WO2015164989A1

WO2015164989A1 - Immunogenic bacterial proteins, pharmaceutical compositions containing same, and vaccines against shiga-toxin-producing escherichia coli

Info

Publication number: WO2015164989A1
Application number: PCT/CL2015/000026
Authority: WO
Inventors: David Arturo MONTERO FORERO; Felipe Antonio DEL CANTO FUENTES; Juan Carlos SALAZAR GARRIDO; Roberto Mauricio VIDAL ALVAREZ
Original assignee: Universidad De Chile
Priority date: 2014-04-29
Filing date: 2015-04-24
Publication date: 2015-11-05
Also published as: CL2014001126A1

Abstract

There are different strains of Shiga toxin-producing Escherichia coli (STEC),associated with foodborne diseases, which can produce hemolytic uremic syndrome (HUS). The invention corresponds to antigens as vaccines, which are recognised by the serum of patients with SUH and recognised both by IgG and IgA.

Description

IMMUNOGENIC BACTERIAL PROTEINS, PHARMACEUTICAL COMPOSITIONS CONTAINING THEM, AND VACCINES AGAINST ESCHERICHIA COLI SHIGATOXIN PRODUCER

TECHNICAL FIELD

The present invention relates to antigens, immunogenic proteins, and pharmaceutical compositions that can be used as vaccines against different strains of Escherichia coli that produce shigatoxins.

BACKGROUND

Different strains of shigatoxin-producing Escherichia coli bacteria (STEC) are appearing globally as a zoonotic pathogen, and as one of the main causes of diseases associated with food consumption. STEC is an etiologic agent of acute diarrhea, dysentery, and hemolytic uremic syndrome (HUS). Cattle is the main STEC reservoir, and the consumption of contaminated meat, undercooked, is the main route of infection. 0157.Ή7 is the STEC serotype most frequently associated with sporadic outbreaks and severe cases, but there are other STEC serogroups, such as 026, O103, 0111 and 0113 that have also been implicated as sources of such outbreaks. The bacterium colonizes the human colon and there it synthesizes and releases shigatoxins (Stx), the main virulent factor involved in infectious pathologies. The presence of other virulence factors such as the intimal outer membrane protein and its Tir receptor, both encoded in the enterocyte scaling locus (LEE), are considered risk factors in the development of HUS.

HUS has had a high incidence in children under 5 years of age and represents the most severe consequence of an STEC infection, often associated with microangiopathic hemolytic anemia, thrombocytopenia, and acute renal failure, which sometimes progresses to chronic renal failure. The treatment of HUS is preferably sustenance, since there is no specific therapy for STEC. Antibiotics are contraindicated, as they promote the production and release of Stx, thus increasing the risk of developing HUS. Therefore, because the most negative effects of an STEC infection are those resulting from Stx activity, efforts in the development of therapies have focused on obtaining compounds that bind to the Stx toxin and block its effects. However, the treatments tested so far have not been successful or are still maintained in stages of evaluation of its clinical efficacy in humans.

Vaccine candidates have been tested in animal models with varying levels of success, although the lack of an animal model that reproduces precisely the clinical profile of the infection is a considerable barrier. Vaccine candidates have considered recombinant versions of Stx, intimina, EspA, chimera proteins constructed by fusing the A and B subunits of Stx1 and Stx2, and also the use of non-virulent strains of 0157: 1-17.

Until now, the most promising vaccine candidate tested in humans is based on the covalent fusion of a specific polysaccharide of E. co / 0157: H7 and the exotoxin A of Pseudomonas aeruginosa (0157-rEPA). A Phase II clinical trial is carried out to evaluate said vaccine. However, all these efforts have focused mainly on serogroups 0157, neglecting serogroups other than 0157, and as a result, such vaccines could provide incomplete coverage against STEC.

In addition, the characterization of STEC antigens has focused on serogroup 0157. Humoral immune responses have been described in patients infected with these bacteria against lipopolysaccharide (LPS), Stx, and flagellin (FliC - H7). Antigens encoded in the LEE locus, apart from intiminates and Tir, include EspA, EspB, and EspD. While the identification of these proteins could lead to a vaccine with improved coverage, protection would still be limited mainly to positive STEC-LEE serogroups. Therefore, it is important to identify antigens and immunogenic proteins that are conserved in a wide variety of STECs, independent of the serogroup, in order to provide a broader coverage with a vaccine.

The use of immunoproteomic techniques (2D-PAGE, mass spectrometry, and Western blot) using serum from infected patients has proven useful in identifying proteins synthesized in vivo, during periods of infection, which stimulates the host's humoral immune response.

PRIOR ART

In order to compare the present invention with relevant documents previously published, a search of the state of the art was performed, the result of which is summarized below.

WO2011080505 describes a method for bacteriophage production, where said Bacteriophages can be used in industrial vaccine production. In particular, the use of E. coli as a form of phage production is indicated, however, it is considered a "deficient" E. coli strain of some genes.

