains (CEB, Iberian, COL and CA) grew exponentially longer at an optical density of 0.5 at 600 nm. The inoculum used was adjusted to approximately 100,000 bacteria. Muller Hinton broth, inoculum of bacteria, growth amounts of purified anti-PBP 2a monoclonal antibody were added to test tubes or 24-well plates. The plate or tube was incubated at 37 ℃ for 12 hours. After this period, the presence or absence of haze in the sample can be observed. The minimum inhibitory concentration is the minimum concentration of antibody that is capable of inhibiting the growth of a bacterial inoculum (100,000 bacteria).

9. And (3) in vivo protection detection:

9.1. lethal dose and LD of MRSA strains ₅₀ Measurement of (2)

Lethal doses and LD of the MRSA strains (CEB, Iberian, WB79CA, and COL) were performed according to the method of Reed-Muench (23)₅₀The measurement of (1). Several groups of 8-week old female Balb/C mice were observed for 7 days by inoculating increasing bacterial doses via the intraperitoneal route. Animals survived this period and were euthanized according to established animal welfare guidelines.

9.2 quantitative detection of systemic infection and bacterial Kidney

The MRSA strains (CEB, Iberian and WB79 CA) grow in the exponential growth phase (DO)₆₀₀0.6) in DO₆₀₀1 × sterile PBS in 0.5 washes and suspensions, corresponding to approximately 2 × 10⁸Bacteria. This concentration was calculated by dilution and plating on BHI agar plates containing 10 micrograms of methicillin per mL. Female 8-week-old Balb/C mice received an intraperitoneal dose of 400 micrograms of purified monoclonal antibody against PBP2a on the first day. On day six, animals were euthanized and the kidneys were aseptically excised. The kidneys were then rendered into uniformly distributed microparticles in 1mL sterile Luria broth, which was successfully dissolved in a series of 10. One hundred microliters of each dilution was inoculated onto BHI agar plates containing 10 micrograms/mL of benzyl isoxazoline, and cultured at 37 ℃The resulting clones were counted for 24 hours and the total dilution used was calculated.

9.3. Survival detection

MRSA strains were grown under the same conditions as described above, with a pre-established LD₅₀The inoculum adjustment was performed. Female 8-week-old Balb/C mice received an intraperitoneal dose of 500 micrograms of purified monoclonal antibody against PBP2a on the first day. On the following day, the animals plus control group were given an approximate 2.5 to 6.0 x 10⁸The dose of bacteria (a) is infected by the intraperitoneal route, according to the LD of each strain₅₀The animals were observed for 10 days and survivors were euthanized.

10. Comparison of monoclonal antibodies against PBP2a with in vivo protective studies for renal quantitation of vancomycin

Detection I

Four groups of female 8-week-old Balb/C mice (4 animals per group) received 6.0 x 10 via the intraperitoneal route⁷Infectious dose of individual bacteria. Animals receiving purified Monoclonal Antibodies (MAB), vancomycin, a dose of MAB + vancomycin, and a negative control group were according to the following groups:

group 1: MAB (400 microgram) (day one)

Group 2: vancomycin (150 microgram, intramuscular route, 12/12 hours)

Group 3: vancomycin + MAB (350 microgram) (one day after infection)

Group 4: control group

The first dose of antibody and vancomycin is 4 hours after administration of the infectious agent. Animals were euthanized on day four, kidneys were aseptically excised, and quantitative identification of kidneys was given as described above.

Detection II

This assay was performed in the same way as the previous one, but lower infectious agents (7.0 ×) were administered 10⁶Individual bacteria), group 1 was treated with 500 micrograms of purified monoclonal antibody; group 2 was treated with vancomycin (150 micrograms, intramuscular route, 12/12 hours; 5 doses); group 3 was treated with vancomycin +500 micrograms of monoclonal antibody; group 4 was the control group (untreated animals).

11. Recognition of Complementarity Determining Regions (CDRs) of the light and heavy chains of a monoclonal antibody against PBP2a

11.1. Extraction of mRNA from hybridoma cells

10mL of centrifugation (pellet) of cell culture medium of hybridoma producing monoclonal antibody mRNA extraction was performed using RNeasy Minikit kit (Qiagen).

11.2. Obtaining cDNA

Reverse transcriptase reaction: complementary DNA was obtained using M-MLV reverse transcriptase kit (Invitrogen) according to the instructions.

11.3. Amplification of VH and VL chains by Polymerase Chain Reaction (PCR)

The reaction was carried out using the following priming sequences (primers).

11.4. Sequencing of the light and heavy chains of the anti-PBP 2a monoclonal antibody

Sequencing:

I. amplification of

This step is carried out using the previous start sequence, defined by SEQ ID NO: 18 to SEQ ID NO: 39.

Sequencing

An ABI Prism 3100 gene analyzer (Genetic Analyzer (Hitachi)) was used.

11.5. Sequence analysis and identification of light and heavy chains of CDRs

The DNA sequences obtained were analyzed with the aid of the DNA Star program (DNA Star program) and translated into amino acid sequences (ExPASy site-translation program) by means of the algorithms of Kabat's and Chotia's as a subsequent analysis to identify the light and heavy chains of the CDRs.

Determination of association and dissociation constants of monoclonal antibodies (clones 10 and 38 by Surface Plasmid Resonance (SPR) (Biacore) method)

SPR measurements were performed using a CM-5 sensor in a Biacore X. The binding reagent and HBS-EP buffer (10 mM hepes, 150 mM NaCl, 3 mM EDTA, 0.005% P20 [ Tween 0, pH 7.4]) were obtained from GE health concern.

Example 2

1 acquisition of murine anti-PBP 2a monoclonal antibody

A group of animals was immunized according to the immunization method described previously. The results of the evaluation by ELISA are depicted in figure 1.

After the degree of fusion (fusion of 90-LATAM), suspensions from 96-well plates (hybridomas) were analyzed via ELISA detection. From this total, five best samples were selected, and the cells (clones) were expanded. The resulting suspension was then analyzed again by ELISA. The results obtained by ELISA were confirmed by immunoblotting against purified recombinant protein (PBP2a) r and confirming positive samples. The final results are in table I.

FIG. 1 shows the results obtained from an enzyme immunoassay (ELISA) for the production of antibodies against PBP2a in the sera of vaccinated animals. Each jumping wave corresponds to a 1:100 dilution of serum from the vaccinated animal. The positive control serum is a column of horns]. First post per jump: pre-immune serum; a second column: sera after the fourth immunization; and a third column: sera after the fifth immunization.

Table I: clone fused to 90: final balance

From the selected clones, the 77-38 clone was subjected to a recloning process to confirm the stability of the cells secreting the monoclonal antibody. From 50 assay wells, all showed positive results by ELISA assay. These clones were expanded and stored in liquid nitrogen on a LATAM (Laboratory of monoclonal antibody technology) apparatus.

2 growth, production and purification of monoclonal antibodies

Normalization was performed according to the procedure described previously. The productivity obtained was about 4 mg of monoclonal antibody per 100mL of suspension after the purification procedure. The results obtained are as follows.

Referring to fig. 2, we can see a polyacrylamide gel with a crude sample and a purified sample. This figure 2 shows native polyacrylamide gel electrophoresis images of suspended samples before (column 1) and after (column 2) purification by High Performance Liquid Chromatography (HPLC) on MAb SelectSure resin. Columns 3 to 9 correspond to the purified sample fragments obtained. The arrow indicates a size of approximately 150 kDa.

3 functional Properties of monoclonal antibodies

3.1 in vitro PBP2a immunoblot recognition

To investigate the antibody recognition ability at the target site of pathogenic bacteria (PBP2a), immunoblot assays were performed with bacteria (CEB and COL MRSA) that characterized PBP2a, MSSA (methicillin-sensitive staphylococcus aureus) strain (not characterized PBP2a), vancomycin-resistant enterococci strain (not characterized PBP5), transpeptidase with low affinity to β -lactam antibiotics (homologous to PBP2a), escherichia coli strain (which produces recombinant proteins) as negative control. The results obtained showed that the antibody was able to recognize a protein with a molecular weight of approximately 76kDa in MRSA and enterococcus strains, corresponding to the size of PBP2a and PBP 5. Neither the methicillin-sensitive Staphylococcus aureus (PBP2 a-negative) protein nor the E.coli strain protein seen monoclonal antibody reactivity. The results are seen in fig. 3 (immunoblots against MRSA and MSSA).

The results of immunoblot detection of MRSA and MSSA lysates and suspensions containing anti-PBP 2a monoclonal antibody are shown in fig. 3.

1 molecular weight marker (Kaleidoscope);

2 MSSA samples grown in exponential growth phase;

3 and 4 MRSA samples grown in exponential growth phase;

MSSA grown in stationary growth phase (overnight); and

6 and 7 MRSA samples grown in stationary growth phase. The left arrow points to the molecular weight of PBP2 a.

3.2 recognition of PBP2a on the cell surface-flow cytometry

The purpose of flow cytometry is to confirm the ability to target recognition of native forms of bacteria by monoclonal antibodies. In the aforementioned detection (immunoblotting), we observed that targeted recognition of proteins was via a denaturing process, which occurs during the separation of proteins by denaturing polyacrylamide gel electrophoresis. We again analyzed negative control strains (MSSA, PBP2a negative) and MRSA strains (CEB) in exponential and stationary growth phases. The results obtained show that the antibodies are able to target-recognize the bacterial surface under both of the aforementioned conditions. The presence of staphylococcus aureus surface protein a was unable to inhibit antibody binding to PBP2 a. The results obtained can be seen in fig. 4.

