ES2547474A1 - Device and procedure for inspection of elements in fuse boxes (Machine-translation by Google Translate, not legally binding) - Google Patents

Abstract

Device and procedure for inspection of elements in fuse boxes. The present invention discloses a device (1) and method to verify the correct arrangement of fuses in a fuse box (2) and, consequently, its correct electrical connection. Said device (2) comprises a tray intended to receive fuse boxes and a mechanism (10) for generating and reading said boxes in which the mechanism for generating and reading said boxes comprises: A) a guide die (100) arranged parallel on a tray; B) a wave generator; y C) a wave reader; Wherein both the wave generator and the wave reader are connected to the guide matrix (100) with displacement capability between at least two points of said guide matrix (100). (Machine-translation by Google Translate, not legally binding)

Description

DESCRIPTION

Live attenuated vaccines of Staphylococcus aureus.

FIELD OF THE INVENTION

Live attenuated Staphylococcus aureus live bacteria vaccines are provided. The methods by which such vaccines can be obtained are also provided.

BACKGROUND OF THE INVENTION

Staphylococcus aureus (S. aureus) is a ubiquitous pathogenic agent that is considered part of the normal microbiota of the human being, in fact it is found in the skin of the healthy individual but sometimes when skin defenses decrease it can cause disease. The main 10 risk groups are hospitalized or immunocompromised patients. Today, about 2 billion people in the world have been colonized by this microorganism.

Humans are a natural reservoir of S. aureus. Between 30 and 50% of healthy adults are colonized, and between 10 and 20% remain persistently colonized. This colonization occurs selectively in the nostrils or nostrils (20-40%, in 15 adults), intertriginous folds, perineum, armpits and vagina. Colonization by S. aureus occurs with higher rates in:

1. People with type 1 diabetes;

2. Intravenous drug users;

3. patients with hemodialysis; twenty

4. surgical patients; and in

5. people with AIDS.

Staphylococci are disseminated, normally, by the performance of common domestic and community activities such as making the bed, dressing or undressing and by certain human groups that constitute true biological vectors. In this sense, health personnel 25 is one of the main biological vectors of dissemination of this type of bacteria. Food handlers also favor the spread of enterotoxigenic S. aureus, contributing to the development of food poisoning.

Since 1970, a gradual increase in the incidence of nosocomial infections due to S. aureus has been detected. Thus, during the period from 1990 to 1992, S. aureus was considered one of the main etiological agents of pneumonia acquired in hospitals. A considerable increase has been detected today, probably due to antibiotic pressure, of S. aureus strains with resistance to different antimicrobial drugs. Among them, methicillin-resistant staphylococcus and vancomycin-resistant staphylococcus. For this reason there is a need to increase the existing prophylactic and / or therapeutic arsenal against this bacterium.

BRIEF DESCRIPTION OF THE INVENTION

The present invention provides bacterial strains of S. aureus auxotrophic for D-alanine, useful as vaccines in human subjects, sufficiently avirulent (attenuated) to avoid unacceptable pathological effects, without substantially any chance of reversing a virulent wild type strain and capable of inducing a sufficient level of immunity in the host to induce acceptable protection against a S. aureus infection in a human subject.

Therefore, a first aspect of the present invention relates to an in vitro method for the production of live attenuated strains of Staphylococcus aureus comprising the following steps:

to. Provide a bacterial strain of Staphylococcus aureus capable of expressing alanine racemase and D-amino acid transaminase, and

b. Inactivate at least one of the genes encoding the alanine racemases enzymes and the genes encoding the D-amino acid transaminase, such that such inactivation results in said bacterial strain being auxotrophic for D-alanine.

In a preferred embodiment of the first aspect of the invention, the method comprises the inactivation of the alr1 gene encoding the alanine racemase 1 enzyme and the dat gene encoding the D-amino acid transaminase, such that the bacterial strain is no longer capable of expressing the functional Alr1 alanine racemase and the Dat functional amino acid transaminase Dat.

In another preferred embodiment of the first aspect of the invention, the method comprises the inactivation of the alr1 and alr2 genes encoding the alanine racemases enzymes and the dat 15 gene encoding the D-amino acid transaminase, such that the bacterial strain no longer is capable of expressing the functional Alr1 alanine racemase, the functional Alr2 alanine racemase and the Dat functional amino acid transaminase Dat.

A second aspect of the invention relates to a live attenuated strain of Staphylococcus aureus obtained or obtainable by the method of the first aspect of the invention or of any of its preferred embodiments.

A third aspect of the invention relates to an attenuated live strain of Staphylococcus aureus, characterized in that said strain comprises inactivated one or more genes encoding the alanine racemase enzyme and the genes encoding the D-amino acid transaminase, such that the strain The bacterial is no longer capable of expressing a functional alanine racemase and a functional D-amino acid transaminase, and in which the inactivation of said genes results in said bacterial strain being auxotrophic for D-alanine.

In a preferred embodiment of the third aspect of the invention, said strain is characterized by inactivation of the loci alr1 and dat, preferably loci alr1, alr2 and dat.

A fourth aspect of the invention relates to a composition comprising the bacterial strain 30 as defined in the second or third aspect of the invention or in any of its preferred embodiments. Preferably, said composition is a pharmaceutical composition that optionally comprises pharmaceutically acceptable carriers as well as excipients and / or other active ingredients, more preferably said composition is a vaccine that optionally comprises pharmaceutically acceptable carriers as well as excipients, adjuvants and / or other active ingredients. Pharmaceutically acceptable adjuvants, excipients and vehicles that can be used in said compositions are carriers known to those skilled in the art. The compositions provided by this invention may be provided through any route of administration, for which said composition will be formulated in the appropriate dosage form and with the excipients that are pharmacologically acceptable for the route of administration chosen.

A fifth aspect of the invention relates to the composition of the fourth aspect of the invention, for use in therapy.

A sixth aspect of the invention relates to the use of the bacterial strain as defined in the second or third aspect of the invention or in any of its preferred embodiments, for the preparation of a medicament for prophylactic treatment.

against a Staphylococcus aureus infection in a mammal. Alternatively, the sixth aspect of the invention relates to the bacterial strain as defined in the second or third aspect of the invention or in any of its preferred embodiments, for use in the prophylactic treatment against infection by Staphylococcus aureus in a mammal, preferably in a human. 5

A seventh aspect of the invention relates to an antibody or fragment thereof selected from the group consisting of F (ab ') 2, Fab', Fab, Fv, "r IgG" and Fc, obtained or obtainable after immunization of a mammal with the live attenuated auxotrophic bacterium for D-alanine as defined in the second or third aspect of the invention or in any of its preferred embodiments. 10

An eighth aspect of the present invention relates to the use of antibodies or fragments thereof of the seventh aspect of the invention, for the preparation of a medicament for use in passive immunization therapy against Staphylococcus aureus in a mammal, preferably In a human subject. Alternatively, the eighth aspect of the invention relates to antibodies or fragments thereof of the seventh aspect of the invention, for use in passive immunization therapy against Staphylococcus aureus in a mammal, preferably in a human subject.

A ninth aspect of the present invention relates to the use of antibodies or fragments thereof of the seventh aspect of the invention, for the preparation of a medicament for therapeutic treatment (after clinical manifestation of the infection) of an infection of Staphylococcus aureus in a mammal, preferably in a human subject. Alternatively, the ninth aspect of the invention relates to the antibodies or fragments thereof of the seventh aspect of the invention, for use in the therapeutic treatment (after clinical manifestation of the infection) of a Staphylococcus aureus infection in a mammal. , preferably in a human subject. 25

A tenth aspect of the invention relates to an in vitro method for the production of a pharmaceutical composition, preferably a vaccine, comprising live attenuated strains of Staphylococcus aureus, comprising the following steps:

to. Provide a bacterial strain of Staphylococcus aureus capable of expressing alanine racemase and D-amino acid transaminase; 30

b. Inactivate at least one of the genes encoding the alanine racemase enzymes and the genes encoding the D-amino acid transaminase, such that such inactivation results in said bacterial strain being auxotrophic for D-alanine; Y

C. Generate a pharmaceutically acceptable composition, preferably a vaccine, comprising said live attenuated bacterium where said composition preferably comprises a pharmaceutically acceptable carrier and excipients as well as adjuvants in the event that said composition is a vaccine.

In a preferred embodiment of the tenth aspect of the invention, the method comprises inactivating the alr1 gene encoding the alanine racemase enzyme and the dat gene encoding the D-amino acid transaminase, such that the bacterial strain is no longer capable of express the functional Alr1 alanine racemase and the Dat functional amino acid transaminase Dat.

In another preferred embodiment of the tenth aspect of the invention, the method comprises the inactivation of the alr1 and alr2 genes encoding the alanine racemases enzymes and the dat gene encoding the D-amino acid transaminase, such that the bacterial strain is no longer be

capable of expressing the functional Alr1 alanine racemase, the functional Alr2 alanine racemase and the Dat functional amino acid transaminase Dat.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 schematically shows the general structure of the bacterial wall of Gram-positive bacteria (teicoic and lipoteic acids not shown). PG, 5 peptidoglycan. M, plasma membrane.

Figure 2 schematically shows the metabolic pathways involved in the formation of D-alanine. The alr1, alr2 and dat genes encode the Alr1, Alr2 and Dat proteins, respectively.