WO2011080595 describes E. coli proteins that can be useful as carriers of polysaccharides to generate immune response. In particular, this document focuses on eliciting an immune response against certain polysaccharides and the function of £ proteins. coli is to boost said immune response.

WO2010068 13 describes a vaccine for prevention of chlamydial infections, where OmpT is among the immunogenic fragment options, however there is no mention of shigatoxin-producing strains.

WO2010010983 describes the production of Gram negative bacteria, which have been modified to express outer membrane proteins (in vesicles) with foreign epitopes. In particular, within the epitopes the Omp family is mentioned. More particularly, it is indicated that the epitopes considered are specifically antigens of viruses or microorganisms that cause infectious diseases in humans (Claim 7). Escherichia spp. Bacteria is specifically mentioned. among others (claim 8). Finally, the vesicles that carry the antigens are used as bio-nano particulate vaccines, however it does not mention shigatoxin-producing strains.

Finally, in the detected documents related to the present invention, no documents were found that specifically mention vaccines against E. coli producing Shiga toxin (or shigatoxins), so that the present invention is novel and inventive.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1: PME and Western blot profiles with sera from patients with HUS and control patients. (TO). SDS-PAGE of PME. 12% polyacrylamide, silver tincture. (B, C). Western blot of SDS-PAGE in 12% polyacrylamide with a serum mixture of SUH patients (B) and controls (C) (dilution 1: 3,500); secondary human anti-IgG antibodies (dilution 1: 5,000). (M). Protein size scale, indicates the molecular sizes of several proteins (Line 1). STEC 026: H11. (Line 2). STEC O103. (Line 3). STEC 0113: H21. (Line 4). STEC 0157: H7. (Line 5). E. coli HS commensal strain. Arrowheads with numbers corresponding to the proteins described in Table 1. Figure 2: 2D-PAGE profiles of PME from HS of STEC and E. coli. PH range 4 - 7. 12% polyacrylamide. Coomassie blue G-250 tincture. The images show the immunogenic proteins identified using MALDI-TOF TOF. (TO). STEC 026: H11. (B). STEC O103. (C). STEC 0113.Ή21. (D). STEC 0157: H7. (AND). E. coli HS. (F). Difference between PME profiles of STEC and E. coli HS strains. A unique PME profile was identified for STEC strains (darker spots) superimposed on the PME profile of E. coli HS strain. Some of these proteins were only present in STEC strains (boxes). The analysis was performed using the BioNumerics Software version 6.6. The scale bar indicates the molecular weights in kDa.

Figure 3: Immunogenic proteins identified with Western blot of 2D-PAGE. 2D-PAGE in 12% polyacrylamide (left) and Western blot (right) of PME from STEC strains using a serum mixture of patients with HUS (dilution 1: 3,500). Secondary anti-human IgG antibodies (1: 5,000 dilution). (TO). FliC (STEC 0103). (B). FliC (STEC 0157: H7). (C). FliC (0113: H2). (D). OmpC, OmpF, OmpA, NmpC, OmpT, and Hek (STEC 0113: H2). (AND). L-asparaginase II (STEC 0113.H2). The scale bar indicates the molecular weights in kDa. The arrow under each gel or Western blot indicates the pH range of the separation.

Figure 14: PME profiles of transformed E. coli BL21 (DE3) and Western blot strains of recombinant proteins with serum from patients with HUS and control. (TO). SDS-PAGE of PME in 12% polyacrylamide. Coomassie blue G-250 tincture. (B). Western blot of serum SUH (dilution 1: 3,500) and secondary antibodies against human IgG (dilution 1: 5,000). (C). Western blot of SUH serum (1: 3,500 dilution) and secondary anti-human IgA antibodies (1: 2000 dilution). (D). Western blot with control serum (dilution 1: 3500) and secondary antibodies against human IgA (dilution 1: 2,000). (AND). Western blot of control serum (dilution 1: 3,500) and secondary antibodies against human IgA (dilution 1: 2,000). (M). Protein size scale (one). E. coli BL21 (DE3) / pET15C. (2). E. coli BL21 (DE3) / pET15C_OmpT (3). E. coli BL21 (DE3) / pET15C_Cah. The arrows and numbers correspond to the proteins indicated in Table 4. ^* immature or degraded forms of rCah. The weight of molecular weight standards is indicated.

There are different strains of Escherichia coli that produce shigatoxin (STEC), and constitute a group of emerging zoonotic pathogens worldwide, associated with foodborne diseases, and that can produce Hemolytic Uremic Syndrome (HUS).

The invention corresponds to the use of bacterial proteins, such as vaccines, which are recognized by serum of patients who studied with HUS and recognized by both IgG as IgA in humans.

In the description given below of the present invention, reference is made to antigens, immunogenic proteins, immunogenic External Membrane Proteins (PME) associated and conserved in STEC, where these terms can be used in an equivalent manner to refer to the same subject. , unless otherwise stated.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to antigens, immunogenic proteins, and pharmaceutical compositions comprising them, useful for use as vaccines against shigatoxin-producing E. coli bacteria. In a particular embodiment, the immunogenic proteins and pharmaceutical compositions of the present invention can be administered to a mammal, bird or other animal, as well as humans.