FIG. 4 shows the results of flow cytometry of MRSA (CEB) and MSSA bacteria cultured with a monoclonal antibody against PBP2a and labeled with Phycoerythrin (PE). (1) Is MSSA bacteria; (2) MRSA in stationary growth phase; (3) MRSA in exponential growth phase. The MRSA population exhibited a right shift corresponding to the growth of cells labeled with fluorescent conjugates.

4 evaluation of protection conferred by monoclonal antibodies against PBP2a

4.1 in vitro protection assay

Determination of Minimum Inhibitory Concentration (MIC)

These assays are aimed at evaluating the ability of antibodies to bind to a target immediately in a closed system. Positive results are very significant as the monoclonal antibodies of ultimate choice for therapy, as they mean that the antibodies are able to target recognition, blocking bacterial growth without the aid of the host immune system, i.e. without the need for binding effects of the host innate and acquired modulated immune systems, such as complementary activity and opsonization mechanisms. CEB, COL and IBIOA (Iberian) MRSA strains were evaluated, wherein similar MIC values (approximately 500. mu.g) were found in the evaluation conditions. These data indicate that the antibodies required for blocked growth do not differ regardless of the genetic background of the different MRSA strains. These results can be seen in fig. 5.

FIG. 5 shows purified monoclonal antibodies against PBP2a and vancomycin for 10 of different MRSA strains⁵Detection of inoculum of bacteria in vitro protection (MIC-minimum inhibitory concentration). The absence of turbidity indicates no bacterial growth under the conditions of the assay.

1A CEB MRSA (Brazilian epidemic clone) + 250mg of antibody;

2A CEB MRSA +500 mg antibody;

CEB MRSA + 750 mg of antibody;

4A and 6A negative control of CEB MRSA strains.

COL MRSA + 250mg antibody;

COL MRSA +500 mg antibody;

COL MRSA + 750 mg antibody;

4B and 6B negative control of COL MRSA strains.

1C Albizia (Iberian) MRSA (European epidemic clone-EEC) + 250mg of antibody;

EEC MRSA +500 mg antibody;

EEC MRSA + 750 mg antibody;

4C and 6C negative control of EEC MRSA strains.

CEB MRSA + 150 mg of vancomycin;

2D CEB MRSA + 300 mg vancomycin;

3D CEB MRSA +500 mg vancomycin;

4D CEB MRSA + 750 mg vancomycin;

5D and 6D negative control.

4.2. Detection of in vivo protective power

4.2.1. Determination of lethal and sublethal doses for CEB, Iberian, WB79CA and COL MRSA strains:

to assess the role of in vivo protection, these assays are necessary, and in two used models-kidney quantification by sub-lethal dose, and survival assay after systemic infection with larger bacterial inoculants-can lead to approximately 50% of animal deaths (LD)₅₀). The route of choice is intraperitoneal based on ease of administration and no loss. A modified Reed-Muench method was used to perform this assay, with two animals (dose of infecting bacteria at growth concentration) per condition being used to determine LD₅₀Andlethal dose, three different doses were tested, and once we know in advance the average lethal dose of the staphylococcus aureus COL MRSA strain, which is the first genomically sequenced MRSA clone, the staphylococcus aureus COL MRSA strain, was used as a reference strain to study this pathogen. However, it shows little toxicity, requiring higher doses of infection relative to other MRSA clones that cause infection in animals. Therefore, it is not used in the detection of this protective force.

4.2.2. Kidney protection following systemic infection with sublethal doses

These assays evaluate the ability of antibodies to detect a reduction in the presence of vital organ (kidney) bacteria following systemic infection using a renal quantification model following infection. Three independent tests of virulent MRSA strains from different genetic backgrounds achieved a reduction in number of up to 3 logs (1000-fold). In these tests, the animals were pre-dosed with 500 micrograms of antibody. The protection conferred by a lower antibody dose (250 micrograms) was evaluated in the test with the CA-MRSA strain, where a reduction in the number of bacteria was observed, but below the protection obtained in the 500 micrograms dose. These results can be seen in fig. 6A, 6B and 6C.

Fig. 6A shows the results of renal quantification assays for animals suffering from a systemic infection with a sub-lethal dose of MRSA CEB strain, treated with monoclonal antibody against PBP2a and not treated with monoclonal antibody against PBP2 a. In the horizontal strip portion: is a record of the concentration of isolated bacteria in the kidney of each untreated animal. In checkered mode: is a record of the number of bacteria isolated from the kidney of each animal treated with antibody. And (3) quantitative determination of bacteria: controlling: c1 is 2000 bacteria; c2: 29, 000 bacteria: c3: 220, 000 bacteria: c4: 52,000 bacteria (75,750 bacteria on average). Treated (protected) animals: p1: 20 bacteria; p2, P3 and P4: 10 bacteria (12.5 bacteria on average). Reduction in the number of bacteria recovered from treated animals compared to untreated animals: 6060 times.

FIG. 6B shows the results of renal quantification assays for animals suffering from systemic infection with a sub-lethal dose of the Epibian (Iberian) MRSA strain (European epidemic clone strain), treated with monoclonal antibody against PBP2a and not treated with monoclonal antibody against PBP2 a. In the horizontal strip portion: is a record of the concentration of isolated bacteria in the kidney of each untreated animal. In a large lattice mode: is a record of the number of bacteria isolated from the kidney of each animal treated with antibody. And (3) quantitative determination of bacteria: controlling: c1 is 210,000 bacteria; c2: 44, 000 bacteria: c3: 300, 000 bacteria: c4: 290,000 bacteria (211,000 bacteria on average). Treated (protected) animals: p1: 80 bacteria; p2: 200 bacteria; p3: 10 bacteria; and P4: 60 bacteria (average 87.5 bacteria). Reduction in the number of bacteria recovered from treated animals compared to untreated animals: 2420 times.

Fig. 6C shows the results of renal quantification assays for animals suffering from systemic infection with a sub-lethal dose of WB79CA-MRSA strain (brazilian population strain), treated with monoclonal antibody against PBP2a and not treated with monoclonal antibody against PBP2 a. Five first stripes (in xx, horizontal skip, and large lattice patterns): is a record of the concentration of isolated bacteria in the kidney of each untreated animal. First strip (in xx): relative to the evaluation of animals that died prior to euthanasia. Strip 6, strip 7, strip 8 and strip 9 (in horizontal jump and checkerboard pattern): record of the number of bacteria isolated in the kidneys of animals treated with 250mg of monoclonal antibody against PBP2 a. Strip 10, strip 11, strip 12 and strip 12 (in the triangle): record number of bacteria isolated in kidney of each animal treated with 500mg of antibody. The square bars (in the 5 th, 9 th and 13 th bars) indicate the average obtained respectively. And (3) quantitative determination of bacteria: controlling: c1 is 650,000 bacteria; c2: 26, 000 bacteria: c3: 17, 000 bacteria: c4: 500,000 bacteria (measurement of dead animals) (average 231,000 bacteria). Animals treated with 250mg of antibody: p1: 0 bacteria; p2: 5,400 bacteria; p3: 830 bacteria; and P4: 10 bacteria (average 1, 560 bacteria). Animals treated with 500mg antibody: p1: 80 bacteria; p2: 0 bacteria; p3: 210 bacteria; p4: 80 bacteria (average 92.5), reduction in the number of bacteria recovered in animals treated with 250mg of antibody compared to untreated animals: 149 times. Animals treated with 500 mg: 2,497 times.

4.2.3. Survival detection

In this test type, we evaluated animals that were able to cause 50% or more (LD)₅₀) Dead bacteria load post-infection animals gain protection from antibodies. The protective power against these three strains in a renal quantification assay was measured. In these three independent assays, significant reductions in (i) survival time and (ii) survival rate have been observed in animals treated with anti-MRSA monoclonal antibodies.

In the detection of CEB MRSA strains, 70% of the treated animals survived, compared to only 10% of the control (untreated). In the detection of the Iberian MRSA strain, the results obtained were similar, with 60% protection in treated animals and 100% mortality in control. In the detection of the CA MRSA strain, 100% protection was seen compared to 70% of the control. These results are all visible in fig. 7A, 7B and 7C.

FIG. 7A shows the transperitoneal approach (LD)₅₀) Administration of 2.3X 10⁸Survival curves for treated (unprotected) and untreated (control) animals following infection with individual (CEB MRSA) bacterial doses.

FIG. 7B shows the transabdominal approach (~ LD)₅₀) Administration of 4.2X 10⁸Survival curves for treated (unprotected) and untreated (control) animals following infection with individual (Iberian MRSA) bacterial doses.

FIG. 7C shows the trans-abdominal approach (~ LD)₅₀) Administration of 1.1X 10⁹Survival curves for treated (unprotected) and untreated (control) animals following infection with a WB79CA-MRSA bacterial dose.

4.2.4. Comparative study of the protective power conferred by monoclonal antibodies compared to vancomycin

Vancomycin is the first antibiotic to treat acute infections caused by MRSA, and comparative studies on protective power were conducted in a different model than ever. In this study, animals were exposed to infection and antibiotics or monoclonal antibodies were administered all four hours after infection. This study was performed in three different groups: one treated with vancomycin, the other treated with monoclonal antibody, and a third administered simultaneously with the antibiotic + antibody combination. The dose of vancomycin is adjusted and administered in a similar manner as administered to humans (500 mg every 12 hours).