Figure 3 shows the multiple alignment of the amino acid sequences of the Staphylococcus aureus 132 racemalan alanines (ALR1_STAPH132 and ALR2_STAPH132), 10 Mycobacterium tuberculosis ATCC 25618 (ALR_MYCTU), Streptocuccus pneumoniae serotype 4 (ALR_STRPN monocyte (ALR_STRPN monocyte) and Bacillus subtilis 168 (ALR1_BACSU and ALR2_BACSU) using the Omega Clustal program (1.2.1). The amino acids conserved in all alanine racemases are shown shaded in black and amino acids with similar physicochemical properties are indicated with a gray background. fifteen

Figure 4 shows the screening of the colonies resulting from the second recombination event during the construction of the triple mutant Δdat / Δalr1 / Δalr2 of S. aureus 132. The individual colonies were selected from TSB agar with 5 mM D-alanine and inoculated into the same position on TSB agar plates with and without 5 mM D-alanine and in the presence or absence of erythromycin (Ery, 10 µg / mL) and X-Gal (150 µg / mL). Colonies with the Δdat / Δalr1 / Δalr2 20 genotype are sensitive to erythromycin and grow exclusively on D-alanine plaques.

Figure 5 shows the growth of the mutants Δdat, Δdat / Δalr1, Δdat / Δalr2 and Δdat / Δalr1 / Δalr2 of S. aureus 132 in liquid medium TSB in the presence (panel A) or not (panel B) of 5 mM of D -alanine, after 24 h of incubation at 37 ° C with stirring. The double Δdat / Δalr1 and triple Δdat / Δalr1 / Δalr2 mutant strains show normal growth in TSB 25 supplemented with 5 mM D-alanine, but no visible growth is observed in the absence of this compound. However, the wild S. aureus 132 strains and the double mutant Δdat / Δalr2 normally grow in the presence or absence of D-alanine.

Figure 6 shows the PCR confirmation of gene deletions in the mutants Δdat, Δdat / Δalr1, Δdat / Δalr2 and Δdat / Δalr1 / Δalr2 of S. aureus 132 (wild strain). A. The 30 oligonucleotides alr1EXT-F and alr1EXT-R were used to originate alr1-EXT fragments with 1600 bp from the strains carrying the wild locus alr1 or a fragment of 451 bp from strains carrying the mutant locus Δalr1. The oligonucleotides alr1-F and alr1-R were used to generate an internal fragment of the gene alr1 (alr1-INT) with 491 bp only from the strains carrying the wild locus alr1. B. The oligonucleotides alr2EXT-F and alr2EXT-R were used to originate alr2-EXT fragments with 1521 bp from the strains carrying the wild locus alr2 or a fragment of 469 bp from strains carrying the mutant locus Δalr2. The oligonucleotides alr2-F and alr2-R were used to generate an internal fragment of the alr2 gene (alr2-INT) with 519 bp only from the carrier strains of the wild locus alr2. C. The oligonucleotides datEXT-F and datEXT-R were used to originate fragments dat-EXT with 40 1892 bp from the strains carrying the wild locus dat or a fragment of 1049 bp from strains carrying the mutant locus Δdat. D. The oligonucleotides dat-F and dat-R were used to generate an internal fragment of the dat (dat-INT) gene with 540 bp only from the strains carrying the wild lot dat. M, molecular weight standard.

Figure 7 shows the growth and viability assays of the S. aureus 132 wild 45 and triple mutant strains Δdat / Δalr1 / Δalr2. The mutant strain Δdat / Δalr1 / Δalr2 has normal growth in TSB culture medium supplemented with 5 mM D-alanine, but is not able to grow without the exogenous contribution of this compound. However, the wild strain grows in shape

normal in TSB medium with and without the addition of D-alanine. A. Turbidity of the crop. B. Viability of the crop.

Figure 8 shows the determination of the minimum concentration of D-alanine necessary for the growth of the mutant Δdat / Δalr1 / Δalr2 of S. aureus 132 on TSB agar medium. Cultures of the wild strain and triple mutant grown in TSB medium supplemented with 10 mM D-5 alanine to an OD600nm of 0.2 were seeded (100 µL) in TSB agar without D-alanine (0 mM) or with D- Alanine at concentrations from 0.005 mM to 10 mM as indicated in the figure. A. Wild strain S. aureus 132. B. Triple mutant Δdat / Δalr1 / Δalr2 of S. aureus 132.

Figure 9 shows morphological alterations in the triple mutant S. aureus 132 Δdat / Δalr1 / Δalr2 (panel B) with respect to its wild counterpart (panel A) in the presence of 10 different concentrations of D-alanine. The images were taken with a scanning electron microscope at two different scales (5 or 20 μm).

Figure 10 shows different atypical morphologies, progressive degeneration of the cell wall and lysis of the mutant strain S. aureus 132 Δdat / Δalr1 / Δalr2 when maintained in the absence of D-alanine. As a control, the wild strain is shown with its cell wall intact. 15 The images were taken with a transmission electron microscope at different scales as specified by horizontal bars. Black arrows indicate intact bacterial cells with normal morphology; dashed arrows indicate fragmented cells without bacterial cell wall, lysed cells and dispersed genetic material.

Figure 11 shows the evaluation of the stability of the auxotrophic phenotype in the strain of S. 20 aureus 132 Δdat / Δalr1 / Δalr2. The graph shows the number of colonies of the mutant strain recovered in TSB agar (open symbols) and TSB supplemented with 5 mM D-alanine (closed symbols) over time, when this strain was grown in liquid medium TSB supplemented with 5 mM D-alanine at 37 ° C with stirring (210 rpm) for 11 days. A. CFU / mL. B. Log UFC / mL. * P <0.0001, according to Student's t-test. 25

Figure 12 shows the survival percentage of BALB / c mice infected intraperitoneally with wild-type S. aureus 132 (A) and the mutant triple strain Δdat / Δalr1 / Δalr2 (B), carried out in a sepsis model acute to determine lethal doses (DL). A. DL100 for the wild strain equal to 5X, 1X being approximately equal to 1 x 107 CFU. B. DL100 for the mutant strain equal to 250X compared to the wild type strain. Survival was monitored for 14 days.

Figure 13 shows the Log10 of the end-point titers of IgG antibodies produced against the isogenic Δspa strain of S. aureus 132 in BALB / c mice (n = 5) at day 14 (one dose) and 21 (two doses) post-vaccination with different doses of the mutant strain Δdat / Δalr1 / Δalr2 of S. aureus 132, and in control mice, not vaccinated. Vaccination dose: (A) 1 x 106 CFU, (B) 35 1.5 x 107 CFU, (C) 5 x 107 CFU. Antibody titers were determined by an indirect ELISA. P-value, according to the Mann-Whitney U test. * P <0.05 and ** P <0.005 compared to the group of unimmunized mice.

Figure 14 shows the percentage of weight loss over time (panel A) and bacterial load in the spleen and liver of BALB / c mice (n = 5) (panel B) at 43 hours after infection. with a inoculum of 2 x 107 CFU of the wild-type S. aureus strain 132. The mice were pre-immunized on days 0 and 14 with the mutant strain Δdat / Δalr1 / Δalr2 (providing a dose of approximately 1.5 x 107 CFU), or not vaccinated (control group administered with saline on the same days). P-value according to the Mann-Whitney U test. In panel B each point represents the individual bacterial load of a mouse's spleen or liver. 45 The average value of each group is represented by a horizontal line.

Figure 15 shows the survival percentage of BALB / c mice (n = 5) after intraperitoneal infection with a dose of 2 x 107 CFU of the wild type S. aureus strain 132.

vaccinated mice were immunized on days 0 and 14 with the S. aureus strain Δdat / Δalr1 / Δalr2 (dose of approximately 1.5 x 107 CFU) and challenged on day 21 with the wild strain. In the unvaccinated mice a saline solution was also administered on days 0 and 14, and they were infected with the wild strain on day 21. ** P = 0.0046 survival of the group of vaccinated mice compared to the unvaccinated group. P value, according to the 5 Mantel-Cox test (Log-rank test). The survival of the mice was followed for 15 days.

Figure 16 shows the survival percentage of BALB / c mice (n = 6) after being challenged with a dose of approximately 6 x 107 CFU of the wild type S. aureus strain 132. The mice were passively immunized with serum of mice vaccinated with the S. aureus Δdat / Δalr1 / Δalr2 strain or with serum from non-immunized mice, in the control group. * P = 0.0008 survival of mice passively immunized with the serum of vaccinated mice compared to that of non-immunized mice. P value, according to the Mantel-Cox test (Log-rank test). The survival of the mice was followed for 7 days.

Figure 17 shows the cross-reactivity (Log10) of the IgG antibodies produced by BALB / c mice (n = 6) on the 7th day after the second shot of vaccine 15 (approximate dose of 1.5 x 107 CFU) and in control mice (not vaccinated) against 2 strains of S. aureus strains of different origin: USA300LAC (human origin) and RF122 (bovine origin). Antibody titers were determined by an indirect ELISA. * P = 0.0179 and P = 0.0357, respectively, compared to the group of non-immunized mice. P-value according to the Mann-Whitney U test. twenty

DESCRIPTION OF THE INVENTION

DEFINITIONS

In the context of the present invention the term "D-alanine" is understood as the compound or molecule with molecular formula C3H7NO2, molecular weight 89.09 (g / mol) and which is presented in the form of D-enantiomer of the alanine . Its systematic name is "D-alanine", but it can also be designated (not limited to) "D-Ala", "338-69-2" "(R) -Alanine", "D-2-amino propanoic acid" , "(2R) -2-aminopropanoic", "D-alpha form of alanine", "(R) -2-amino propanoic acid" and HD-Ala-OH. Its nomenclature in the IUPAC (International Union of Pure and Applied Chemistry) system is (2R) -2-amino propanoic acid and its identifier in the “PubChem Compound” database is 71080. 30

In the context of the present invention the term "alanine racemase" is understood as the protein that catalyzes the interconversion reaction of L-alanine to D-alanine, necessary for the synthesis of the bacterial wall. Its EC (Enzyme Commission number) identifier is 5.1.1.1. This protein is easily and invariably identified in the nucleotide or amino acid sequence databases by its EC code, which refers to an enzyme whose catalytic activity is L-alanine = D-alanine, since its designation can be variable .