An advantage of the present invention is that the antigens, immunogenic proteins, and pharmaceutical compositions comprising them are not limited to LEE positive strains (Enterocyte Scaling Locus), so that the protection conferred by a vaccine made on the basis of the antigens , immunogenic proteins, and pharmaceutical compositions comprising them of the present invention are broader in that it covers negative LEE strains, which similarly produce shigatoxins.

Thus, the present invention identifies antigens, and immunogenic proteins that in one embodiment are immunogenic External Membrane Proteins (PME) associated and conserved in STEC. In another embodiment said immunogenic PMEs associated and conserved in STEC are used as targets for the development of therapies that help decrease infections related to this pathogen.

In a first aspect, the invention relates to antigens, immunogenic proteins, immunogenic External Membrane Proteins (PME) associated and conserved in STEC.

In a specific embodiment, the immunogenic PMEs associated and conserved in STEC are obtained from particular STEC strains, such as for example STEC 026: H11, 0103 and 0157: H7. Specifically, these STEC strains have been frequently associated with disease in humans and especially with complications of infection such as HUS. Additionally, to ensure coverage in case of negative strains for LEE, a negative LEE strain is also incorporated, which has been reported as causing SUH, said strain corresponds to 0113: H11. In another particular embodiment, to identify antigens, and immunogenic proteins that in one embodiment are immunogenic External Membrane Proteins (PME) associated and conserved in STEC, and prevent the generation of cross-reactivity against E.coli strains that are part of the microbiota commensal (beneficial), the E. coli HS commensal strain was used.

In yet another embodiment, PME extracts are used, since in Gram negative bacteria, it is these proteins that primarily interact with host components mediating adhesion, colonization and invasion processes.

In a particular embodiment, the immunogenic proteins that in one embodiment are immunogenic External Membrane Proteins (PME) associated and conserved in STEC are flagelins (FliC), belonging to STEC strains. In another embodiment the immunogenic proteins are porins. In yet another embodiment, the immunogenic proteins are proteins associated with at least one STEC serotype but absent in E. coli HS.

In a more specific embodiment, porine immunogenic proteins are specifically OmpC, OmpF and OmpA. In an even more specific embodiment, the OmpC, OmpF and OmpA proteins show immunodominance for SUH sera and weak seroreactivity for control sera. In a particular embodiment, the immunogenic protein is L-asparaginase II which is seroreactive for SUH sera.

In yet another more specific embodiment, the immunogenic proteins of the present invention are EF-Tu, Ag43, Cah, OmpT, Hek and, NmpC, all associated with one or more STEC serotypes studied and absent in E. coli HS.

In an even more specific embodiment, the immunogenic protein considered in the present invention OmpT has an amino acid sequence with an identity percentage of at least 80% with respect to SEQ ID NO: 1, more preferably 85%, even more preferably a 90%, and even more preferably 95%, even more preferably 98%, even more preferably 99%.

In an even more specific embodiment, the immunogenic protein considered in the present invention OmpT has a nucleotide sequence with an identity percentage of at least 80% with respect to SEQ ID NO: 2, more preferably 85%, even more preferably a 90%, and even more preferably 95%, even more preferably 98%, even more preferably 99%.

In an even more specific embodiment, the immunogenic protein considered in the The present invention Cah has an amino acid sequence with an identity percentage of at least 80% with respect to SEQ ID NO: 3, more preferably 85%, even more preferably 90%, and even more preferably 95%, even more preferably 98%, even more preferably 99%.

In an even more specific embodiment, the immunogenic protein considered in the present invention Cah has a nucleotide sequence with an identity percentage of at least 80% with respect to SEQ ID NO: 4, more preferably 85%, even more preferably a 90%, and even more preferably 95%, even more preferably 98%, even more preferably 99%.

In an even more specific embodiment, the immunogenic protein considered in the present invention Ag43 has an amino acid sequence with an identity percentage of at least 80% with respect to SEQ ID NO: 5, more preferably 85%, even more preferably a 90%, and even more preferably 95%, even more preferably 98%, even more preferably 99%.

In an even more specific embodiment, the immunogenic protein considered in the present invention Ag43 has a nucleotide sequence with an identity percentage of at least 80% with respect to SEQ ID NO: 6, more preferably 85%, even more preferably a 90%, and even more preferably 95%, even more preferably 98%, even more preferably 99%.

In an even more specific embodiment, the immunogenic protein considered in the present invention Hek has an amino acid sequence with an identity percentage of at least 80% with respect to SEQ ID NO: 7, more preferably 85%, even more preferably a 90%, and even more preferably 95%, even more preferably 98%, even more preferably 99%.

In an even more specific embodiment, the immunogenic protein considered in the present invention Hek has a nucleotide sequence with an identity percentage of at least 80% with respect to SEQ ID NO: 8, more preferably 85%, even more preferably a 90%, and even more preferably 95%, even more preferably 98%, even more preferably 99%.