The results obtained show that the number of bacteria present in the kidneys of animals treated with antibiotics or antibodies decreases to an extent of approximately 15-fold three days after the infection has occurred. However, a 4617-fold reduction was seen in the group of animals receiving antibiotic and antibody treatment.

Based on these results, we can conclude that the protection obtained by the antibody agent corresponds to 5 vancomycin doses and that the simultaneous combined administration of vancomycin and antibody is very effective in reducing the bacterial load observed in the kidneys of infected animals. This study was again repeated with slightly reduced infectious doses, in order for us to be able to better observe the protective effect of anti-MRSA monoclonal antibodies used alone and in combination with vancomycin (see figure 8).

In the implementation of a second test at a lower infectious dose, the results obtained confirm the initial conclusions. The protection obtained with the monoclonal antibody resulted in an 89-fold reduction, which is higher than the protection obtained with treatment with 5 vancomycin doses (35-fold reduction). However, the most significant reduction results were seen in the antibody + vancomycin treated group, resulting in a 450-fold reduction.

FIG. 8A shows a graph consisting of 6.0 x 10⁷(CEB MRSA) bacteria infected animals were treated with anti-PBP 2a monoclonal antibody, vancomycin, and antibody + vancomycinBacterial counts in kidney of treated and untreated animals were combined with administration of the agent. Treatment started 4 hours after infection. Vancomycin was administered every 12 hours (5 doses). Column 1, column 2, column 3, column 4 and column 5 (jumping wave): record of bacterial concentration recovered from untreated animals (control). C1: 7,000,000; c2: 295,000, respectively; c3: 380,000; c4: 3,200,000 (average: 2,718,750 bacteria). Column 6, column 7, column 8, column 9 and column 10 (checkerboard pattern): record of recovered bacterial concentration in animals treated with 400mg of monoclonal antibody against PBP2 a. P1: 4,200; p2: 310,000; p3: 330,000; p4: 90,000 (183,550 bacteria on average). Columns 11, 12, 13, 14 and 15 (spherical): animals were treated with vancomycin. P1: 110,000; p2: 58,000, P3: 500,000, P4: 21,000 (172,250 bacteria on average), columns 16, 17, 18, 19 and 20 (triangles): record of recovered bacterial concentration in animals treated with antibody (300 mg) + vancomycin. P1: 1,100, P2: 700, P3: 450, P4: 90 (on average 585 bacteria).

FIG. 8B shows a graph consisting of 7.0 x 10⁶(CEB MRSA) bacterial infected animals were dosed with a monoclonal antibody against PBP2a (columns 7 to 12), vancomycin (columns 13 to 18), antibody + vancomycin (columns 19 to 24) in combination and untreated (columns 1 to 6) animals were dosed with bacterial numbers in the kidneys. Treatment was started 4 hours after infection. Vancomycin was administered every 12 hours (5 doses). Columns 1 to 6: record of recovered bacterial concentration in untreated animals (control). C1: 6,000, C2: 1,000, C3: 500, C4: 118,000, and C5: 1,000 (an average of 25,220 bacteria). Columns 7 to 12: record of recovered bacterial concentration in animals treated with 500mg of monoclonal antibody against PBP2 a. MB1: 450, MB2: 200, MB3: 100, MB4: 20, MB5:0 (284 bacteria on average). Columns 13 to 18: vancomycin-treated animals. VC1: 100, VC2: 700, VC3: 0, VC4: 0, VC5: 2800 (720 bacteria on average). Columns 19 to 24: record of recovered bacterial concentration in animals treated with antibody (500 mg) + vancomycin in combination. MBV1: 130, MBV2: 20, MBV3: 10, MBV4: 80, MBV5: 20 (56 bacteria on average).

5. Affinity assay

Results of affinity assay of monoclonal antibody clones 10 and 38

Method for measuring affinity in urine:

clone (clone) 10: 1.03/1.46 (read treated/untreated DOs) = 70.5%;

clone (clone)38: 1.00/1.21 = 82.6%.

Method for determination of affinity of ammonium thiocyanate:

clone (clone)10 (DOs):

control group 1.12.

Thiocyanate-treated samples (Thiocyanate-treated samples):

2 M = 0.046; 1.5 M = 0,047; 1 M = 0.107; 0.75 M = 0.483; 0.5 M = 0.602; 0.375 M = 0.684；

affinity ratio: 2.47;

clone (clone)38 (DOs):

control group 1.22.

Thiocyanate-treated samples (Thiocyanate-treated samples):

2M = 0.056; 1.5M = 0.062; 1M = 0.129; 0.75M = 0.648; 0.5M = 0.758; 0.375 M = 0.793；

affinity ratio: 4.40.

it can be seen that in both tests, the affinity ratio of clone 38 was higher than that of clone 10. Further, according to both methods, it was confirmed that the antibody of clone 10 was required to be used more than 50 times as much as the antibody of clone 38 in order to achieve DO close to 1.0.

6. Determination of the Association and dissociation constants of monoclonal antibodies (surface plasmid response method [ SPR ] [ BIAcore ]) clones 10 and 38)

The results obtained by the SPR method confirmed the preliminary results for antibody affinity, in which clone 38 again showed higher affinity than clone 10. Based on the data shown in table II, we found that clone 38 showed a 450-fold higher affinity than clone 10.

This affinity is mainly due to its higher association rate, which is about 100 times higher than that of clone 10. Based on the very high affinity of clone 38, it measured parameters close to the measurement threshold of the device. However, based on experimental planning and implementation concerns, we obtained excellent adjustment of experimental data using the Langmuir's model, which indicates that the obtained data is authentic.

FIG. 9 shows the interaction between recombinant PBP2a (antigen) and monoclonal antibody clone 38 (FIG. 9A) and clone 10 (FIG. 9B). The smoke-like curves represent SPR data according to the concentration of the correct key point. All samples were analyzed in replicates and the 1:1 Langmuir's theoretical model for each curve was shown to be black under each curve. The answering unit is represented in the vertical axis and time in seconds is represented in the horizontal axis. The line closest to the horizontal axis represents the base line of each sample (negative control).

TABLE II

Dissociation and interaction constants between clone 10 and clone 38 of antigen (PBP2a) and monoclonal antibody

7. Determination of Complementarity Determining Regions (CDRs) of the light and heavy chains of a monoclonal antibody against PBP2a

After mRNA extraction in hybridoma cells of the clone producing the antibody used (clone 38), cDNA was obtained and PCR reactions were performed using this material with different light and heavy chain alleles. Sequencing is facilitated by using material obtained from the same start sequence (defined by SEQ ID NO: 18 to SEQ ID NO: 39) in the PCR reaction. Light chain 391 and heavy chain 310 were determined in three different sequencers. Using the Kabat's and Chotia's algorithms we obtained a confirmation of the CDRs of the light and heavy chains, which is the object of the claims appended to this application.

We will present the sequences of the light and heavy chains of the CDRs below.

6-CDR1 light chain amino acid

RSSQSIGHSNGNTYLE

7-CDR2 light chain amino acids

KVSNRFS

8-CDR3 light chain amino acid

FQGSYVPLT

9-CDR1 light chain DNA

cgcagcagccagagcattggccatagcaacggcaacacctatctggaa

10-CDR2 light chain DNA

aaagtgagcaaccgctttagc

11-CDR3 light chain DNA

tttcagggcagctatgtgccgctgacc

12-CDR1 heavy chain amino acids.

GFSITSSSSCWH

13-CDR2 heavy chain amino acids

RICYEGSISYSPSLKS

14-CDR3 heavy chain amino acids

ENHDWFFDV

15-CDR1 heavy chain DNA

ggctttagcattaccagcagcagcagctgctggcat

16-CDR2 heavy chain DNA

cgcatttgctatgaaggcagcattagctatagcccgagcctgaaaagc

17-CDR3 heavy chain DNA

gaaaaccatgattggttttttgatgtg

Complete data

Further additional tests were carried out to carry out the development of the present invention. These will be elucidated below by way of examples.

Example 3

A second study using CEB MRSA strains, using the same method described in example 1, item 7.2, assisted vortex agitation with two pulses, 15 seconds each, after each wash to aid in the dissociation of staphylococcus aureus clumps and increase the number of PBP2a exposed to antibodies.

In control samples (i pure bacteria, not exposed to monoclonal antibodies; and ii pure bacteria plus FITC or PE markers) and samples treated with monoclonal antibodies, the markers FITC (fluorescein isothiocyanate)) and PE (phycoerythrin) were detected after labeling with analytically available PE or FITC. Read in linear mode in a facsimiur instrument.

Fig. 10 is a graph of flow cytometry analysis of MRSA samples in the presence of FITC-labeled anti-PBP 2a antibody. Curve (x) corresponds to the unlabeled sample and curve (y) corresponds to the labeled sample.

The results obtained show that approximately 22% of the labelled population was detected by the instrument, confirming the recognition of the anti-PBP 2a antibody for the target present on the bacterial surface (PBP2a) (fig. 10).

Example 4

The inventors have also investigated the protection conferred by the monoclonal antibody against PBP2a of methicillin-resistant staphylococcus aureus against enterococci. According to what has been described hereinbefore, the antibody recognizes a protein present in enterococci, possibly PBP 5-a transpeptidase with low affinity for β -lactams, present in all enterococci strains, having a molecular weight of about 76kDa (237 amino acids). This enzyme exhibits homology to PBP2a of MRSA according to the following comparison of related gene sequences (ClustalW).