In the context of the present invention the term "D-amino acid transaminase" is understood as the protein that catalyzes the interconversion reaction of D-alanine and 2-oxoglutarate to pyruvate and D-glutamate. Its EC (Enzyme Commission number) identifier is 2.6.1.21. This protein is easily and invariably identified in the nucleotide or amino acid sequence databases by its EC code, which refers to an enzyme whose catalytic activity is D-alanine + 2-oxoglutarate = pyruvate + D-glutamate, and that your designation may be variable.

In the context of the present invention, the term "auxotroph for D-alanine" is understood as the lack of a functional metabolic pathway generated by the substance D-alanine, on which the bacterium so named depends on its growth, due to the inability to synthesize this compound.

In the context of the present invention the term "Alr1" is understood as synonymous with the term "alanine racemase 1".

In the context of the present invention the term "Alr2" is understood as synonymous with the term "alanine racemase 2".

In the context of the present invention the term "Dat" is understood as a synonym for the term "D-amino acid transaminase."

In the context of the present invention the term "alr1" is understood as a nucleotide gene or sequence that encodes a alanine racemase protein. Depending on the strain of Staphylococcus aureus, the chromosomal genes encoding the alanine racemase protein may be referred to (without being limited to) alr1 or alr, or be identified only by their chromosomal locus.

In the context of the present invention the term "alr2" is understood as a nucleotide gene or sequence that encodes an alanine racemase protein. Depending on the strain of Staphylococcus aureus, the chromosomal genes encoding the alanine racemase protein may be referred to (without being limited to) alr2 or dal, or be identified only by their chromosomal locus.

In the context of the present invention the term "dat" is understood as a nucleotide gene or sequence that encodes an alanine racemase protein. Depending on the strain of Staphylococcus aureus, the chromosomal genes encoding the D-amino acid transaminase protein may be referred to (not limited to) dat, or be identified only by their chromosomal locus.

In the context of the present invention the term "inactivation" is understood as blocking the expression of a particular gene or a protein, either through molecular modification or negative regulation of one or both. Molecular modification includes the use of conventional recombinant DNA techniques which in turn include: substitution of one or more nucleotides, insertion of one or more nucleotides, partial or total deletion of a gene, disruption by induced mutagenesis Chemically or radiation induced. Negative regulation of the expression of a gene or protein includes transcriptional and posttranscriptional gene silencing.

In the context of the present invention, the term "Staphylococcus aureus" is defined as any microorganism belonging to the domain "Bacteria", phylum "Firmicutes", class "Bacilli", order "Bacillales", family "Staphylococcaceae", genus " Staphylococcus ”and species“ S. aureus. " The microorganisms thus defined are characterized as facultative anaerobic Gram-positive cocci.

In the context of the present invention, the term "Δalr1" is defined as the absence of locus 35 alr1 in the chromosome of strain S. aureus 132.

In the context of the present invention, the term "Δalr2" is defined as the absence of the alr2 locus on the chromosome of the S. aureus strain 132.

In the context of the present invention, the term "Δdat" is defined as the absence of the dat locus on the chromosome of the S. aureus strain 132. 40

In the context of the present invention, the term "double mutation Δalr1 / Δalr2" is defined as the simultaneous absence of locus alr1 and alr2 in the chromosome of strain S. aureus 132.

In the context of the present invention, the term "double mutation Δdat / Δalr1" is defined as the simultaneous absence of the locus dat and alr1 in the chromosome of strain S. aureus 132.

In the context of the present invention, the term "double mutation Δdat / Δalr2" is defined as the simultaneous absence of the dat and alr2 locus in the chromosome of the S. aureus strain 132.

In the context of the present invention, the term "triple mutation Δdat / Δalr1 / Δalr2" is defined as the simultaneous absence of locus dat, alr1 and alr2 on the chromosome of strain S. aureus 132. 5

In the context of the present invention the term "132" refers to any bacterial strain with the same designation and which belongs to the domain "Bacteria", phylum "Firmicutes", class "Bacilli", order "Bacillales", family "Staphylococcaceae" , genus "Staphylococcus" and species "S. aureus. " This is a clinical strain resistant to methicillin (Vergara-Irigaray et al., Infection and Immunity, 2009, 77: 3978-3991), and was used in this invention as a model to generate an auxotrophic mutant of Staphylococcus aureus.

In the context of the present invention, the term "132Δspa" refers to a microorganism with the same designation and belonging to the domain "Bacteria", phylum "Firmicutes", class "bacilli", order "Bacillales", family "Staphylococcaceae" , genus "Staphylococcus" and species "S. aureus". The microorganism thus defined is a strain of S. aureus 132 with the spa 15 gene deleted (Vergara-Irigaray et al., Infection and Immunity, 2009, 77: 3978-3991).

In the context of the present invention, the term "USA300LAC" refers to any bacterial strain with the same designation and which belongs to the domain "Bacteria", phylum "Firmicutes", class "Bacilli", order "Bacillales", family "Staphylococcaceae" , genus "Staphylococcus" and 20 species "S. aureus. " The microorganism thus defined is a methicillin resistant epidemic strain of S. aureus causing infections in the community (described in Diep et al., Lancet, 2006, 367: 731-739).

In the context of the present invention the term "RF122" refers to any bacterial strain with the same designation with the sequence type MLST 151 (ST151) and clonal complex 151 (CC151) and which belongs to the domain "Bacteria", phylum " Firmicutes ”, class“ Bacilli ”, order“ Bacillales ”, family“ Staphylococcaceae ”, genus“ Staphylococcus ”and species“ S. aureus. " The microorganism thus defined is a strain of S. aureus isolated from cow that had clinical signs of mastitis (described in Herron-Olson L et al., 2007, PloS One 2: e1120).

In the context of the present invention the term "ED133" refers to any bacterial strain 30 with the same designation with the sequence type MLST 133 (ST133) and clonal complex 133 (CC133) and which belongs to the domain "Bacteria", phylum " Firmicutes ”, class“ Bacilli ”, order“ Bacillales ”, family“ Staphylococcaceae ”, genus“ Staphylococcus ”and species“ S. aureus. " The microorganism thus defined is a strain of S. aureus causing mastitis in sheep and recovered in France (described in Guinane et al., 2010, Genome Biol Evol 2: 454-466). 35

In the context of the present invention the term "ED98" refers to any bacterial strain with the same designation with the sequence type MLST 5 (ST5) and clonal complex 5 (CC5) and which belongs to the domain "Bacteria", "Firmicutes" ”, Class“ Bacilli ”, order“ Bacillales ”, family“ Staphylococcaceae ”, genus“ Staphylococcus ”and species“ S. aureus. " The microorganism thus defined is a strain of S. aureus adapted to poultry and recovered from a sick chicken (described in Lowder et al., 2009, PNAS 106: 19545-19550).

In the context of the present invention the term "DC10B" refers to any bacterial strain with the same designation and which belongs to the domain "Bacteria", phylum "Proteobacteria", class "Gammaproteobacteria", order "Enterobacterials", family "Enterobacteriaceae" , genus "Escherichia" and species "E. coli. " The microorganism thus defined is a strain of E. coli DH10B with a deletion of the dcm gene and is an ideal host for the construction of recombinant plasmids that are subsequently introduced directly into S. aureus by electroporation (described in Monk et al. , 2012, mBio 3 (2): e00277-11).

In the context of the present invention, the term "auxotrophic bacterial strains for D-alanine" is understood as any bacterial strain unable to produce a functional and / or active form of the alanine racemase enzyme and the D-amino acid transaminase enzyme. This deficiency may be due to: blocking the expression of its coding genes, post-translational modifications and post-translational modifications that affect both the enzymatic activity, the allosteric regulation or the cellular location of these enzymes.

In the context of the present invention the term "passive immunization" is used to refer to the administration of antibodies or fragments thereof, to an individual with the intention of conferring immunity to that individual.

In the context of the present invention, the expression "therapeutically effective amount" refers to the amount of antibodies of the invention or of attenuated bacterial strains of the invention that allow producing the desired effect. Pharmaceutically acceptable adjuvants and vehicles that can be used in said compositions are the carriers known to those skilled in the art. The compositions provided by this invention may be provided by any route of administration, for which said composition will be formulated in the appropriate pharmaceutical form and with the pharmacologically acceptable excipients for the route of administration chosen.

In the context of the present invention, the term "vaccine" refers to an antigen preparation used to establish the response of the immune system to a disease. Additionally, this term includes the so-called therapeutic vaccines whose purpose is to treat the disease once the first clinical symptoms of it have manifested.