In an even more specific embodiment, the immunogenic protein considered in the present invention NmpC has an amino acid sequence with an identity percentage of at least 80% with respect to SEQ ID NO: 9, more preferably 85%, even more preferably 90%, and even more preferably 95%, even more preferably 98%, even more preferably 99%.

In an even more specific embodiment, the immunogenic protein considered in the present invention NmpC has a nucleotide sequence with an identity percentage of at least 80% with respect to SEQ ID NO: 10, more preferably 85%, even more preferably a 90%, and even more preferably 95%, even more preferably 98%, even more preferably 99%.

In an even more specific embodiment, the immunogenic protein considered in the present invention L-asparaginase II has an amino acid sequence with an identity percentage of at least 80% with respect to SEQ ID NO: 11, more preferably 85%, even more preferably 90%, and even more preferably 95%, even more preferably 98%, even more preferably 99%.

In an even more specific embodiment, the immunogenic protein considered in the present invention L-asparaginase II has a nucleotide sequence with an identity percentage of at least 80% with respect to SEQ ID NO: 12, more preferably 85%, even more preferably 90%, and even more preferably 95%, even more preferably 98%, even more preferably 99%.

In a specific embodiment, the immunogenic proteins of the present invention are preferably reactive only for SUH sera.

In a second aspect, the invention relates to pharmaceutical compositions comprising antigens, immunogenic proteins, immunogenic External Membrane Proteins (PME), associated and conserved in STEC.

In a particular embodiment, the pharmaceutical composition of the present invention comprises at least one of the antigens or antigenic proteins of the present invention, at least one pharmaceutically acceptable adjuvant, and optionally at least one pharmaceutically acceptable excipient and at least one preservative.

In a more specific embodiment, the adjuvants can be organic adjuvants, such as oil-based adjuvants, virosomal adjuvants; as well as inorganic adjuvants, such as aluminum salts, among others.

In a particular embodiment, the pharmaceutical composition of the present invention It comprises at least about 1 g / ml to about 500 pg / ml of at least one of the immunogenic proteins of the present invention for a unit dose. More preferably between 5 g / ml to about 250 pg / ml

In another embodiment, the pharmaceutical composition of the present invention comprises at least about 2 g / ml to about 1,000 pg / ml of at least two of the immunogenic proteins of the present invention for a unit dose. More preferably between 10 Mg / ml to about 500 g / ml

In another embodiment, the pharmaceutical composition of the present invention comprises at least about 3 Mg / ml to about 1,500 g / ml of at least three of the immunogenic proteins of the present invention for a unit dose. More preferably between 15 iglm \ to about 750 g / ml

In another embodiment, the pharmaceutical composition of the present invention comprises at least about 4 pg / ml to about 2,000 pg / ml of at least four of the immunogenic proteins of the present invention for a unit dose. More preferably between 20 μg / ml to about 1 000 pg / ml.

As for the amounts of each immunogenic protein present in each pharmaceutical composition, it should be understood that the ranges indicated above indicate the minimum and maximum for a given formulation, but all sub-ranges between said minimum and maximum are also considered within the invention. . For example, they are considered sub-ranges with increases or decreases of 50 pg / ml, increases or decreases of 25 g / ml, increases or decreases of 10 pg / ml, increases or decreases of 5 pg / ml, increases or decreases of 2.5 pg / ml, increases or decreases of 1 g / ml, or increases or decreases of 0.1 g / ml.

In particular, the preferred immunogenic proteins for use in the pharmaceutical composition of the present invention are Cah, OmpT, Hek, NmpC, EF-Tu and L-asparaginase II.

Various examples made to illustrate the operation of the present invention are provided below, however, they should not be construed as limiting the scope of the present invention, which is given by the statement of claims. EXAMPLES

EXAMPLE 1: Differences between external membrane protein profiles of STEC and HS strains of E. coli.

Multiple protein bands were observed when PME was analyzed using SDS-PAGE, some of which apparently were only present in STEC strains (Figure 1A). However, it was not possible to discriminate all bands, making it difficult to identify each protein. Therefore, we perform a 2D-PAGE to better discriminate between PMEs of similar molecular weight, ensuring a definitive identification. Because the literature suggests that most PMEs of £. coli are best identified at a pH between 4 to 7, the proteins were separated in this pH range into 2D gels, stained with Coomassie G-250 blue, photographed and analyzed using Bionumerics 2D software, with an average of 47 points detected by each strain (Figures 2A-E). The software also allowed a global PME profile to be performed for the STEC strains analyzed, where the profiles of HS £ strains were superimposed. coli diners to identify unique proteins associated with STEC strains (Figure 2F).

EXAMPLE 2: External membrane immunogenic proteins associated with STEC.