PBP5Efas MERSNRNKKSSKNPLILGVSALVLIAAAVGGYYAYSQWQAKQELAEAKKTATTFLNVLSK 60

PBP5efam -----KHGKNRTGAYIAG--AVILIAAAGGGYFYYQHYQETQAVEAGEKTVEQFVQALNK 53

PBP2a ------------MKKIKIVPLILIVVVVGFGIYFYASKDKEINNTIDAIEDKNFKQVYKD 48

* ::::... * : * : * :. ..

PBP5Efas QEFDKLPSVVQEASLKKNGYDTKSVVEKYQAIYSGIQAEGVKASDVQVKKAKDNQYTFTY 120

PBP5efam GDYNKAAEMTSKKAANKSALSEKEILDKYQNIYGAADVKGLQISNLKVDKKDDSTYSFSY 113

PBP2a SSY-----------ISKSDNGEVEMTERPIKIYNSLGVKDINIQDRKIKKVSKNKKRVDA 97

.: .*. . .: :: **.. .:.:: .: ::.* ... .

PBP5Efas KLSMSTPLGEMKDLSYQSSIAKKGDTYQIAWKPSLIFPDMSGNDKISIQVDNAKRGEIVD 180

PBP5efam KAKMNTSLGELKDLSYKGTLDRNDGQTTINWQPNLVFPEMEGNDKVSLTTQEAARGNIID 173

PBP2a QYKIKTNYGNIDRN-VQFNFVKEDGMWKLDWDHSVIIPGMQKDQSIHIENLKSERGKILD 156

: .:.* *::. : .: ::.. : *. .:::* *. ::.: : :: **:*:*

PBP5Efas RNGSGLAINKVFDEVGVVPGKLGSGAEKTANIKAFSDKFGVSVDEIN---QKLSQGWVQA 237

PBP5efam RNGEPLATTGKLKQLGVVPSKLGDGDEKTANIKAIASSFDLTEDAIN---QAISQSWVQP 230

PBP2a RNNVELANTGTHMRLGIVPKNVS-----KKDYKAIAKELSISEDYINNKWIKIGYKMIPS 211

**. ** . .:*:** ::. . : **::..:.:: * ** :. : .

PBP5Efas DSFVPITVASEPVTELPTG--AATKDTESRYYPLGEACAINR-VYGTITAEDIEKN--PE 292

PBP5efam DYFVPLKIIDGATPELPAG--ATIQEVDGRYYPLGEAAAQLIGYVGDITAEDIDKN--PE 286

PBP2a FHFKTVKKMDEYLSDFAKKFHLTTNETESRNYPLEKATSHLLGYVGPINSEELKQKEYKG 271

* .:. . .::. : ::.:.* *** :* : * *.:*::.::

PBP5Efas LSSTGVIGKTGLERAFDKELRGQDGGSLVILDDK-ENVKKALQTKEKKDGQTIKLTIDSG 351

PBP5efam LSSNGKIGRSGLEMAFDKDLRGTTGGKLSITDAD-GVEKKVLIEHEVQNGKDIKLTIDAK 345

PBP2a YKDDAVIGKKGLEKLYDKKLQHEDGYRVTIVDDNSNTIAHTLIEKKKKDGKDIQLTIDAK 331

.. . **:.*** :**.*: * : * * . :.* :: ::*: *:****:

PBP5Efas VQQQAFAIFDKRPGSAVITDPQKGDLLATVSSPSYDPNKMANGISQKEYDAYNNNKDLPF 411

PBP5efam AQKTAFDSLGGKAGSTVATTPKTGDLLALASSPSYDPNKMTNGISQEDYKSYEENPEQPF 405

PBP2a VQKSIYNNMKNDYGSGTAIHPQTGELLALVSTPSYDVYPFMYGMSNEEYNKLTEDKKEPL 391

.*: : : ** . *:.*:*** .*:**** : *:*:::*. :: . *:

PBP5Efas STFKTARFATGYAPGIITGAIGLDAGTLKPDEELEINGLKWQKDKSWGGYFATRVKEAS- 470

PBP5efam STFKISRFATGYAPGMITAAIGLDNGTIDPNEVLTINGLKWQKDSSWGSYQVTRVSDVS- 464

PBP2a STQKLNKFQITTSPGILTAMIGLNNKTLDDKTSYKIDGKGWQKDKSWGGYNVTRYEVVNG 451

:* :**** *::*. ***: *:. . *:* ****.***.* .** . ..

PBP5Efas PVNLRTALVNSDNIYFAQQTLRMGEDKFRAGLNKFIFGEELDLPIAMTPAQISNEDKFNS 530

PBP5efam QVDLKTALIYSDNIYTAQETLKMGEKKFRIGLDKFIFGEDLDLPISMNPAQISNEDSFNS 524

PBP2a NIDLKQAIESSDNIFFARVALELGSKKFEKGMKKLGVGEDIPSDYPFYNAQISNKN-LDN 510

::*: *: ****: *: :*.:*..**. *:.*: .**:: .: *****:: ::.

PBP5Efas EILLADTGYGQGQLLISPIQQATMYSVFQNNGTLVYPKLVLDKETKK-KDNVISANAANT 589

PBP5efam DILLADTGYGQGELLINPIQQAAMYSVFANNGTLVYPKLIADKETKD-KKNVIGETALQT 583

PBP2a EILLADSGYGQGEILINPVQILSIYSALENNGNINAPHLLKDTKNKVWKKNIISKENINL 570

:*****:*****::**.*:* ::**.: ***.: *:*: *.:.* *.*:*. :

PBP5Efas IATDLLGSVEDPSGYVYNMYNPNFSLAAKTGTAEIKDKQDTDGKENSFLLTLDRSNNKFL 649

PBP5efam IVPDLREVVQDVNGTAHSLSALGIPLAAKTGTAEIKEKQDVKGKENSFLFAFNPDNQGYM 643

PBP2a LNDGMQQVVN--KTHKEDIYRSYANLIGKSGTAELKMKQGESGRQIGWFISYDKDNPNMM 628

: .: *: . .: * .*:****:* **. .*:: .:::: : .* :

PBP5Efas TMIMVENSGENGSATDISKPLIDYLEATIK---------- 679

PBP5efam MVSMLENKEDDDSATKRASELLQYLNQNYQ---------- 673

PBP2a MAINVKDVQDKGMASYNAKISGKVYDELYENGNKKYDIDE 668

::: :.. *: :. . : :

The sequences corresponding to PBP5 in enterococcus faecalis (enterococcus E. faecalis (Efas)) and enterococcus frequens (Efam)) with MRSA PBP2A were compared. The marked sequence (bold-PBP 5; underlined-PBP 2a) corresponds to the PBP2a region used for the generation of the monoclonal antibody. The amino acids corresponding to the active center of the enzyme are marked in italics.

Thus, in vitro protection assays, lethal doses and LD₅₀The in vivo assays (sub-lethal dose for kidney quantification and survival assay for lethal dose) were performed in a murine model (Balb/C mice) with enterococcus strains, as previously performed with MRSA. These results are visible in the corresponding reports.

1.1. Detection of protective Effect in vitro

The purpose of this assay was to evaluate the in vitro protective effect of antibody administration against the VRE enterococcus strain.

In vitro protection assay (MIC), monoclonal antibody (90/DA5/CB5/AA3 hib 77) purified from clone 38 against Enterococcus clinical strain (Enterococcus f. clinical strain (VRE)), Richet laboratories.

Conditions are as follows:

purifying the suspended substance by HPLC of SelecSure MAB resin, separating out, and freeze-drying;

antibody quantitation (Lowry's method) 3.5 mg/mL;

the inoculum is VRE strain;

preincubation of 1 VRE population at 20 mL of Lb and vancomycin (10 mg/mL) at 37 ℃, 160 rpm;

inoculum, 400 mL of pre-inoculum in 20 mL Lb, 200-mL erlenmayer, 37 ℃, 160 rpm;

reading DO after 7 hours_600nmIs 0.7;

quantitative determination of 5.5X 10⁸bacteria/mL.

Detection conditions are as follows:

inoculum of 5.5X 10⁵ bacteria；

Antibody concentration: 300, 400, 500, 600, and 700 mg of antibody;

1mL of Luria broth;

cell culture dish, 24 wells;

positive control: luria broth + bacterial inoculum;

negative control l Luria broth;

incubation for 18 hours at 37 ℃.

Fig. 11 shows the results of the evaluation of the protective effect imparted by the antibody. In fig. 11, we have:

A. 300 mg of antibody;

B. 400mg of antibody;

C. 500mg of antibody;

D. 600 mg of antibody;

E. negative control;

F. a positive control;

G. 700 mg of antibody.

1.2. Vancomycin-resistant enterococcus faecium(s) entered via the intraperitoneal routeEnterococcus faecium) LD of₅₀And determination of lethal dose

The scheme is as follows:

female 7-week-old Balb/C animals with an average body weight of 20 grams;

day 1-pre-inoculation: 1 VRE strain population in 10mL Lb broth and vancomycin (10 mg/mL), 50 mL Falkow, grown at 37 ℃ at 160 rpm;

day 2-inoculation 1mL of pre-inoculum in 50 mL Lb broth/vancomycin (250-mL erlenmayer) -4 vials, 37 ℃, 160 rpm, grown until OD_600nm = 0.80；

Centrifuge for 10 minutes at 4000 rpm, suspend in 1 Xsterile PBS, OD 1.2.