DETAILED DESCRIPTION OF THE INVENTION

Live attenuated vaccines are based on microorganisms that have previously been limited in their pathogenic capacity, but retain their ability to induce a cellular and / or humoral immune response mimicking a natural infection, but without causing the disease. Once the animal or human has been vaccinated, the entry of the microbial pathogen into the host rapidly induces a "redial" of the previous immunity that can control the further growth of the microorganism before the infection assumes clinically significant proportions. In these types of vaccines, depending on the level of attenuation, there is a danger that the host may contract the disease, even if a protective immune response is generated. Therefore, it is necessary to guarantee the genetic stability of the attenuated strain by eliminating any possibility of this strain reverting to the virulent wild strain. In addition, it is crucial that the vaccine causes a sufficient level of immunity (protection) in the host. 35

The auxotrophic bacterial strains for D-alanine (D-Ala) have lost their ability to produce D-alanine, a necessary component for the synthesis of bacterial wall peptidoglycan and substituent in membrane-associated wall and lipoteicic teicoic acids. As already mentioned, for use as vaccines these strains must be sufficiently attenuated, but they must also be able to generate an effective immune response and induce lasting protection in the host. The latter is not simple. In fact, the international patent application WO 99/25376 describes an attenuated strain of Listeria monocytogenes auxotrophic for D-alanine, which also contains DNA encoding a heterologous antigen (an HIV-1 antigen) and the method for use as a vaccine. The experimental examples provided in this patent application simply state that attenuated mutants of L. monocytogenes auxotrophs for D-alanine induce a cellular immune response, but do not consider an antibody-mediated immune response (humoral immunity) that is capable of confer cross protection and therefore, generate a broad immune response for mostly extracellular pathogens, such as

Staphylococcus aureus, and toxin producers. In addition, the possible protective effect of the L. monocytogenes auxotrophic mutant for D-Ala was determined from the count of bacteria in the spleen of pre-immunized mice that were subsequently infected with the wild strain of Listeria. However, no survival trials were conducted, which would be key to measuring the protective efficacy of the vaccine against acute and lethal bacterial infections. Likewise, the authors recognize that the Listeria auxotrophic mutant provides low protection when administered in the absence of D-alanine, or what is the same, they must supplement the initial inoculum of the mutant with D-alanine in order to reach the same level of protection than the wild strain, thereby reducing the lethal dose of the mutant, seriously limiting the safety of the vaccine. 10

In addition, the dose of the attenuated strain of Listeria monocytogenes auxotrophic for D-alanine necessary to achieve an acceptable level of protection (2 x 107 CFU), and determined by counting the bacterial load present in the spleen, is very close to the LD50 when injected in the presence of D-Ala (7 x 107 CFU), which seriously limits its use as a vaccine. A comparable level of protection is already achieved by inoculating the parental strain at a dose 15 100 times lower than the LD50 (1 x 104 CFU). The administration of a dose 100 times lower (2 x 105 CFU), which would be within acceptable safety limits, reduces the effectiveness of the vaccine in terms of protection by 50% (Thompson et al., Infect Immun, 1998, 66: 3552-3561). All these data show that the supplementation of D-alanine in the inoculum of the attenuated strain seriously determines that the administration of the vaccine is safe and would increase the risk of undesirable pathological effects in the host.

In contrast, the data collected in the present patent application show that the Staphylococcus aureus auxotroph strain for D-alanine is highly attenuated in a murine model of systemic infection, presenting a lethal dose 100 (DL100; minimum dose necessary to reduce survival of the 0% mice) significantly higher than the DL100 that the wild strain has and, in addition, is able to provide a significant level of protection in mice against death in survival trials without the need to add a D-alanine supplement in the initial inoculum. Likewise, it was demonstrated that this vaccine candidate is capable of significantly inducing an antibody-mediated immune response in mice immunized with two shots of the vaccine and that said antibodies are capable of recognizing heterologous strains of S. aureus, or what is the same , it is revealed that the vaccine has the ability to generate a broad immune response in the host. These antibodies can, in turn, be used to passively immunize mice, conferring protection against death from an acute lethal disease caused by S. aureus, as also included in the present patent application. For example, immunization Passive with antibodies directed against tetanus toxin, it is a treatment that could save the patient's life in an acute infection with Clostridium tetani (also a gram-positive bacterium). In addition, it has been shown that the auxotrophic bacterial strain for D-Ala has substantially no chance of reverting to the virulent wild strain.

Likewise, the authors of the present invention have demonstrated that the pre-immunization of the 40 mice with an auxotrophic mutant for D-alanine of the S. aureus species produces a significant reduction in the spleen and liver bacterial load of mice infected with a lethal dose of S. aureus.

Humoral immunity (antibody-mediated) is the main specific protective response against extracellular bacteria, such as S. aureus. The polysaccharides of the cell walls 45 and of the capsules of these microorganisms (both Gram-positive and Gram-negative) constitute one of the most immunogenic components thereof and are the prototype of independent T antigen. These antigens stimulate B cells that generate a specific immunoglobulin (Ig) M and G response against surface antigens (polysaccharide or protein) and bacterial toxins, which stimulate effector mechanisms 50 to favor their phagocytosis via cell (monocytes) , macrophages and neutrophils). For other

In part, the Ig produced neutralize bacterial toxins by preventing them from binding to their target cells, promoting their phagocytosis. Some bacterial toxins (superantigens) can also unspecifically stimulate T cells, and consequently large amounts of cytokines and inflammatory mediators are released that end with the production of toxic shock syndrome. The toxin of the toxic chock syndrome of S. aureus (TSST) is an example of these superantigens.

In turn, intracellular bacteria such as Mycobacterium and Listeria monocytogenes, are capable of surviving and replicating within host cells and even within phagocytes, and since they are in a niche inaccessible to circulating antibodies, their elimination requires immune mechanisms other than already seen for extracellular bacteria. 10

Therefore, it is clear that the type of immune response generated by a vaccine - which can be mediated by cells or antibodies or both, can affect the level of protection against a particular microorganism, if it is an extracellular or intracellular pathogen . It cannot be foreseen that a vaccine composed of a strain of Listeria monocytogenes auxotrophic for D-alanine can generate a response that protects against an infection of extracellular origin.

Another significant difference between S. aureus and Listeria monocytogenes that can affect the type of immune response is the constitution of its wall.

The bacterial cell wall is a highly organized and complex structure that allows bacteria to interact with the surrounding environment, gives them their characteristic shape and provides them with mechanical protection. Peptidoglycan, which constitutes the basic structure of the cell wall of eubacteria, is a copolymer formed by an alternating sequence of N-acetylglucosamine and N-acetylmuramic acid linked by β-1,4 bonds. The peptidoglycan layer is substantially thicker in Gram-positive bacteria (20 to 80 nm) than in Gram-negative bacteria (7-8 nm) and constitutes about 90% of the dry weight of 25 Gram-positive bacteria versus 10% in Gram-negative bacteria. In addition to peptidoglycan, the cell wall of Gram-positive bacteria is composed of teicoic acids, which are present in small quantities and embedded in the wall of the bacteria as acidic polysaccharides. The term "teicoic acids" refers to wall polymers that contain glycerol phosphate or ribitolfosphate units. These polyols are linked by phosphate esters and often have other sugars and D-alanine attached. Some of these glycerol-containing acids are bound to membrane lipids of Gram-positive bacteria and due to this intimate association with lipids, these teicoic acids are also called lipoteic acids.

As illustrated in Figure 1, D-alanine is one of the main components of the peptidoglycan cell wall and therefore D-alanine is necessary for cell wall synthesis. Two types of enzymes catalyze the formation of D-alanine (see Figure 2):

1. Alanine racemase, Alr (EC 5.1.1.1), an enzyme that catalyzes reversible racemization between alanine enantiomers (L-alanine and D-alanine); Y

2. D-amino acid transaminase, Dat (EC 2.6.1.21), an enzyme that catalyzes the reversible transamination reaction between pyruvate and D-glutamate to give D-alanine and 2-oxoglutarate.

D-alanine, once synthesized, is initially incorporated into the peptide chain linked to the N-acetylmuramic acid residues of peptidoglycan as a D-alanyl-D-alanine dipeptide. Four. Five

In the peptidoglycan of the S. aureus cell wall there is an L-lysine in the third position of the peptide chain that is bound by N-acetylmuramic acid, and there is a bridge

of cross-linking of pentaglycins between D-alanine and L-lysine of the peptide chains of two adjacent glycan layers. In Listeria there is a diaminopimelic residue in place of the L-lysine and the 5 glycines that serve as a cross-linking bridge between adjacent glycan chains are not present. Additionally, S. aureus has two types of teicoic acids (TAs): wall teicoic acids (WTAs) and lipoteicoic acids (LTAs) that contain D-alanine residues as substituents. In Listeria, these D-alanine ester substituents have only been described in LTAs. The WTAs associated with peptidoglycan are highly variable in structure and are often characteristic of species and there are even specific strain variations. In general, membrane-associated LTAs have less structural and compositional diversity than WTAs. 10

Therefore, in S. aureus, D-alanine not only participates in the cross-linking of peptidoglycan but also forms part of the WTA and LTA. Initially the D-alanine residues are incorporated into the LTAs and from here they are transferred to the WTAs of S. aureus; therefore, the composition of the wall between both pathogens is different. The presence of these D-alanine esters in TAs seems to have some role in the regulation of autolytic activity, binding of Mg2 + in the cell wall, susceptibility of the bacterium to nisin, defensins, etc., phagocyte death, biofilm formation , adhesion to epithelial cells, etc. In S. aureus TAs esterified with D-alanine protect against cationic antimicrobial peptides produced by the host.

Therefore, it is not possible to conclude from the data on L. monocytogenes exposed above, that the auxotrophic bacterial strains for S. aureus D-alanine are sufficiently avirulent (attenuated) to avoid unacceptable pathological effects, and that at their they may induce a sufficient level of immunity in the host without presenting substantially any chance of reverting to a virulent wild type strain.

Therefore, one embodiment of the invention relates to live attenuated bacteria of the S. aureus species suitable as vaccine candidates that are no longer capable of producing D-alanine.

Live attenuated bacteria for use according to the invention as vaccine candidates can be obtained in several ways, some of these ways are clearly evidenced in example 2 of the present invention entitled "Construction and characterization of single, double mutant strains and triple S. aureus without alanine racemase and / or D-amino acid transaminase ”.

Consequently, a first aspect of the invention relates to a new platform technology for the design and production of vaccines based on live attenuated auxotrophic bacterial strains for D-alanine belonging to the S. aureus species. This new platform technology has potential in a wide variety of bacterial strains belonging to that species.

Therefore, an embodiment of the first aspect of the present inventions refers to an in vitro method for the production of live attenuated strains of Staphylococcus aureus comprising the following steps:

to. Provide a bacterial strain of Staphylococcus aureus capable of expressing alanine racemase and D-amino acid transaminase, and

b. Inactivate at least one of the genes encoding the alanine racemase enzymes and the genes encoding the D-amino acid transaminase, such that such inactivation results in said bacterial strain being auxotrophic for D-45 alanine.