The PMEs separated by SDS-PAGE and 2D-PAGE were transferred to nitrocellulose membranes and subjected to a Western blot assay using a serum mixture of patients with HUS (SUH serum) and a mixture of control patients serum (control serum ), all obtained during convalescence phase. An ELISA test showed that IgG and IgA concentrations did not differ significantly between sera. The SUH serum was recognized by several of the PME, while the control serum was recognized only by a minority of the PME or showed very weak reactivity (Figures 1 B and 1C respectively). Interestingly, some of the proteins associated with STEC (that is, those present only in STEC strains), were recognized by the SUH serum and not by control serum, while others were reactive to both sera. STEC-associated proteins that were seroreactive to SUH serum were extracted from SDS-PAGE and 2D-PAGE gels for identification by MALDI-TOF TOF. Other proteins with molecular weights between 32 and 37 kDa were also extracted, despite being found in all PME profiles, if they were strongly seroreactive with SUH serum. Table 1 lists the immunogenic proteins analyzed using MALDI-TOF / TOF.

A total of twelve immunogenic proteins were identified, of which some were present in multiple PME STEC profiles. The OmpC, OmpF, and OmpA porins, ubiquitous in £ coli, were recognized by both SUH and control sera, although seroreactivity was weaker in the last group. The L-asparaginase II protein was observed only in profiles O103 and 0113: H21 STEC; However, its molecular weight and isoelectric point coincide with the OmpA protein present in other strains studied, possibly masking its presence. Recent studies in our laboratory using anti-L-asparaginase II antibodies suggest that this protein is present in all PME profiles. However, it is notable that this protein was seroreactive only with SUH serum. On the other hand, several flagellar antigens (FliC) were strongly recognized by both SUH serum and control serum. Interestingly, the Ag43 (a43), OmpT, Hek, NmpC, and EF-Tu proteins, which were present in PME profiles of STEC strains but not in E. coli HS commensal strains, were recognized only by SUH serum (Figures 1 B, 1 C and 3), and therefore were classified as immunogenic proteins associated with STEC.

EXAMPLE 3: Bioinformatic analysis of immunogenic proteins.

A bioinformatic analysis was performed using PSORTb to predict the subcellular location of immunogenic proteins, where it was indicated that EF-Tu and L-asparaginase II are cytoplasmic and periplasmic protein, respectively. All other proteins were located in the outer membrane of the bacterial surface.

EXAMPLE 4: Presence of ompT, ag43, and can in STEC strains versus E. coli diners.

Since OmpT is present in the four STEC strains analyzed, and apparently cah is conserved in this pathogen, a PCR analysis was used to determine the ompT, ag43, and cah genes in STEC and commensal strains of E. coli from human feces. . Interestingly, ompT and cah were present in 98% and 64% respectively of STEC isolates, compared with only 36% and 9% of diner isolates. An exact two-tailed Fisher test with a level of significance of 95% determined that the frequency of gene detection for ompT (p <0.0001) and cah (p <0.001) were significantly higher in STEC when compared to isolates from diners The detection frequency of ag43 was significantly higher (p <0.01) in human isolates other than 0157 when compared to commensal isolates (Table 3). These data clearly demonstrate that omT and cah are highly conserved in STEC strains and preferably absent in E. coli commensal strains.

EXAMPLE 5: Recombinant proteins rOmpT and rCah are serum reactive of 6 SUH patient for IgG and IgA.

Recombinant techniques were used to obtain the OmpT protein (rOmpT), in order to confirm the identity suggested by the MALDI-TOF / TOF analysis as well as its immunogenicity. Moreover, because the cah gene is conserved in STEC, recombinant techniques were used to obtain the Cah protein (rCah), in order to determine its probable seroactivity in SUH and control sera. Proteins were produced in £ coli BL21 (DE3) and were partially purified using PME extraction. Interestingly, the PME profile for the strain expressing cah showed 3 different bands, which are not observed if it is obtained from the same strain with the empty plasmid. One of these bands had the expected weight of Cah (92 kDa), while the other two could represent immature or degraded forms of Cah, according to MALDI-TOF / TOF (Figure 4A and Table 4). The Western blot assay determined that rOmpT is recognized by IgG and IgA in SUH and control sera, although seroreactivity is weak in the last group. Interestingly, rCah is recognized by IgG and IgA only from SUH serum, and therefore, Cah was included as an immunogenic protein associated with STEC in the present invention (Figures 4B-E). These results indicate that OmpT and Cah are synthesized in vivo during STEC infections in humans and generate a strong humoral immune response.

Table 1

Immunogenic proteins identified with MALDI-TOF / TOF

Asparaginase II 34000

^A The numbers correspond to proteins shown in Figure 1. Probability of mistakenly assigning protein identity. ^C EI isoelectric point and theoretical molecular weight were determined using the ExPASy Proteomics tools Server UniProt Knowledgebase (http://us.expasy.org/). ^D The prediction made using the PSORTb v3.0 program (http://www.psort.org/psortb/index.html). OM: External membrane, pl: Isoelectric point. Mw: Molecular Weight

Table 2

Presence of ag43 and cah in STEC and avirulent strains of E. coli according to genomic sequence analysis