Quantitative determination 2.1X 10⁸bacteria/mL

A. 60 microliter (1.5X 10)⁷)；

B. 300 microliter (6.5 x 10)⁷)；

C. 900 microliter (reduced to 300 microliter/dose) (1.5X 10)⁸)；

D. 4.5 mL (reduced to 300. mu.L/dose) (6.5X 10)⁸)；

E. 9.0 mL (1.2 x 10⁹Bacteria) (reduced to 300 μ l/dose);

F. 45.0 mL (6.5 x 10⁹individual bacteria) to 300 microliters/dose);

observations of the animals were performed from day 2 to day 10 of the test.

The results are shown in FIG. 12, which includes a lethal dose of 1.2X 10⁹Individual bacteria, LD₅₀Is 6.5 x 10⁸And (4) bacteria.

Renal bacteria quantification of animals surviving on day 7:

A (1.5 x 10⁷) No bacteria growth;

B (6.5 x 10⁷) No bacteria growth;

C (1.5 x 10⁸) 3100 bacteria;

D (6.5 x 10⁸) 2.8X 104 bacteria.

1.3. In vivo protection test-survival test-lethal dose, systemic infection in murine models by administration via the intraperitoneal route

The purpose of this assay was to evaluate the resistance of the anti-PBP 2a monoclonal antibody to a lethal dose of enterococcus faecium strain (Enterococcus faecium(VRE)) resulting linesProtection in vivo of systemic infection.

1. Antibodies (purified in suspension in cell culture in serum media)

Precipitated and lyophilized purified samples (HPLC selectsure MAB), resuspended and filtered before use.

Quantitative determination (Lowry's method) 1.0 mg/mL.

2. Mouse model: female, 8-week old Balb/C animals weighing from 23 to 25 grams.

3. The scheme is as follows:

group A (6 animals) 650 mg of antibody (350 mg + 300 mg)

Grade B (6 animals) control group (alkaline dosing)

4. Preparation of bacterial inoculum:

VRE strain:

pre-inoculation, day 1,10 mL BHI broth and 10 mg/mL vancomycin, ON, 37 ℃, 160 rpm;

inoculation, day 3 300 mL of Pre-inoculum in 30 mL of BHI broth and vancomycin, DO₆₀₀At 1.31, centrifuge for 10 min, 4,000 rpm, suspend in sterile 0.5x PBS, adjust to OD = 1.10, quantify dilutions and plates (2.0 x 10)⁸bacteria/mL), inoculation 12 mL, centrifugation suspended in 300 mL, IP route (-2.2X 10)⁹Individual bacteria).

And (3) time table:

day 1 IP vaccination with antibody (350 mg);

day 2 IP vaccination with antibodies (300 mg), systemic infection in the afternoon (IP, 250 mL of bacterial solution-2.2X 10)⁹Individual bacteria);

days 2 to 13: observing the animal;

the results are shown in fig. 13. Only 2 treated animals died (66.6% protection). All animals in the control group died the next day.

1.4. Detection of in vivo protective Effect-vancomycin-resistant enterococcus faecium in murine models by systemic infection by the Abdominal route

For the purpose of evaluating the strain of enterococcus and enterococcus faecium: (E. faecium (VRE) strain), efficacy of in vitro protection of monoclonal antibodies against PBP2a and PBP 5.

And (3) detection:

1. antibodies (purified in suspension in cell culture in serum media)

-purified sample precipitation, lyophilization and resuspension (AffiPrep protein a Biorad/HPLC selectsure MAB);

quantitative determination (Lowry's method) 1.5 mg/mL;

2. murine models female, 8 week old Balb/C animals weighing from 19 to 23 grams.

3. The scheme is as follows:

group A (4 animals) 500 micrograms of antibody (within 2 months, day 1, day 2);

group B (4 animals): unprotected control group.

4. Preparation of bacterial inoculum:

iberian MRSA strain:

pre-inoculation, 10mL Lb Broth ON, 37 ℃, 120 rpm

Inoculation of 200. mu.l of Pre-inoculum in 20 mL of Lb Broth, DO₆₀₀At 0.80, centrifuge for 10 min, 4,000 rpm, resuspend in 0.5x sterile PBS, adjust to OD = 0.51, quantify dilutions and plates (2.4 x 10)⁸Individual bacteria/mL); 500 mu of inoculumThe liter, IP pathway (2.4X 10)⁸Individual bacteria).

And (3) time table:

day 1, 250 micrograms of antibody was inoculated intraperitoneally;

day 2 250 micrograms of antibody were inoculated intraperitoneally and infected systemically (intraperitoneally, 500 micrograms of bacterial solution);

day 6, euthanasia, quantitative determination of bacteria in the kidney.

Based on the above results, we can specify the following:

detection of in vitro protection-determination of minimum inhibitory concentration:

a total of 700 micrograms of antibody was able to block the growth of 550,000 bacteria. These values are higher than the MIC obtained from MRSA strains, which is about 500 micrograms.

Detection of in vivo protection:

measurement of in vivo protective Effect-systemic infection of vancomycin-resistant enterococcus faecium with lethal dose administered in murine models by intraperitoneal route

Animals received 500 micrograms of monoclonal antibody, the intraperitoneal route (IP), suffered from systemic infection, the IP route, with 2.4x 10⁸The bacterium of (1). After the fourth day, they were euthanized and the kidneys were excised for bacterial quantification. Treated animals presented an average of 87.5 bacteria per animal, and when the control group (untreated infected animals) presented an average of 211,000 bacteria per animal.

Survival assay for lethal dose administration

Animals received 650 micrograms of monoclonal antibody (IP route), suffered systemic infection, observed daily for 10 days. Control (untreated) animals died the next day after infection; two of the treated animals (6) died the next day; other animals remained alive until the end of the experiment. The survival rate reaches 66.6 percent.

Thus, we have demonstrated that anti-PBPa monoclonal antibodies to MRSA show cross-protection against enterococci. However, the dose required to confer protection is higher than the dose used under similar conditions to combat MRSA. This is probably due to the fact that the ability of the antibody to recognize PBP5 is lower than the same titer of PBP2a that it has been developed to recognize.

Thus, the current invention described herein, a monoclonal antibody against PBP2A, is capable of specifically binding to PBP2a and homologous sequences-bacteria carrying this protein or similar substances (MRSA, MRSE, and enterococci and any other pathogenic bacteria having proteins homologous to PBP2a) are capable of causing infections.

It is worth emphasizing that the products of the invention can be applied in any aspect of the infection caused by this pathogen, once these infections become a global problem, against the tests carried out against the predominantly known prevalent MRSA clonal strains.

The literature relevant to the inventors' technical field, cited in the current illustrative report, is listed below.

1. Clinical and economic analysis of methicillin-resistant Staphylococcus aureus infections (Clinical and environmental analysis of methicillin-resistant Staphylococcus aureus) by Kopp, BJ. Nix DE, Armstrong EP.Staphylococcus aureus infections. The Annals of Pharmacotherapy); 38:1377-82. 2004.

2. Is the epidemiological and microbiological alteration of Boyce, JM. methicillin-resistant Staphylococcus aureus? (Are the epidemic and microbiology of methicillin-resistantStaphylococcus aureus changing？)JAMA; 279(8):623-4. 1998.

3. Hunt, C, Dionne M, et. al, Four pediatric cases of death (Four pediatric deaths from communality-acquired methicillin-resistant Staphylococcus aureus) obtained in the U.S. population of Minnesota and North DakotaStaphylococcus aureusin Minnesota and North Dakota), 1997-1999, the weekly journal of disease and Mortality reported by the U.S. CDC (morbid and mortuality weekly report-CDC USA), 48(32), 707-10.1999.

4. O' Brien, FG; Pearman, JW; Gracey, M; Riley, TV. Grubb, WB. Hospital outbreaks of strains of methicillin-resistant Staphylococcus aureus (Community strain of methicillin-resistant)Staphylococcus aureusinnovated in a domestic outbreak) Journal of clinical microbiology (Journal of Clin Microbiol) 37(9): 2858-62.1999.

5. Reduced susceptibility of staphylococci to vancomycin and other glycopeptides (characterisation of vancomycin and other glycopeptides) by Tenover, FC, Lancaster, MV, Hill, BC, Stedward, CD, Stocker, SA, Hancock, GA, et. alstaphylococciwith reduced microorganisms to microorganisms and other microorganisms, Journal of clinical microbiology 36(4) 1020-27.1998.

6. Lutz, L; Matos, SB; Kuplich, N; Machado A; Barth AL. Características laboratoriais de Staphylococcus aureus isolado de paciente que n?o respondeu ao tratamento com vancomicina. Primeiro Encontro de Controle de Infec??o Hospitalar do MERCOSUL. 1999.

7. Decreased susceptibility of Cosgrove, SE, Carroll, KC, Perl, TM. Staphylococcus aureus to vancomycin (Staphylococcus aureuswith reduced susceptibility to vancomycin) clinical infectious disease (Clin infection Diseases) 39(4) 539-45.2004.

8. National prevalence of methicillin-resistant Staphylococcus aureus in healthcare facilities (National prediction of methicillin resistance)Staphylococcusaureus in inpatients at US health care facilities), 2006. Am J Infect Control. 35(10):631-7. 2007.

9. YAMAUCHI, M. Japan struck by resistant Staphylococcus aureus. British Medical Journal. 306:740. 1993.

10. Methicillin-resistant Staphylococcus aureus (Methicillin-resistant) in U.S. Hospital, Panlilo, AL; CURVER, DH; GAYNES, RPStaphylococcus aureus in US hospitals), 1975-1991. Infection Control and Hospital Epid. 13:582-86. 1992.