In a preferred embodiment of the first aspect of the invention, the method comprises the inactivation of the alr1 gene encoding the alanine racemase enzyme and the dat gene encoding the D-amino acid transaminase, such that the bacterial strain is no longer capable of expressing the alanine racemase Alr1 functional and the D-amino acid functional transaminase Dat.

In another preferred embodiment of the first aspect of the invention, the method comprises the inactivation of the alr1 and alr2 genes encoding the alanine racemases enzymes and the dat gene encoding the D-amino acid transaminase, such that the bacterial strain no longer is capable of expressing the functional Alr1 alanine racemase, the functional Alr2 alanine racemase and the Dat functional amino acid transaminase Dat.

Such inactivation may be an insertion, a deletion, a substitution or a combination thereof, provided that inactivation leads to the absence of expression of at least one alanine racemase and a functional D-amino acid transaminase. A alanine racemase and / or a functional D-amino acid transaminase are understood as proteins that have the regulatory characteristics of wild-type proteins. Therefore, a alanine racemase and / or a D-amino acid transaminase that are defective are therefore unable to participate in the synthesis of D-alanine and are considered non-functional proteins.

 A second aspect of the invention relates to a live attenuated strain of Staphylococcus aureus obtained or obtainable by the method of the first aspect of the invention or of any of its preferred embodiments.

A third aspect of the invention relates to an attenuated live strain of Staphylococcus 20 aureus, characterized in that said strain comprises inactivated one or more genes encoding the alanine racemase enzyme and the genes encoding the D-amino acid transaminase, such that the Bacterial strain is no longer capable of expressing a functional alanine racemase and a functional D-amino acid transaminase, and in which the inactivation of said genes results in said bacterial strain being auxotrophic for D-alanine. 25

In a preferred embodiment of the third aspect of the invention, said strain is characterized by inactivation of the loci alr1 and dat, preferably loci alr1, alr2 and dat.

Such inactivation may be an insertion, a deletion, a substitution or a combination thereof, provided that inactivation leads to the absence of expression of at least one alanine racemase and a functional D-amino acid transaminase. A alanine racemase and / or a functional D-amino acid transaminase are understood as proteins that have the regulatory characteristics of wild-type proteins. Therefore, a alanine racemase and / or a D-amino acid transaminase that are defective are therefore unable to participate in the synthesis of D-alanine and are considered non-functional proteins.

A fourth aspect of the invention relates to a composition comprising the bacterial strain as defined in the second or third aspect of the invention or in any of its preferred embodiments.

In addition and as already stated throughout the text, due to their attenuated but immunogenic character in vivo, bacterial strains, as defined in any of the second or third aspects in any of their preferred embodiments, are very suitable. as a basis for live attenuated vaccines. Accordingly, preferably, said composition is a pharmaceutical composition that optionally comprises pharmaceutically acceptable carriers as well as excipients and / or other active ingredients.

In addition, and in connection with the use as a vaccine of the bacterial strains of the invention mentioned in the preceding paragraph, the present invention also relates, as a fifth aspect of the invention, to said compositions, in particular, compositions of live attenuated vaccines comprising auxotrophic mutant bacterial strains as

defines in any of the second or third aspects of the invention or in any of its preferable embodiments.

These compositions are especially suitable for the protection of animals and humans against infection with the wild-type form of the auxotrophic mutant bacterial strain. Such animals can be selected from the group consisting of placental 5 (including humans), marsupials and monotremes. Such pharmaceutical compositions, in particular vaccine compositions, comprise an immunogenically effective amount of live attenuated bacteria as defined in any of the second or third aspects of the invention or in any of its corresponding preferred embodiments. 10

In addition to an immunogenically effective amount of live attenuated bacteria described above, a pharmaceutical composition, in particular, a vaccine, according to the present invention also contains a pharmaceutically acceptable carrier. Such a vehicle can be as simple as water, but it can, for example, also comprise culture medium in which the bacteria were grown. Another suitable vehicle is, for example, a physiological saline concentration solution.

The dosage useful to administer will vary depending on the age, weight of the vaccinated animal and the mode of administration.

The pharmaceutical composition, in particular the vaccine, may comprise any dose of bacteria, sufficient to elicit an immune response. Doses ranging from 103 to 20 1010 bacteria are, for example, very suitable doses.

Optionally, one or more compounds having adjuvant activity for the vaccine can be added. Adjuvants are non-specific stimulators of the immune system. They improve the host's immune response to the vaccine. Examples of adjuvants known in the art are complete and incomplete Freund's adjuvant, vitamin E, non-ionic block polymers, muramylipeptides, ISCOM (immune stimulating complexes, cf. for example European patent EP 109,942), saponin, mineral oil , vegetable oil and Carbopol. Adjuvants, especially suitable for application in the mucosa are, for example, heat labile E. coli toxin (LT) or cholera toxin (CT). Other suitable adjuvants are for example aluminum hydroxide, aluminum phosphate or aluminum oxide, oily emulsions (for example Bayol F® or Marcol 52®), saponins or solubilized vitamin E.

Therefore, preferably, the vaccines according to the present invention comprise an adjuvant.

Other examples of pharmaceutically acceptable carriers or diluents useful in the present invention include stabilizers such as SPGA, carbohydrates (eg, sorbitol, mannitol, starch, sucrose, glucose, dextran), proteins such as albumin or casein, agents containing proteins such as bovine serum or skim milk and buffers (for example phosphate buffer). Especially when such stabilizers are added to the vaccine, the vaccine is very suitable for freeze drying. Therefore, preferably, the vaccine is in a lyophilized form. 40

For administration to animals or humans, the vaccine according to the present invention can be given intranasally, intradermally, subcutaneously, orally, by aerosol or intramuscularly. For application to poultry, the wing membrane and administration of eye drops are very suitable. The medicament, pharmaceutical composition or vaccine of the invention can be used both in asymptomatic patients and in those who have already shown symptoms of the disease.

A further aspect of the invention relates to an antibody or fragment thereof selected from the group consisting of "Fab", "F (ab ') 2 fragments," Fv "fragments, single chain Fv fragments or" scFv "," diabodies "and" bispecific antibodies "(Bab), (hereinafter, from the antibody or fragment thereof of the invention) or which can be obtained after immunization of a mammal with the live attenuated auxotrophic bacterium 5 for D-alanine which is defined in any of the second or third aspects of the invention or in any of its corresponding preferred embodiments.

 In a preferable embodiment of this aspect of the invention, the mammal used for immunization is selected from the group consisting of placentals (including humans), marsupials and monotremes. 10

A further aspect of the invention relates to the antibodies or fragments thereof of the invention for use in therapy, in particular for use in passive immunization.

A further aspect of the invention relates to a pharmaceutical composition, in particular, a vaccine composition, comprising an antibody or fragment thereof of the invention. fifteen

In a preferable embodiment of this aspect of the invention, the pharmaceutical composition further comprises an adjuvant and / or a pharmaceutically acceptable carrier or excipient. As in the previous case, for the administration to animals or humans of the antibody or fragment thereof of the invention, the pharmaceutical composition, in particular the vaccine, can be given, inter alia, intradermally, intradermally, subcutaneously, orally, by spray or intramuscularly. For application to poultry, the wing membrane and administration of eye drops are very suitable. The drug, pharmaceutical composition or vaccine can be used both in asymptomatic patients and in those who have already shown symptoms of the disease.

The purpose of the following examples is simply to illustrate the invention and they do not serve to limit it.

EXAMPLES

Example 1. Analysis and identification of nucleotides and amino acid sequences of genes encoding the alanine racemase enzyme of S. aureus 132

The authors of the present invention conducted a search for genes encoding the alanine racemase and D-amino acid transaminase enzymes in complete S. aureus genomes using the Protein Database (UniProtKB) and the Base Search Tool data. Two amino acid sequences corresponding to Alr1 and Alr2 proteins were identified. These sequences were compared with each other, and with other alanine racemases protein sequences present in other bacterial species, by means of the Omega Clustal alignment tool.

As a result of the previous analysis, two candidate genes for possible alanine racemases were found in the genome of S. aureus 132: the gene we call alr1 that encodes a 382 amino acid protein called Alr1 and the alr2 gene that encodes a 361 amino acid protein called Alr2 . The dat gene encoding a D-amino acid 40 transaminase, Dat of 282 amino acids was also identified.

Figure 3 shows the multiple alignment of the amino acid sequences of the amass racemases of S. aureus 132 (ALR1_STAPH132 and ALR2_STAPH132) and the alanines racemases of M. tuberculosis ATCC 25618 (ALR_MYCTU), S. pneumoniae serotype 4 (ALR_STRPN), Listeria monocytogenes serotype 4b (ALR_LISMC) and B. subtilis 168 (ALR1_BACSU and 45 ALR2_BACSU). The Alr1 and Alr2 proteins of S. aureus 132 have 28.5% of each other.

identity at the amino acid sequence level. The Alr1 and Alr2 proteins have an identity of 39.3% and 23.9% with the alanine racemases Alr1 and Alr2 of B. subtilis 168, respectively.

Example 2. Construction and characterization of single, double and triple mutant strains of S. aureus without alanine racemases and / or D-amino acid transaminase

In order to generate the deletion mutants, a homologous double recombination was carried out using the thermosensitive pMAD vector. First, the suppression of the D-amino acid transaminase (Dat) was performed and this mutant called Δdat was used as the basis for generating the double mutants and the triple mutant, independently.