%

No. Access Size Identity /

Strain Gen Percentage ^B

Similar GenBank (AA)

d ^A

STEC

0157: 1-17 str. EDL933 AAG55356.1 cah 1005 - -

AAG55766.1 cah 1005 100/100 5574

026: H11 str. 11368 BAI24655.1 cah 1005 100/100 5568

^To EMBOSS 6.3.1: (http://mobyle.pasteur.fr/cg¡-bin/portal.py?#forms::matcher). ^B BLASTN algorithm (http: //blast.ncbi.nlm. Nih.gov/Blast.cgi?CMD=Web&PAGE_TYPE=BlastHome). * indicates a gene with a deletion. AA: amino acids

Table 3

Detection frequency for ag43, Cah, and OmpT in 181 E. coli isolates from different sources, determined using PCR

Number

No. (%) of positive isolates for

Isolated origin of

each gene

isolated

ag43 cah ompT

£ coli stool diner 4 (36)

11 3 (27) 1 (9)

human

STEC 0157: H7 of humans 48 1 (2) 48 (98) ^D 48 (100) °

STEC 026: 1-111 of humans 28 28 (100) ^D 28 (100) ° 28 (100) °

Other non-0157 of humans 49 32 (65) ^A 16 (33) 49 (100) °

Total no 0157 77 60 (78) ^B 44 (57) ^B 77 (100) °

125

STEC of humans 125 61 (49) 92 (74) ^D

(100) °

STEC of animals 45 26 (58) 17 (38) 41 (91) ^c Total STEC of isolates 170 87 (51) 109 (64) ^c 166 (98) ^D

^A P <0.05. ^B P <0.01. ^U P <0.001. ^U P <0.0001. Fisher's two-tailed exact test (compared to diner isolates).

Table4

Recombinant proteins identified using MALDI-TOF TOF

^A The numbers correspond to the proteins in Figure 4. ^B Probability of incorrectly assigning the protein identity

Discussion

The present invention was made with PME extracts, since in Gram-negative bacteria these proteins play an important role in the interaction with host components, through adhesion, colonization, and invasion. It has been reported that exposure to enterobacterial PME stimulates the humoral immune response in infected patients, making them suitable candidates for vaccines against Gram negative pathogens.

The immunogenic proteins of the present invention can be classified into three groups, namely flagelins (FliC) of STEC strains that are strongly recognized in both SUH sera and control sera; a second group of OmpC, OmpF, and OmpA porins, which show immunodominance in SUH sera and weak reactivity in control sera; and the third group that includes the protein L-asparaginase II, also present in the PME profiles, but interestingly it only shows reactivity in SUH serum. This third group includes the Ag43, Cah, OmpT, Hek, NmpC, and EF-Tu proteins, usually associated with one or more STEC serotypes but absent from E. coli HS.

In addition, in the present invention a recombinant rOmpT protein was developed, where through Western blot using SUH sera and control sera, it could be determined that rOmpT is recognized by IgG and IgA of both sera, although the response is weak in serum. control. This result contradicts the initial observation that OmpT is not recognized by control serum. Without wishing to be limited by scientific theories, it is postulated that this reactivity derives from a non-specific reaction due to the abundance of the recombinant protein. In addition, these results indicate that both OmpT and Cah are proteins synthesized in vivo during STEC infections in humans and generate a strong humoral immune response.

Finally, it should be noted that the Ag43, Cah, OmpT, Hek, NmpC, EF-Tu, and L-asparaginase II proteins have not been characterized until this invention as immunogenic in STEC frames. Therefore, the present invention represents the first disclosure of these new antigens for the pathotype. Evidence that the cah and ompT genes are conserved in STEC but not in £ coli fecal commensals suggests that both Cah and OmpT may confer protection against a wide range of STEC serogroups and minimal cross reactivity with the commensal microbiota. Because STEC colonize the mucosa, the humoral response with IgA is a requirement to eliminate the pathogen. The reactivity of Cah and OmpT for IgA from SUH serum, together with the other results shown in the present invention, allows the antigens of this invention to be identified as ideal immunogens for STEC vaccines.

Methods used in performing the above examples

Different methods used during the different stages of development of the present invention are described below.

EXAMPLE 6: Bacterial strains, growth conditions, and plasmids

The bacterial and plasmid strains used in the present invention are indicated in Table 5. STEC strains correspond to clinical isolates that were characterized by PCR and identified by the Institute of Public Health of Chile and the Laboratory of Enteropathogens, of the Microbiology Program and Mycology, from the School of Medicine of the University of Chile. The E. coli commensal strain used was HS. A total of 170 STEC and £ 11 were isolated. coli commensals of human feces (PCR negative for virulence factors of known diarrhea patotypes), described in Table 5, which were used for frequency detection of ompT, ag43, and cah genes.

Table 5

Strains and plasmids

Serogroup-

Strain Origin of the isolate Reference

Serotype

Serogroup-

Strain Origin of the isolate Reference

Serotype

E. coli (Studier F, Moffatt B. Use of BL21 (DE3) bacteriophage T7 RNA

polymerase to direct selective high level expression of cloned genes. Journal of molecular biology. 986; 189: 113-30)

The E. coli strains were grown in Luria Bertani medium (LB, 10 g / L Tryptone, 10 g / L NaCl and 5 g / L yeast extract) at 37 ° C for 18 h without shaking. Bacteriological agar was added in a final concentration of 1.5% (w / v) so as to prepare the solid medium. The culture medium was supplemented with ampicillin (100 pg / mL) and 1 mM of isopropyl β-ϋ-1- thiogalactopyranoside (IPTG). To perform alpha complementation, ampicillin (100 pg / mL), IPTG (50 g / mL), and X-Gal (5-bromo-4-chloro-3-indoll-pD-galactopyranoside) were added to the culture medium ( 50 pg / mL).