11. LOWRY, F. infection with Staphylococcus aureus (Staphylococcus aureus.) New England Journal of medicine, 339: 520-32.1998.

12. FARR, BM Prevention and control of methicillin-resistant Staphylococcus aureus infections (Prevention and control of methicillin-resistant Staphylococcus aureus)Staphylococcus aureus infections.) Current Opinion Infectious Diseases. 17:317-22. 2004.

13. TIEMERSMA, EW, BRONZWA, ER, SLAM, et. al. Methicillin-resistant Staphylococcus aureus (Methicillin-resistant) in EuropeStaphylococcus aureus in Europe), 1999-2002. Emerging Infectious Diseases. 10:1627-34, 2004.

14. Use of molecular epidemiology for the monitoring of the nosocomial spread of methicillin-resistant Staphylococcus aureus in the university Hospital, BERETTA, ALRZ; TRABASSO, P.; STUCCHI, RB; MOLETTI, ML, 1991 to 2001 (Use of molecular epidemiology to monitor the nosocomial diagnosis of methicillin-resistant Staphylococcus aureus)Staphylococcus aureus in an University hospital)from 1991-2001. Braz. Journal of Medical and Biological Research. 37:1345-51. 2004.

15. Pannuti, CS; GRINBAUM, RS. overview of Hospital infections in Brazil (An overview of nosocomial Infection Control in Brazil.) Infection Control and Hospital Epidemiology (Infection Control and Hospital Epidemiology.) 16(3): 170-74.1995.

16. RESENDE, EM, COUTO, BRGM, STARLING, CEF, M Lo DENA, CM, Prevalence of Hospital infections in general hospitals in Belo Olympate, Infection Control and Hospital Epidemiology (Infection Control and Hospital Epidemiology), 19(11), 872-76.1998.

The Emergence and resurgence of methicillin-resistant Staphylococcus aureus (Emergene and resurgence of Methicillin-resistant Staphylococcus aureus) is a public health threat (Grundmann H., Aires de Souza M., Boyce J., and Tiemers J., methicillin-resistant Staphylococcus aureus)Staphylococcus aureusas a public street health) Lancet (Lancet) 2006.368: 874-85.

17. GUIGNARD, B; ENTENZA, JM; MOREILLON P. Beta-lactams against methicillin-resistant Staphylococcus aureus. Curr. Opin Pharmacol. 5(5):479-89. 2005.

18. SENNA, JP, ROTH, DM, OLIVEIRRA, JS, MACHADO, DC, SANTOS DS. Protective immune response against methicillin-resistant Staphylococcus aureus (Protective immune response) in murine models using DNA vaccinesStaphylococcus aureus in a murine model using a DNA vaccine approach). Vaccine. 21:2661-66. 2003.

19. DNA vaccines of the sequence OHWADA, A, SEKIYA, M, HANAKI, H, ARAI, KK, HIRAMATSU, K, FUKUCHI, Y. mecA evoke an antibacterial immune response against methicillin-resistant Staphylococcus aureus (DNA vaccination by mecA sequence vaccine against an antibacterial animal vaccine against methicillin-resistant Staphylococcus aureusStaphylococcus aureus.) Antimicrob Agents and Chemother. 44:767-74. 1999.

20. Suitable therapeutic methicillin-resistant Staphylococcus aureus (MRSA) strainsClinical features of infection (Clinical ailments of unpropropriated methicillin-resistantStaphylococcus aureus infections). J. Infect. 57(2):110-5. 2008.

Sequence comparison of the mecA genes isolated from methicillin-resistant Staphylococcus aureus and Staphylococcus epidermidis (Sequence comparison of mecA genes isolated from methicillin-resistant Staphylococcus aureus)S. aureus and S. epidermidis.) Gene. 94:137-38. 1990.

21. Evaluation of humoral immune response in murine models of naked DNA vaccine against methicillin-resistant Staphylococcus aureus (Evaluation of the humanized immune response in BALB/c microbial immune with a bound DNA vaccine-methicillin-resistantStaphylococcus aureus.) Genetics Molecular Research. 5(3):503-12. 2006.

22. Production of YOKOYAMA, w. monoclonal antibodies: induction of an immune response (Production of monoclonal antibodies: induction of immune responses), p.2.5.4-2.5.8. In: J.E. Coligan, A.M. Kruisbeam, D.H. Margulies, E.M. Shevach and W. Strober (ed.) Current Protocols In Immunology, vol.1. Wiley and Sons, Hoboken, N.J. 1995.

23. REED, LJ AND MUENCH H. Am. J. Hyg. 27:493-97. 1938.

24. KABAT, EA; WU, TT; BILOFSKY, H; M REID-MULLER, PERRY, H. protein sequences of immunological interest (Sequence of proteins of immunological interest) National Institutes of Health, Bethesda. 1993.

25. CHOTIA, C, LESK, AM, TRAMONTANO, A, LEVITT, M, SMITH-GILL, PADLAN, EA, DAVIES, D, TULIP, WR. confirmation of immunoglobulins in the hypervariable region (relationships of immunoglobulin hypervariable regions) Nature, 342: 877-883.1989.

26. Clinical significance of GOULD IM. methicillin-resistant Staphylococcus aureus (The clinical diagnosis of methicillin-resistant Staphylococcus aureus)Staphylococcus aureus.) J. Hospital Infections, 61(4):277-82. 2005.

27. ANDREMONT, A; TIBON-CORNILLOT, M. Le triomphe des bactéries, la fin des antibiotiques? Ed Max Milo, 1 ed, 2007.

28. Silverstein, Arthur M. Paul Ehrlich's receptor immunology. San Diego: Academic Press, 2002.

29. LIM, D; STRINADKA NC. Structural basis for beta lactam resistance of PBP2a isolated from methicillin-resistant Staphylococcus aureus (Structural basis for the beta lactam resistance of PBP2a from methicillin-resistant microorganismsStaphylococus aureus. )Nat Struct Biol. 9(11): 870-6. 2002.

30. Molecular analysis of the regulators and the balance of the expression of virulence factors of the MCPAKKYIRIAKOU, H; VAZ, D; SIMOR, A; LOUIE M; MCGAVIN MJ. epidemic methicillin-resistant Staphylococcus aureus (Molecular analysis of the access gene regulator (ag) locations and balance of viral factor expression in epidemic metallic-resistorStaphylococcus aureus. )J Infect Dis. 181(3):2400-4. 2000.

31. Spread of a multidrug resistant Staphylococcus aureus clone in Brazil (Geogranic spot of epidemic multiresistant) regionStaphylococcus aureus clone in Brazil. )) J Clin Microbiol. 33(9):2400-4.1995.

32. Spread of a multi-antibiotic resistant Staphylococcus aureus clone of DA SILVA, COIMBRA MV, TEIXEIRA, LA, RAMOS, RL, PREDARI, SC, CASLLO, L, FAMIGLIETTI A, VA, C, KLAN, L, FIGUEIREDODO AM. in Argentina two major cities ((Spread of the Brazilian epidemic clone of a multiresistant MRSA in two cities in argentina.)) J Med Microbiol.49 (2) 187-92.2000.

33. Spread of a clone of Staphylococcus aureus (MRSA) mainly resistant to methicillin by SenNA, JP, PINTO, CA, MATEOS, S, QUINTANA, A, SANTOS DS. in the Userried and Nanzilian hospitals ((Spread of a dominant methicillin-resistant)Staphylococcus aureus (MRSA) clone between Uruguayan and South Brazil Hospitals. J Hospital)) Infect. 53(2):15607. 2003.

34. MELTER, O, SANTOS SANCHES, I, SCHINDLER, J, AIRES DE SOUZA, M, KOV a ROVA, V, ZEMLICKOV a, H, DE LENCASTRE H, clone type of Methicillin-resistant Staphylococcus aureus in the Czech republicStaphylococcus aureus clonal types in the Czech Republic.)) J Clin Microbiol, 37(9):2798-803. 1999.

35. DA SILVA COIMBRA MV, SILVA-CARVALHO MC, WISPLINGHOFF H, HALL GO, TALLENT S, WALLICE, S, EDMOND, MB, FIGUEIREDO, AM, WEINZEL, RP. methicillin-resistant Staphylococcus aureus (Clonal batch of methicillin-resistant Staphylococcus aureus) spread over a large area of the U.S.Staphylococcus aureus in a large geographic area of the United States. ))J Hosp Infect. 53(2):103-10. 2003.

36. NYGARD, TK, DELEO, FR, VOYICH, JM. human-related methicillin-resistant Staphylococcus aureus skin infections: (Community-associated methicillin-resistant) recognizing toxic factorsStaphylococcus aureus skin infections: advances toward identifying the key virulence factors. ))Curr Op Infect Dis. 21(2):147-52. 2008.

37. DIEP, BA; CHAMBERS, HF; GRABER, CJ; SZUMOWSKI, JD; MILLER, LG; HAN, LL; CHEN, JH; LIN, F; et. al. Emergence of multidrug-resistant, community-associated, methicillin-resistant Staphylococcus aureus clone USA300 in men whohave sex with men. Ann Intern Med. 148(4):249-57. 2008.

38. REICHERT, JM; DEWITZ, MC. Anti-infective monoclonal antibodies: perils and promise of development. Nat Rev Drug Discov. 5(3):191-5. 2006.

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40. GOFFIN, C; GHUYSEN JM. Multimodular penicillin-binding proteins: an enigmatic family of orthologs and parologs. Microbiol Mol Biol Rev. 62(4):1079-93. 1998.