For the construction of these mutants, approximately 1000 bp fragments corresponding to the 10 regions of DNA located in front (upstream) and behind (downstream) of the dat, alr1 and genes were cloned separately into the plasmid pMAD. alr2. The resulting recombinant plasmids are used to eliminate the chromosomal genes dat, alr1 and alr2, respectively, located on the chromosome of the wild type S. aureus 132 strain.

S. aureus 132 is used in the present invention as a model organism of species 15 "Staphylococcus aureus" to generate auxotrophic mutants for D-alanine. This is a clinical strain resistant to methicillin (MRSA) (Vergara-Irigaray et al., 2009, Infection and Immunity, 77: 3978-3991).

The upstream fragment of the alr1 gene was obtained by PCR amplification using the combination of oligonucleotides alr1UP-F (MluI) and alr1UP-R (NotI), and was subsequently digested by the restriction enzymes MluI and NotI. The downstream fragment of the alr1 gene was obtained by PCR amplification using the oligonucleotides alr1DN-F (NotI) and alr1DN-R (BglII), followed by digestion with NotI and BglII enzymes. The digested upstream and downstream fragments were cloned into the pMAD vector previously linearized with the MluI and BglII enzymes, generating the construction called pMAD_UP / DOWN_alr1. 25

The same strategy was followed for the construction of plasmids pMAD_UP / DOWN_alr2 and pMAD_UP / DOWN_dat. The upstream and downstream fragments of the arl2 gene were amplified using the oligonucleotide pairs alr2UP-F (MluI) / alr2UP-R (NotI) and arl2b-DN-F (NotI) / alr2DN-R (BglII), respectively. In the same way, the upstream and downstream fragments of the dat gene were amplified using oligonucleotide pairs 30 datUP (MluI) F / datUP (NotI) R and datDOWN (NotI) F / datDOWN (BglII) R, respectively.

The three constructions pMAD_UP / DOWN_alr1, pMAD_UP / DOWN_alr2 and pMAD_UP / DOWN_dat were introduced in E. coli DC10B by electroporation. Transforming colonies were selected on LB agar supplemented with ampicillin (100 µg / mL) and X-Gal (150 µg / mL). After incubation at 37 ° C for 18 h, ampicillin-resistant blue colonies were subjected to PCR to check for the presence of pMAD_UP / DOWN_alr1, pMAD_UP / DOWN_alr2 or pMAD_UP / DOWN_dat with oligonucleotide combinations alr1Ext-F / alr1Ext -R, alr2Ext-F / alr2Ext-R or datExtF / datExtR, respectively.

The pMAD_UP / DOWN_dat construct was extracted from E. coli DC10B and transformed directly by electroporation into the specific strain of S. aureus 132. The selection of 40 colonies of S. aureus 132 containing the recombinant plasmid was performed on TSB agar supplemented with erythromycin (10 µg / mL) and X-Gal (150 µg / mL) after 24-48 h of incubation at 30 ° C.

To remove the dat chromosomal gene from the S. aureus 132 strain, a blue colony of S. aureus 132 containing pMAD_UP / DOWN_dat was inoculated into 3 ml of TSB with 10 µg / mL of 45 erythromycin and cultured at 30 ° C for 2 h . Then, the culture was incubated at 43.5 ° C, a non-permissive temperature for plasmid pMAD replication, leading to integration

of pMAD_UP / DOWN_dat on the bacterial chromosome by means of a single recombination. After 6 hours, serial dilutions of the culture were seeded on TSB agar plates supplemented with erythromycin (10 µg / mL) and X-Gal (150 µg / mL) and subsequently incubated at 43.5 ° C for 18 h. Several of the resulting colonies were transferred to 2 ml of TSB without antibiotic and incubated for 24 h at 30 ° C in order to induce a second recombination event that leads to excision of plasmid pMAD_UP / DOWN_dat from the chromosome. The selection of white colonies, which no longer contain pMAD_UP / DOWN_dat, was carried out by sowing serial dilutions on TSB agar plates with X-Gal (150 µg / mL) and without antibiotic. Each selected white colony was inoculated on TSB agar supplemented or not with erythromycin (10 µg / mL) and X-Gal (150 µg / mL). The suppression of the dat gene (Δdat) in erythromycin-sensitive white colonies was checked by PCR using the oligonucleotide pairs datExtF / datExtR, datseqF / datseqR and datF / datR.

To generate the double mutants Δalr1 / Δdat or Δalr2 / Δdat, plasmids pMAD_UP / DOWN_alr1 or pMAD_UP / DOWN_alr2 were introduced by electroporation into mutant strain S. aureus Δdat and the same protocol described above was carried out. In this case, recovery of the double mutant may require the exogenous addition of D-alanine (5 or 10 mM) to the TSB medium or TSB agar. The absence of the loci alr1 (or alr2) and dat in the wild-type S. aureus 132 genome was confirmed by PCR using the following oligonucleotides: alr1Ext-F / alr1Ext-R, alr1UP-Fseq / alr1DN-Rseq, alr1-F / alr1-R (or alr2Ext-F / alr2Ext-R, alr2UP-Fseq / alr2DN-Rseq, alr2-F / alr2-R), and datExtF / datExtR, datseqF / datseqR and datF / datR. twenty

The triple mutant Δalr1 / Δalr2 / Δdat was generated by electroporation introducing plasmid pMAD_UP / DOWN_alr2 into mutant strain S. aureus Δdat / Δalr1 and following the protocol for the allelic exchange system described above. Also in this case, the recovery of the triple mutant Δalr1 / Δalr2 / Δdat required the exogenous addition of D-alanine (5 or 10 mM) to the TSB medium. Figure 4 depicts the colony screening method performed for the selected erythromycin sensitive white colonies as a result of the second recombination event for the triple mutant Δalr1 / Δalr2 / Δdat.

Culture of the different mutants in medium with and without D-alanine revealed that the single deletion of the dat gene or the double deletion of the dat and alr2 genes does not affect bacterial growth. However, the double mutant Δalr1 / Δdat and the triple mutant Δalr1 / Δalr2 / Δdat require the presence of D-alanine in the medium for growth (Figure 5).

Figure 6 shows the confirmation by PCR of the different deletions in the four mutants of S. aureus 132. The results obtained show that the simultaneous suppression of the genes alr1, alr2 and dat generates in this strain an inability to grow without the presence of D-Alanine Also, the double mutant Δalr1 / Δdat does not grow visibly in the absence of 35 D-alanine.

Therefore, it is worth noting that the inactivation of the alanine racemases and D-amino acid transaminase proteins produce an auxotrophy for D-alanine and that the method for obtaining auxotrophic mutants belonging to S. aureus species is independent of the selected strain. 40

Example 3. Effect of D-alanine on the growth and viability of the triple mutant (Δdat / Δalr1 / Δalr2)

To evaluate the growth curve and the viability of the triple mutant of S. aureus 132 Δalr1 / Δalr2 / Δdat compared to the wild type, both strains were inoculated in exponential growth phase (1: 200 dilution) in 100 mL of supplemented TSB or not with 5 mM of 45 D-alanine, and incubated at 37 ° C with constant agitation (210 rpm). The optical density of each culture and its bacterial concentration were determined at the initial moment of inoculation and then every hour until a total of 8 h of incubation. The optical density was evaluated by measuring aliquots of each flask at DO600nm while the

Bacterial concentration (CFU / mL) was calculated by seeding 1:10 dilutions on TSB agar plates. All cultures were performed in triplicate.

Growth and viability curves were performed to assess the effect of the absence of D-alanine in the medium over time for wild and triple mutant S. aureus 132 strains Δalr1 / Δalr2 / Δdat. 5

The strain Δdat / Δalr1 / Δalr2 shows normal growth in TSB culture medium supplemented with 5 mM D-alanine, but is not able to grow without the exogenous contribution of this compound (Figure 7A). However, the wild-type strain grows normally in TSB medium with and without the addition of D-alanine. It was observed that the viability of the triple mutant in TSB medium decreased approximately 2 logarithmic units at 8 h of incubation 10 due to the limitation of D-alanine in the culture medium (Figure 7B).

Example 4. Determination of the minimum concentration of D-alanine necessary for the growth of the mutant Δdat / Δalr1 / Δalr2 of S. aureus 132 on TSB agar medium

To determine the minimum concentration of D-alanine necessary for the growth of the triple mutant (Δdat / Δalr1 / Δalr2) samples were taken from exponential phase cultures of the wild strain S. aureus 132 and the triple mutant grown in TSB medium supplemented with 10 mM D-alanine up to an OD600nm of 0.2. The bacteria were collected by centrifugation and washed with TSB medium, resuspended in the original volume with TSB medium and subsequently seeded (100 µL) on TSB agar without D-alanine (0 mM) or with increasing concentrations of D-alanine from 0.005 mM at 10 mM, as indicated in Figure 8. The plates were incubated at 20 37 ° C for 24 h and bacterial growth was observed. The wild-type parental strain showed a confluent growth in TSB agar without D-alanine (Figure 8A). With respect to the triple mutant (Δdat / Δalr1 / Δalr2) an absence of colonies was observed at concentrations below 0.05 mM D-alanine, dispersed colonies at 0.1 mM D-alanine and a confluent growth on the plates a concentrations above 0.5 mM D-alanine. 25

Example 5. Morphological analysis of wild and triple mutant S. aureus 132 strains Δdat / Δalr1 / Δalr2 by electron microscopy

For the taking of photomicrographs by scanning electron microscopy (SEM), the S. aureus 132 wild and triple mutant strains were cultured for 16 h at 37 ° C in TSB supplemented with 10 mM D-alanine. Bacteria were collected by centrifugation, washed twice with 0.9% NaCl and finally resuspended in 1 mL of TSB culture medium. 50 μL of each culture was inoculated in 5 mL of TSB supplemented with increasing concentrations of D-alanine: 0; 0.01; 0.1; 1 and 10 mM and incubated at 37 ° C for 3 h with constant stirring (180 rpm). The bacterial cultures obtained were centrifuged and the sediments were washed twice with PBS. The cells were then fixed with 4% paraformaldehyde in 0.1 M PBS pH 7.4 for 30 min at room temperature and with stirring. After fixation, the samples were washed again twice with PBS, and dehydrated with a series of increasing concentrations of alcohol (50%, 70%, 90%, 100%) for 15 min each. Finally, the samples were dried at the critical point with CO2 (Bal-Tec CPD 030). A drop of each sample was placed in a sliding cover and fixed on an aluminum support for the gold coating (Bal - Tec SCD 004 Sputter). Samples were observed and photographed on a Jeol JSM-6400 scanning electron microscope.