PME extracts were obtained as indicated in Rivas et al., 2008 (Rivas L, Fegan N, Dykes G. "Expression and putative roles in attachment of outer membrane proteins of Escherichia coli 0157 from planktonic and sessile culture." Foodborne pathogens and disease 2008; 5 (2): 155-64) with minor modifications that are briefly indicated: centrifugation was performed at 25,500 xg for 60 and 40 minutes. The protein concentration in the External membrane fraction was measured using the "Bradford protein assay dye reagent" (Bio-Rad) and standard bovine serum albumin (BSA) reagent according to manufacturer's recommendations. PME extracts were stored at -80 ° C until used.

EXAMPLE 7: Polyacrylamide Denaturant Gel Electrophoresis (SDS-PAGE).

Samples of 8 pg of PME from each strain were separated using 12% polyacrylamide gel with sodium dodecyl sulfate (SDS-PAGE) according to the method described by Laemmli (Laemmli U. "Cleavage of structural proteins during the assembly of the head of bacteriophage T4. "Nature. 1970; 227: 680-685.), using a SE 600 Ruby Standard Dual Cool Vertical Unit (Amersham Biosciences). Precision Standards Plus Protein ™ Kaleidoscope ™ (Bio-Rad) were used as molecular weight markers. The proteins were stained with Coomassie Brilliant Blue G-250 (Bio-Rad) or with the Silver Stain Plus Kit (Bio-Rad).

EXAMPLE 8: Two-dimensional electrophoresis (2D-PAGE).

Preformed strips IPG (13 cm, pH 4-7, linear, GE Healthcare) were rehydrated in 250 pL of DeStreak rehydration solution (GE Healthcare) with an IPG buffer pH 4-7 (1%) (GE Healthcare), DTT ( 10 mM) and 200 pg of PME, for 16 h at room temperature according to manufacturer's recommendations. The rehydrated strips were subjected to isoelectric focusing (IEF) using an Ettan IPGphor Isoelectric Focusing System (GE Healthcare) at 20 ° C with a current limit of 50 μΑ / strip according to the following protocol: 200 V / 1 h, 500 V / 1 h, 1000 V gradient / 1 h, 8000 V gradient / 3: 30 h and finally 8000 V until reaching the focus 20 kVh. After the IEF, the strips were equilibrated for 15 min in 3 mL of buffer I [50 mM Tris-HCI pH 8.8, 6 M urea, 30% glycerol (w / v), 2% SDS (w / v) and DTT 10 mg / mL (w / v)] and then in 3mL of buffer II for 15 minutes. Buffer II differs from I in that it contains iodoacetamide (25 mg / mL) instead of DTT. Then, the strips were applied directly to 12% polyacrylamide gels. The second dimension of vertical SDS-PAGE was performed using SE 600 Ruby Standard Dual Cool Vertical Unit (Amersham Biosciences) according to the manufacturer's instructions. After separation in the second dimension, the gels were stained with Coomassie Brilliant Blue G-250 (Bio-Rad) and photographed, and the images were analyzed using BioNumerics 2D version 6.6 software (Applied-Maths). Precision Plus Protein ™ Kaleidoscope ™ (Bio-Rad) standards were used as molecular weight markers.

EXAMPLE 9: Serum concentrations of IgG and IgA.

A mixture was created from the serum of 10 pediatric patients undergoing convalescence, which they presented with prodromal diarrhea within 20 days prior to the diagnosis of HUS (SUH serum), collected from 1990 to 1993 and from 1999 to 2003. As a control, a mixture was created from serum of 3 patients with no history of HUS or episodes of diarrhea associated with STEC The sera were obtained from several health centers in Santiago, with the informed consent of the patients. All procedures were approved by the Ethics Committee of the School of Medicine of the University of Chile. Table 6 shows relevant characteristics of each patient who donated serum used in this invention. In addition, IgG and IgA concentrations were determined in the mixtures using ELISA with the Protein Detector ELISA kit (Kirkegaard & Perry Laboratories) according to manufacturer's instructions.

Table 6

Serums used in this invention

F: Female; M: Male; m: months; N.R .: Not registered; N.A .: Non-binder

EXAMPLE 10: Transfer and immunodetection of proteins

The PMEs separated by SDS-PAGE and 2D-PAGE were transferred to membranes of nitrocellulose (Millipore) for 60 min at 100 V / 4 ° C using the Mini Trans-Blot Cell (Bio-Rad) system according to the manufacturer's instructions.