41. COURVALIN P. Vancomycin resistance in gram-positive cocci. Clin Infect Dis. 1;42 Suppl 1:S25-34. 2006.

42. BAILIE GR, NEAL D. Vancomycin ototoxicity and nephrotoxicity. Med Toxicol Adverse Drug Exp. 3:376-86. 1988.

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SEQUENCE LISTING

<110> Oswald. Kraus Foundation

Kruski foundation of oswaldo

<120> monoclonal antibody

<130> P1704

<160> 39

<170> PatentIn version 3.3

<210> 1

<211> 668

<212> PRT

<213> Staphylococcus aureus

<220>

<221> MISC_FEATURE

<223> PBP2a amino acid sequence

<400> 1

Met Lys Lys Ile Lys Ile Val Pro Leu Ile Leu Ile Val Val Val Val

1 5 10 15

Gly Phe Gly Ile Tyr Phe Tyr Ala Ser Lys Asp Lys Glu Ile Asn Asn

20 25 30

Thr Ile Asp Ala Ile Glu Asp Lys Asn Phe Lys Gln Val Tyr Lys Asp

35 40 45

Ser Ser Tyr Ile Ser Lys Ser Asp Asn Gly Glu Val Glu Met Thr Glu

50 55 60

Arg Pro Ile Lys Ile Tyr Asn Ser Leu Gly Val Lys Asp Ile Asn Ile

65 70 75 80

Gln Asp Arg Lys Ile Lys Lys Val Ser Lys Asn Lys Lys Arg Val Asp

85 90 95

Ala Gln Tyr Lys Ile Lys Thr Asn Tyr Gly Asn Ile Asp Arg Asn Val

100 105 110

Gln Phe Asn Phe Val Lys Glu Asp Gly Met Trp Lys Leu Asp Trp Asp

115 120 125

His Ser Val Ile Ile Pro Gly Met Gln Lys Asp Gln Ser Ile His Ile

130 135 140

Glu Asn Leu Lys Ser Glu Arg Gly Lys Ile Leu Asp Arg Asn Asn Val

145 150 155 160

Glu Leu Ala Asn Thr Gly Thr Ala Tyr Glu Ile Gly Ile Val Pro Lys

165 170 175

Asn Val Ser Lys Lys Asp Tyr Lys Ala Ile Ala Lys Glu Leu Ser Ile

180 185 190

Ser Glu Asp Tyr Ile Lys Gln Gln Met Asp Gln Asn Trp Val Gln Asp

195 200 205

Asp Thr Phe Val Pro Leu Lys Thr Val Lys Lys Met Asp Glu Tyr Leu

210 215 220

Arg Asp Phe Ala Lys Lys Phe His Leu Thr Thr Asn Glu Thr Glu Ser

225 230 235 240

Arg Asn Tyr Pro Leu Gly Lys Ala Thr Ser His Leu Leu Gly Tyr Val

245 250 255

Gly Pro Ile Asn Ser Glu Glu Leu Lys Gln Lys Glu Tyr Lys Gly Tyr

260 265 270

Lys Asp Asp Ala Val Ile Gly Lys Lys Gly Leu Glu Lys Leu Tyr Asp

275 280 285

Lys Lys Leu Gln His Glu Asp Gly Tyr Arg Val Thr Ile Val Asp Asp

290 295 300

Asn Ser Asn Thr Ile Ala His Thr Leu Ile Glu Lys Lys Lys Lys Asp

305 310 315 320

Gly Lys Asp Ile Gln Leu Thr Ile Asp Ala Lys Val Gln Lys Ser Ile

325 330 335

Tyr Asn Asn Met Lys Asn Asp Tyr Gly Ser Gly Thr Ala Ile His Pro

340 345 350

Gln Thr Gly Glu Leu Leu Ala Leu Val Ser Thr Pro Ser Tyr Asp Val

355 360 365

Tyr Pro Phe Met Tyr Gly Met Ser Asn Glu Glu Tyr Asn Lys Leu Thr

370 375 380

Glu Asp Lys Lys Glu Pro Leu Leu Asn Lys Phe Gln Ile Thr Thr Ser

385 390 395 400

Pro Gly Ser Thr Gln Lys Ile Leu Thr Ala Met Ile Gly Leu Asn Asn

405 410 415

Lys Thr Leu Asp Asp Lys Thr Ser Tyr Lys Ile Asp Gly Lys Gly Trp

420 425 430

Gln Lys Asp Lys Ser Trp Gly Gly Tyr Asn Val Thr Arg Tyr Glu Val

435 440 445

Val Asn Gly Asn Ile Asp Leu Lys Gln Ala Ile Glu Ser Ser Asp Asn

450 455 460

Ile Phe Phe Ala Arg Val Ala Leu Glu Leu Gly Ser Lys Lys Phe Glu

465 470 475 480

Lys Gly Met Lys Lys Leu Gly Val Gly Glu Asp Ile Pro Ser Asp Tyr

485 490 495

Pro Phe Tyr Asn Ala Gln Ile Ser Asn Lys Asn Leu Asp Asn Glu Ile

500 505 510

Leu Leu Ala Asp Ser Gly Tyr Gly Gln Gly Glu Ile Leu Ile Asn Pro

515 520 525

Val Gln Ile Leu Ser Ile Tyr Ser Ala Leu Glu Asn Asn Gly Asn Ile

530 535 540

Asn Ala Pro His Leu Leu Lys Asp Thr Lys Asn Lys Val Trp Lys Lys

545 550 555 560

Asn Ile Ile Ser Lys Glu Asn Ile Asn Leu Leu Thr Asp Gly Met Gln

565 570 575

Gln Val Val Asn Lys Thr His Lys Glu Asp Ile Tyr Arg Ser Tyr Ala

580 585 590

Asn Leu Ile Gly Lys Ser Gly Thr Ala Glu Leu Lys Met Lys Gln Gly

595 600 605

Glu Thr Gly Arg Gln Ile Gly Trp Phe Ile Ser Tyr Asp Lys Asp Asn

610 615 620

Pro Asn Met Met Met Ala Ile Asn Val Lys Asp Val Gln Asp Lys Gly

625 630 635 640

Met Ala Ser Tyr Asn Ala Lys Ile Ser Gly Lys Val Tyr Asp Glu Leu

645 650 655

Tyr Glu Asn Gly Asn Lys Lys Tyr Asp Ile Asp Glu

660 665

<210> 2

<211> 2007

<212> DNA

<213> Staphylococcus aureus

<220>

<221> other characteristic region

<223> mecA Gene nucleic acid encoding PBP2a

<400> 2

ttattcatct atatcgtatt ttttattacc gttctcatat agctcatcat acactttacc 60

tgagattttg gcattgtagc tagccattcc tttatcttgt acatctttaa cattaatagc 120

catcatcatg tttggattat ctttatcata tgatataaac cacccaattt gtctgccagt 180

ttctccttgt ttcattttga gttctgcagt accggatttg ccaattaagt ttgcataaga 240

tctataaata tcttctttat gtgttttatt tacgacttgt tgcataccat cagttaatag 300

attgatattt tctttggaaa taatattttt cttccaaact ttgtttttcg tgtcttttaa 360

taagtgaggt gcgttaatat tgccattatt ttctaatgcg ctatagattg aaaggatctg 420

tactgggtta atcagtattt caccttgtcc gtaacctgaa tcagctaata atatttcatt 480

atctaaattt ttgtttgaaa tttgagcatt ataaaatgga taatcacttg gtatatcttc 540

accaacacct agttttttca tgcctttttc aaatttctta ctgcctaatt cgagtgctac 600

tctagcaaag aaaatgttat ctgatgattc tattgcttgt tttaagtcga tattaccatt 660

taccacttca tatcttgtaa cgttgtaacc accccaagat ttatcttttt gccaaccttt 720

accatcgatt ttataacttg ttttatcgtc taatgttttg ttatttaacc caatcattgc 780

tgttaatatt ttttgagttg aacctggtga agttgtaatc tggaacttgt tgagcagagg 840

ttctttttta tcttcggtta atttattata ttcttcgtta ctcatgccat acataaatgg 900

atagacgtca tatgaaggtg tgcttacaag tgctaataat tcacctgttt gagggtggat 960

agcagtacct gagccataat catttttcat gttgttataa atactctttt gaactttagc 1020

atcaatagtt agttgaatat ctttgccatc ttttttcttt ttctctatta atgtatgtgc 1080

gattgtattg ctattatcgt caacgattgt gacacgatag ccatcttcat gttggagctt 1140

tttatcgtaa agtttttcga gtcccttttt accaataact gcatcatctt tatagccttt 1200

atattctttt tgttttaatt cttcagagtt aatgggacca acataaccta atagatgtga 1260

agtcgctttt tctagaggat agttacgact ttctgtttca ttagttgtaa gatgaaattt 1320

ttttgcgaaa tcacttaaat attcatccat ttttttaacg gttttaagtg gaacgaaggt 1380

atcatcttgt acccaatttt gatccatttg ttgtttgata tagtcttcag aaatacttag 1440

ttctttagcg attgctttat aatctttttt agatacattc tttggaacga tgcctatctc 1500

atatgctgtt cctgtattgg ccaattccac attgtttcgg tctaaaattt taccacgttc 1560

tgattttaaa ttttcaatat gtatgctttg gtctttctgc attcctggaa taatgacgct 1620

atgatcccaa tctaacttcc acataccatc ttctttaaca aaattaaatt gaacgttgcg 1680

atcaatgtta ccgtagtttg ttttaatttt atattgagca tctactcgtt ttttattttt 1740

agatactttt tttattttac gatcctgaat gtttatatct ttaacgccta aactattata 1800

tatttttatc ggacgttcag tcatttctac ttcaccatta tcgcttttag aaatataact 1860

gctatcttta taaacttgtt tgaaattttt atcttcaatt gcatcaatag tattattaat 1920

ttctttatct tttgaagcat aaaaatatat accaaacccg acaactacaa ctattaaaat 1980

aagtggaaca atttttatct ttttcat 2007

<210> 3

<211> 76

<212> PRT

<213> Staphylococcus aureus

<220>

<221> MISC_FEATURE

<223> PBP2a fragment amino acid sequence