At the microscopic level, significant morphological and structural changes were observed in the mutant strain (Δdat / Δalr1 / Δalr2) due to the progressive decrease in the exogenous contribution of D-alanine. Figure 9 shows the obtained photomicrographs that allow visual comparison between the triple mutant and its wild counterpart after growing in TSB medium supplemented with increasing concentrations of D-alanine. Panel B of Figure 9 shows that the S. aureus strain Δdat / Δalr1 / Δalr2 is not able to divide without external addition of D-alanine. Accordingly, bacterial cells detected at 0 mM D-alanine

they correspond mostly to the initial inoculum that was previously cultured in the presence of this compound and as a possible consequence of incomplete cell division, deformed cells and protoplasts were also observed. In the presence of 0.1 mM D-alanine a slightly higher bacterial density is observed, but protoplasts and some elongated forms are still present. Finally, when the medium is supplemented with 10 mM D-alanine, it can be observed that both the density and the cell morphology of the triple mutant is comparable to that presented by spherical bacterial cells of the wild-type strain (panel A), becoming indistinguishable from this one.

For the taking of photomicrographs by transmission electron microscopy (MET), the wild strain and the triple mutant Δdat / Δalr1 / Δalr2 were grown on TSB agar supplemented with 10 10 mM D-alanine for 18 h at 37 ° C. They were then subcultured in Mueller-Hinton agar plates and incubated at 37 ° C for 18 h. After incubation, several colonies were resuspended in PBS buffer. After collecting the cells by centrifugation, the resulting sediment was first washed with cacodylate buffer, and immediately afterwards the cells were fixed in cold 2.5% glutaraldehyde prepared in 0.2 M sodium cacodylate buffer pH 7.4 during 4 h at room temperature. The sediments were washed with cacodylate buffer, dehydrated in acetone and embedded in Spurr (Spurr epoxy inclusion medium). Ultra-thin sections (70 nm) of these samples were obtained and stained with uranyl acetate and lead citrate for observation under a JEOL JEM 1010 electron transmission microscope (80 kV). twenty

Transmission electron microscopy studies show that the cell wall of the triple mutant Δdat / Δalr1 / Δalr2, when maintained in the absence of D-alanine, undergoes progressive destruction as a result of inactivation of alanine racemase and D-amino acid transaminase, producing cell lysis and bacterial death. Figure 10 shows different stages of cell wall degeneration, ranging from the decrease in the murein layer and consequently an increase in cell size (protoplasts) to cells showing various ruptures, lysis and extrusion of the cell wall. intracellular content (especially genetic material). However, the bacterial cells of the wild strain have a normal morphology with the cell wall intact.

Example 6. Evaluation of the stability of the auxotrophic phenotype in the strain of S. aureus 30 Δdat / Δalr1 / Δalr2

To test the irreversibility of the nutritional auxotrophy of S. aureus Δdat / Δalr1 / Δalr2 for the compound D-alanine, the triple mutant strain was grown in 100 mL of TSB supplemented with 5 mM D-alanine under optimal conditions for 11 days at 37 ° C under stirring (180 rpm). Samples of this culture were taken at the beginning of the incubation and on days 1, 3, 5, 7 and 11 for the determination of CFU on TSB agar and TSB agar supplemented with 5 mM D-alanine. All cultures were performed in triplicate. In the hypothetical case of a phenotype reversal, bacterial counts recovered over time should be similar on agar plates, regardless of the presence or absence of the compound in the culture medium. In contrast, significant differences were observed between the bacterial counts obtained when the culture was plated on agar medium with and without D-alanine.

The resulting bacterial counts were significantly higher in the first case (agar plates supplemented with D-alanine) at the initial stage of incubation (day 0) and on days 1, 3, 5, 7 and 11 (see Figure 11 ) (P <0.0001, according to Student's t-test). Although significantly much lower, the recovery of a low number of colonies in the agar plates without D-alanine may be due to a residual growth derived from the accumulation of this compound in the cytoplasm of bacterial cells during growth in supplemented medium. . This difference indicates that the strain Δdat / Δalr1 / Δalr2 remains auxotrophic for D-alanine over time, without the possibility of reversion to the wild phenotype.

Example 7. Determination of the lethal dose (DL) of wild-type 132 and triple mutant S. aureus Δdat / Δalr1 / Δalr2 in BALB / c mice in an acute infection model

The authors of the present invention produce a systemic infection in BALB / c mice by intraperitoneal administration of an inoculum of the wild S. aureus 132 strain and of the triple mutant Δdat / Δalr1 / Δalr2 in saline solution with 3% of porcine gastric mucin. For the preparation of the inoculum, the bacteria were cultured in TSB (wild type) or TSB medium supplemented with 10 mM D-alanine (triple mutant) at 37 ° C with shaking (180 rpm) until reaching an OD600nm of 0.7. The cultures were centrifuged and washed with saline, and finally resuspended in saline with 3% gastric mucin. The corresponding bacterial inoculum of approximately 1 x 107 CFU was called 1X. Bacterial suspensions were adjusted from the above-mentioned inoculum to different doses as necessary and inoculated (250 µl) in BALB / c mice (n = 5) intraperitoneally. The survival of the mice was monitored for 14 days. Lethal doses (DL) were determined according to the survival observed in both cases. DL 100 is defined as the minimum lethal dose to achieve 100% mortality of mice. fifteen

Figure 12A shows different degrees of survival in mice infected with increasing doses of the wild-type S. aureus 132 strain. A gradual decrease in the survival of the mice with respect to the 1X dose was observed, the LD 100 being determined when the dose administered is equal to or greater than 5X for the wild strain.

Figure 12B shows different degrees of survival in mice infected with increasing doses of the triple mutant Δdat / Δalr1 / Δalr2. The DL100 that was determined for this strain was 250X, a very high dose of bacterial inoculum, which indicates that this strain is less virulent and highly attenuated compared to its wild counterpart. In addition, as illustrated in the figure, with an inoculum of the triple mutant strain equivalent to the DL 100 of the virulent parental strain none of the mice succumbed to the infection, or what is the same, a survival of 100 was obtained. %. Therefore, the DL100 of the triple mutant is 50X higher than the DL100 of its wild counterpart. This demonstrates that the triple mutant is a highly attenuated strain with respect to the wild-type parental strain.

Example 8. Quantification of IgG antibodies against S. aureus 132 Δspa by indirect ELISA in sera from BALB / c mice subjected to vaccination with triple mutant S. 30 aureus Δdat / Δalr1 / Δalr2

To assess the immune response to antibody-mediated vaccination, groups of BALB / c mice (n = 5) were immunized on days 0 and 14 by intraperitoneal injection (250 µL) with increasing doses of triple mutant Δdat / Δalr1 / Δalr2 in solution saline. Vaccination dose: (A) 1 x 106 CFU, (B) 1.5 x 107 CFU, (C) 5 x 107 CFU. On days 14 and 21 (7 days 35 after the first and second shot of the vaccine, respectively) serum samples were obtained from all of the mice, together with serum samples from the control mice, injected with saline. To obtain the serum, the mice were anesthetized with sevofluoran and the blood was extracted by puncturing the sub-mandibular vein. The sera were separated from the cells by centrifugation and stored at -80 ° C until analysis.

The quantification of IgG was performed by means of an indirect enzyme linked immunoassay (ELISA). 96-well ELISA plates were coated with the S. aureus 132 Δspa strain (strain deficient in the S. aureus 132 spa gene protein). Therefore, the bacterial antigens are fixed to the bottom of the wells after 18 h of incubation at 4 ° C in 100 mM carbonate-bicarbonate buffer, pH 9.6. Five washings with phosphate buffer buffered saline (PBS) were performed to remove unbound bacteria. Residual site blocking was done in two steps to reduce non-specific interactions with mouse sera. First, the plates were incubated at room temperature for 1 h with 100

µL per well of blocking solution (5% skim milk in PBS) and secondly, at 37 ° C for 1 h with 100 µL of rabbit serum (diluted 1: 1000 in PBS). After 5 washing steps with wash buffer (0.005% Tween 20 in PBS), the plates were incubated overnight at 4 ° C with 100 µL of a series of sera diluted in dilution buffer (DMEM medium with 5- 10% FCS). The next day, 5 washes were performed with 5 wash buffer to remove unreacted antibodies and 100 µL of secondary antibody was added per well (peroxidase-labeled anti-mouse IgG-HRP) diluted 1: 5000 in dilution buffer . Incubation was performed for 1 h at room temperature in darkness. The plates were washed 5 times with wash buffer to remove unreacted labeled anti-antibodies. To carry out the development process, the plates were incubated for 3 min with 100 µL of TMB (HRP-peroxidase substrate). The reaction was stopped with 50 µL of 1 M H2SO4 per well. Colorimetric measurement was performed at 450 nm. To determine the IgG titers in each case, the endpoint titre was evaluated as the maximum serum dilution having a value that exceeds the absorbance of the reading blank (absorbance of the dilution buffer) by 0.1 values. fifteen

Therefore, blood samples collected from each mouse were used to determine the antibody titer (IgG) against S. aureus 132 Δspa by ELISA, and thus measure the ability of the vaccine to generate an antibody-mediated immune response. With the three doses tested, significant titers of IgG were detected in the sera obtained seven days after the second shot of the vaccine (day 21) of the immunized mice with respect to the non-vaccinated mice (figure 13), which demonstrates the ability immunogenic of this strain as a vaccine.