<400> 3

Met Tyr Gly Met Ser Asn Glu Glu Tyr Asn Lys Leu Thr Glu Asp Lys

1 5 10 15

Lys Glu Pro Leu Leu Asn Lys Phe Gln Ile Thr Thr Ser Pro Gly Ser

20 25 30

Thr Gln Lys Ile Leu Thr Ala Met Ile Gly Leu Asn Asn Lys Thr Leu

35 40 45

Asp Asp Lys Thr Ser Tyr Lys Ile Asp Gly Lys Gly Trp Gln Lys Asp

50 55 60

Lys Ser Trp Gly Gly Tyr Asn Val Thr Arg Tyr Glu

65 70 75

<210> 4

<211> 4

<212> PRT

<213> Staphylococcus aureus

<220>

<221> other characteristic region

<223> PBP2a active center amino acid sequence

<400> 4

Ser Thr Gln Lys

1

<210> 5

<211> 228

<212> DNA

<213> Staphylococcus aureus

<400> 5

atgtatggca tgagcaacga agaatataac aaactgaccg aagataaaaa agaaccgctg 60

ctgaacaaat ttcagattac caccagcccg ggcagcaccc agaaaattct gaccgcgatg 120

attggcctga acaacaaaac cctggatgat aaaaccagct ataaaattga tggcaaaggc 180

tggcagaaag ataaaagctg gggcggctat aacgtgaccc gctatgaa 228

<210> 6

<211> 16

<212> PRT

<213> Staphylococcus aureus

<220>

<221> other characteristic region

<223> CDR1 light chain amino acid sequence

<400> 6

Arg Ser Ser Gln Ser Ile Gly His Ser Asn Gly Asn Thr Tyr Leu Glu

1 5 10 15

<210> 7

<211> 7

<212> PRT

<213> Staphylococcus aureus

<220>

<221> other characteristic region

<223> CDR2 light chain amino acid sequence

<400> 7

Lys Val Ser Asn Arg Phe Ser

1 5

<210> 8

<211> 9

<212> PRT

<213> Staphylococcus aureus

<220>

<221> other characteristic region

<223> CDR3 light chain amino acid sequence

<400> 8

Phe Gln Gly Ser Tyr Val Pro Leu Thr

1 5

<210> 9

<211> 48

<212> DNA

<213> Staphylococcus aureus

<220>

<221> other characteristic region

<223> CDR1 light chain DNA sequence

<400> 9

cgcagcagcc agagcattgg ccatagcaac ggcaacacct atctggaa 48

<210> 10

<211> 21

<212> DNA

<213> Staphylococcus aureus

<220>

<221> other characteristic region

<223> CDR2 light chain DNA sequence

<400> 10

aaagtgagca accgctttag c 21

<210> 11

<211> 27

<212> DNA

<213> Staphylococcus aureus

<220>

<221> other characteristic region

<223> CDR3 light chain DNA sequence

<400> 11

tttcagggca gctatgtgcc gctgacc 27

<210> 12

<211> 12

<212> PRT

<213> Staphylococcus aureus

<220>

<221> other characteristic region

<223> CDR1 heavy chain amino acid sequence

<400> 12

Gly Phe Ser Ile Thr Ser Ser Ser Ser Cys Trp His

1 5 10

<210> 13

<211> 16

<212> PRT

<213> Staphylococcus aureus

<220>

<221> other characteristic region

<223> CDR2 heavy chain amino acid sequence

<400> 13

Arg Ile Cys Tyr Glu Gly Ser Ile Ser Tyr Ser Pro Ser Leu Lys Ser

1 5 10 15

<210> 14

<211> 9

<212> PRT

<213> Staphylococcus aureus

<220>

<221> other characteristic region

<223> CDR3 heavy chain amino acid sequence

<400> 14

Glu Asn His Asp Trp Phe Phe Asp Val

1 5

<210> 15

<211> 36

<212> DNA

<213> Staphylococcus aureus

<220>

<221> other characteristic region

<223> CDR1 heavy chain DNA sequence

<400> 15

ggctttagca ttaccagcag cagcagctgc tggcat 36

<210> 16

<211> 48

<212> DNA

<213> Staphylococcus aureus

<220>

<221> other characteristic region

<223> CDR2 heavy chain DNA sequence

<400> 16

cgcatttgct atgaaggcag cattagctat agcccgagcc tgaaaagc 48

<210> 17

<211> 27

<212> DNA

<213> Staphylococcus aureus

<220>

<221> other characteristic region

<223> CDR3 heavy chain DNA sequence

<400> 17

gaaaaccatg attggttttt tgatgtg 27

<210> 18

<211> 33

<212> DNA

<213> Staphylococcus aureus

<400> 18

atggaagctt gctgggtcta caagctgtgg att 33

<210> 19

<211> 30

<212> DNA

<213> Staphylococcus aureus

<400> 19

atggaaatgg cagcctggtc ttattcctct 30

<210> 20

<211> 20

<212> DNA

<213> Staphylococcus aureus

<400> 20

gatgtgaagc ttcaggagtc 20

<210> 21

<211> 20

<212> DNA

<213> Staphylococcus aureus

<400> 21

caggtgcagc tgaaggagtc 20

<210> 22

<211> 20

<212> DNA

<213> Staphylococcus aureus

<400> 22

caggtgcagc tgaagcagtc 20

<210> 23

<211> 20

<212> DNA

<213> Staphylococcus aureus

<400> 23

caggttactc tgaaagagtc 20

<210> 24

<211> 21

<212> DNA

<213> Staphylococcus aureus

<400> 24

gaggtccagc tgcaacaatc t 21

<210> 25

<211> 20

<212> DNA

<213> Staphylococcus aureus

<400> 25

gaggtccagc tgcagcagtc 20

<210> 26

<211> 21

<212> DNA

<213> Staphylococcus aureus

<400> 26

caggtccaac tgcagcagcc t 21

<210> 27

<211> 20

<212> DNA

<213> Staphylococcus aureus

<400> 27

gaggtgaagc tggtggagtc 20

<210> 28

<211> 20

<212> DNA

<213> Staphylococcus aureus

<400> 28

gatgtgaact tggaagtgtc 20

<210> 29

<211> 29

<212> DNA

<213> Staphylococcus aureus

<400> 29

tggacaggga tccagagttc caggtcact 29

<210> 30

<211> 21

<212> DNA

<213> Staphylococcus aureus

<400> 30

gacattgtga tgacccagtc t 21

<210> 31

<211> 21

<212> DNA

<213> Staphylococcus aureus

<400> 31

gatgttttga tgacccaaac t 21

<210> 32

<211> 18

<212> DNA

<213> Staphylococcus aureus

<400> 32

gatattgtga taacccag 18

<210> 33

<211> 21

<212> DNA

<213> Staphylococcus aureus

<400> 33

gacattgtgc tgacccaatc t 21

<210> 34

<211> 21

<212> DNA

<213> Staphylococcus aureus

<400> 34

gatattgtgc taactcagtc t 21

<210> 35

<211> 21

<212> DNA

<213> Staphylococcus aureus

<400> 35

gatatccaga tgacacagac t 21

<210> 36

<211> 21

<212> DNA

<213> Staphylococcus aureus

<400> 36

gacatccagc tgactcagtc t 21

<210> 37

<211> 21

<212> DNA

<213> Staphylococcus aureus

<400> 37

caaattgttc tcacccagtc t 21

<210> 38

<211> 21

<212> DNA

<213> Staphylococcus aureus

<400> 38

caggctgttg tgactcagga a 21

<210> 39

<211> 18

<212> DNA

<213> Staphylococcus aureus

<400> 39

tacagttggt gcagcatc

Claims (2)

1. An isolated monoclonal antibody that itself binds to PBP2a protein in methicillin-resistant bacterial MRSA, characterized in that the CDR1 light chain amino acid sequence is SEQ ID No. 6, the CDR2 light chain amino acid sequence is SEQ ID No. 7; the CDR3 light chain amino acid sequence is SEQ ID NO 8; the CDR1 heavy chain amino acid sequence is SEQ ID NO 12; the CDR2 heavy chain amino acid sequence is SEQ ID NO 13; the CDR3 heavy chain amino acid sequence is SEQ ID NO. 14; or the CDR1 light chain DNA sequence is SEQ ID NO. 9; the CDR2 light chain DNA sequence is SEQ ID NO. 10; the CDR3 light chain DNA sequence is SEQ ID NO: 11; the CDR1 heavy chain DNA sequence is SEQ ID NO. 15; the CDR2 heavy chain DNA sequence is SEQ ID NO 16; the CDR3 heavy chain DNA sequence is SEQ ID NO: 17.

2. A pharmaceutical composition for treating or preventing infection by bacteria containing PBP2a, comprising the monoclonal antibody isolated according to claim 1 and a pharmaceutically acceptable adjuvant, or carrier or excipient.