Example 9. Determination of loss of body weight and bacterial load in spleen and liver in a systemic infection model with the wild strain S. aureus 132 in pre-immunized BALB / c mice with the triple mutant Δdat / Δalr1 / Δalr2 25

To evaluate the efficacy (level of protection) of the Δdat / Δalr1 / Δalr2 strain as a vaccine, BALB / c mice (n = 6) were immunized intraperitoneally (250 μL) with the Δdat / Δalr1 / Δalr2 strain in saline serum at doses of approximately 1.5 x 107 CFU on days 0 and 14. A group of control mice (n = 6) were administered 250 μL of saline in an identical manner at 0 and 14 days. On day 21, the mice were infected with an inoculum of the wild strain S. aureus 30 132 (2 x 107 CFU in saline with 3% porcine gastric mucin) by intraperitoneal injection. In the moment prior to the challenge with the wild strain and at 5, 21, 29 and 43 hours post-challenge, the body weight of each mouse was evaluated individually.

At 43 hours post-infection the mice were sacrificed with sodium thiopental, the spleens were extracted and weighed aseptically, and after being homogenized in saline serum, the CFUs were determined per gram of organ in TSB agar.

Thus, the protective effect of vaccination with the mutant tripe Δdat / Δalr1 / Δalr2 was confirmed when it was observed that preimmunization with this strain protects mice from an abrupt decrease in body weight. In fact, Figure 15A shows how the loss of body weight in the group of vaccinated mice (approximately 5% compared to 40 before the infection) is less than that experienced by mice belonging to the saline group (10-12% ). Likewise, from the count of bacteria in the spleen and liver, the authors of the present invention demonstrated that the administration of the triple mutant strain Δdat / Δalr1 / Δalr2 as a vaccine causes a significant decrease of almost 4 logarithmic units (Log10) (P = 0.0087 and P = 0.0152, respectively, Mann-Whitney U test) in the bacterial load of mice infected with a sub-lethal dose of S. aureus 132 (Figure 15B and 15C).

Example 10. Protection of BALB / c mice against challenge with S. aureus 132 by immunization with the mutant Δdat / Δalr1 / Δalr2

To evaluate the efficacy of the Δdat / Δalr1 / Δalr2 mutant of S. aureus as a vaccine, BALB / c mice (n = 5) were given 250 µl of the strain Δdat / Δalr1 / Δalr2 (1.5 x 107 dose CFU in saline) on days 0 and 14. Control mice were given only saline 5 identically on the same days. Seven days after the second injection, the mice were inoculated with the wild-type S. aureus 132 strain (2 × 10 7 CFU in saline with 3% porcine gastric mucin) in order to establish a lethal systemic infection in both cases. After infection, mice were monitored for 15 days to determine the survival of vaccinated mice compared to control mice (not vaccinated).

As shown in Figure 15, all mice in the vaccinated group survived the challenge, overcoming the infection with 100% survival, while all mice in the unvaccinated group succumbed to the infection in less than 72 h. The differences in survival between the two groups were statistically significant 15 (P <0.0046, according to the Mantel-Cox Log-rank test).

These results demonstrate that vaccination with the strain Δdat / Δalr1 / Δalr2 provides protective immunity against acute infection by the S. aureus strain 132.

Example 11. Protection of BALB / c mice by passive immunization with bodies generated with the Δdat / Δalr1 / Δalr2 vaccine 20

In situations where there are no authorized vaccines, or vaccination failures are detected in current immunization schedules, the use of passive immunization with antibodies may be beneficial when exposure or contact with bacterial pathogens is suspected.

Next, it was determined whether the serum of mice immunized with the strain Δdat / Δalr1 / Δalr2 with a high titer of IgG antibodies could be used to passively immunize 25 previously unvaccinated mice. These sera were obtained from immunized BALB / c mice (n = 5) with three doses of approximately 2 x 10 8 CFU of the triple mutant strain (administered on days 0, 14 and 28). To obtain the serum (day 35), the mice were anesthetized with sodium thiopental and blood was extracted by puncture of the retro-orbital plexus. Likewise, sera were obtained from control mice (n = 5), injected with saline. The 30 sera were separated from the blood cells by centrifugation and stored at -80 ° C until use.

Sera from immunized mice or sera from control mice were administered by intraperitoneal injection (250 µL) to BALB / c mice (n = 6) 3.5 h before infection with a lethal dose of wild S. aureus 132 strain ( 6 x 107 CFU) and the survival of mice 35 was monitored for 7 days.

As shown in Figure 16, 85% of mice passively immunized with serum from vaccinated mice survived the challenge, while 100% of mice that received control serum succumbed to infection (P = 0.0008; Log-rank test of Mantel-Cox).

These results demonstrate that passive serum immunization of mice vaccinated with the strain Δdat / Δalr1 / Δalr2 is capable of conferring a significant level of protection against infection and that the antibodies alone are sufficient to provide protective immunity against infection. due to acute sepsis caused by S. aureus 132 (humoral immunity).

 Four. Five

Example 12. Cross-reactivity assay with IgG antibodies of BALB / c mice immunized with strain Δdat / Δalr1 / Δalr2 against heterologous strains of S. aureus

In order to assess whether the antibodies generated in BALB / c mice in response to the triple mutant Δdat / Δalr1 / Δalr2 are capable of recognizing unrelated S. aureus strains, an indirect ELISA was performed with the sera indicated in example 8 against strains of S. 5 aureus USA300LAC and RF122, and with the pool of sera administered passively described in example 12 against strains ED133 and ED98. Thus, the authors of the present invention assess the ability of the vaccine strain to generate a broad humoral immune response. It should be noted that the S. aureus USA300LAC strain is an MRSA (methicillin resistant) strain of great current clinical importance due to its wide distribution and for being a predominant cause of community acquired infections in healthy adults. On the other hand, strains RF122 (bovine origin), ED133 (sheep) and ED98 (poultry) constitute representatives of clones specifically adapted to animal hosts and cause infections in cattle intended for human consumption.

To assess cross-reactivity, the "upholstery" of plates was performed independently with the mentioned strains and the washing, detection and development steps were performed as described in example 8.

Compared to S. aureus strains USA300LAC and RF122, similar results were obtained as compared to the isogenic S. aureus 132 Δspa strain (Figure 17). Likewise, high titers were obtained (Log101 / endpoint title = 3.8854) against strains ED133 and ED98. Overall, a high capacity of these antibodies was observed to recognize antigenic determinants of heterologous strains of S. aureus, which demonstrates that immunization with the strain Δdat / Δalr1 / Δalr2 not only generates antibodies against the isogenic wild strain, but also It also generates IgG antibodies that recognize other strains with different resistance and virulence patterns, and even from different origins. 25

Claims (12)

1. An in vitro method for the production of live attenuated strains of Staphylococcus aureus comprising the following stages: to. Provide a bacterial strain of Staphylococcus aureus capable of expressing alanine racemase and D-amino acid transaminase, and b. Inactivate at least one of the genes encoding the alanine racemase enzymes and the genes encoding the D-amino acid transaminase, such that such inactivation results in said bacterial strain being auxotrophic for D-alanine. 2. The method of claim 1, wherein the method comprises inactivation of the 10 alr1 gene encoding the alanine racemase enzyme and the dat gene encoding the D-amino acid transaminase, such that the bacterial strain is no longer is capable of expressing the functional Alr1 alanine racemase and the Dat functional amino acid transaminase Dat. 3. The method of claim 1, wherein the method comprises the inactivation of the alr1 and alr2 genes encoding the alanine racemases enzymes and the dat gene encoding the D-amino acid transaminase, such that the bacterial strain already is not able to express the functional Alr1 alanine racemase, the functional Alr2 alanine racemase and the Dat functional amino acid transaminase Dat. 4. A live attenuated strain of Staphylococcus aureus, characterized in that said strain 20 comprises inactivated one or more genes encoding the alanine racemase enzyme and the genes encoding the D-amino acid transaminase, such that the bacterial strain is no longer capable of expressing a functional alanine racemase and a functional D-amino acid transaminase, and wherein the inactivation of said genes results in said bacterial strain being auxotrophic for D-alanine. 25 5. The live attenuated Staphylococcus aureus strain of reividication 4, wherein said strain is characterized by inactivation of loci alr1 and dat. 6. The live attenuated Staphylococcus aureus strain of reividication 4, wherein said strain is characterized by inactivation of loci alr1, alr2 and dat. 7. A composition comprising the bacterial strain as defined in any one of claims 4 to 6. 8. A pharmaceutical composition comprising the bacterial strain as defined in any one of claims 4 to 6. 9. Use of the bacterial strain as defined in any one of claims 4 to 6 or any of the compositions of claims 7 or 8, 35 for the preparation of a medicament for the prophylactic treatment against infection by Staphylococcus aureus in a mammal. 10. Use of the bacterial strain as defined in any one of claims 4 to 6 or any of the compositions of claims 7 or 8, for the preparation of a medicament for the prophylactic treatment against infection by Staphylococcus aureus in a human subject. 11. An antibody or fragment thereof selected from the group consisting of F (ab ') 2, Fab', Fab, Fv, "r IgG", Fc, obtained or obtainable after immunization of a mammal with live attenuated auxotrophic bacteria for D-alanine as defined in any one of claims 4 to 6. 45 12. Use of the antibodies or fragments thereof of claim 11, for the preparation of a medicament for use in passive immunization therapy against Staphylococcus aureus.