After the transfer was completed, the non-specific binding sites available on the membrane were saturated using a blocking solution (Saline in 1X Tris buffer (TBS-1X), 0.03% Tween-20 and 1% BSA) by 1 h at room temperature. Then, the membranes were rinsed 5 minutes 3 times with TBS-T solution (1X TBS, 0.3% Tween-20). The membranes were then incubated with stirring for 1 h in a blocking solution containing the mixtures of SUH or control sera, diluted 1: 3500. After 2 rinses with TBS-T solution for 10 minutes at a time, the membranes were incubated with block solution containing anti-human IgG conjugated with alkaline phosphatase diluted 1: 5000 for 1 hour at room temperature with stirring. The images were revealed using a kit with 5-bromo-4-chloro-3-indolyl phosphate (BCIP) chromogenic substrate (Invitrogen), and the reaction was stopped with distilled water.

The seroreactivity of the rOmpT and rCah recombinant proteins was evaluated using secondary IgG and IgA anti-human antibodies (Invitrogen) conjugated to alkaline phosphatase and diluted 1: 5000.

EXAMPLE 11: Identification of immunogenic proteins using mass spectrometry

The proteins recognized by antibodies of the SUH serum but not of the control serum, or that were present only in STEC strains, were cut from the SDS-PAGE and 2D-PAGE gels stained with "Coomassie Brilliant Blue G-250." Other proteins that demonstrated immunodominance were also obtained from the gels. These proteins were sent for analysis by Mass Spectrometry at the University of Texas Medical Branch in Galveston (Galveston, Texas, United States) for identification by MALDI-TOF / TOF mass spectrometry. Each peptide fingerprint fragment was sent to the MASCOT server (http://www.matrixscience.com) to identify the protein by comparing it with the fragments of other proteins present in that database. The identities of the rOmpT and rCah recombinant proteins were also confirmed by MALDI-TOF / TOF.

EXAMPLE 12: Bioinformatic analysis of the immunogenic proteins identified.

Subcellular localization was predicted using pSORTb version 3.0 (http://psort.org/). Theoretical isoelectric points and molecular weights were determined using the ExPAS Server and Proteomics Server UniProt Knowledgebase (http://us.expasy.org/). The BLASTN algorithm (http://blast.ncbi.nlm.nih.gov) was also used to evaluate the distribution of coding genes for each protein in other £ coli strains and in other species of bacteria.

Gene frequency detection for ompT, ag43, and cah.

EXAMPLE 13: Polymerase chain reaction (PCR) was used to determine the presence of genes.

The amplification reactions were performed in a final volume of 25 pL containing tempered DNA, 0.4 μΜ of each splitter, 5 pL of 5X GoTaq DNA-polymerase buffer, 0.2 μΜ of each dNTP (Fermentas), and 1, 25 U of GoTaq® DNA Polymerase (Promega). The PCR conditions were specific for each amplification. The hybridization temperature was determined as a function of the melting temperature of the splitter, and the duration of the extension stage as a function of the length of the DNA fragment, generally 1 min / kilobase.

Because the main difference between ag43 and cah is a 270 bp deletion in the coding region of the alpha domain of Cah, the splitters were designed to flank this region, and obtain two amplifications with two different molecular weights (Table 7). The frequency of data obtained was subjected to an exact two-tailed Fisher test with a significance level of 95%.

Table 7

Splitters used in the invention

* The underlined portion indicates the cut sites for the restriction enzymes Ndel and Xhol. bp: base pairs

EXAMPLE 14: Generation of rOmpT and rCah recombinant proteins.

The coding sequences of OmpT and Cah were amplified from the strain of E. coli reference 0157: H7 EDL933, using the cleaners described in Table 7, with recognition sites for restriction enzymes Nde \ and Xho \ at terminals 5 'and 3' respectively. The PCR products were linked to the vector pTZ57R / T (Fermentas) according to the manufacturer's instructions to construct the vectors pTZ57R / T_OmpT and pTZ57R / T_Cah. These vectors were used to transform the E. coli DH5a strain in the laboratory, and the clones were selected according to ampicillin resistance and alpha complementation. Sequencing was performed to confirm correct cloning of the coding sequences (Macrogen).

Plasmid DNA was extracted from transformed DH5a E. coli strains and digested with restriction enzymes Nde \ and Xho \. The products were analyzed in 1% agarose gels, and OmpT and Cah were purified. The sequences were linked to the expression vector pET15C (described in Caballero V, et al. Expression of Shigella flexneri gluQ-rs gene is linked to dksA and controlled by a transcriptional terminator. BMC Microbiology. 2012; 12: 226), allowing production of recombinant proteins under the control of the Phage T7 promoter and with a histidine tail at the C-terminal of each protein, to construct the vectors pET15C_OmpT and pET15C_Cah. These vectors were used to transform strains E. coli DH5a and E. coli BL21 (DE3). As a control, both strains were also transformed with an empty pET15C vector. The clones were selected according to ampicillin resistance and confirmed by PCR. Transformed strains of E. coli BL21 (DE3) were grown in LB medium supplemented with 100 pg / mL of ampicillin for 10 h at 37 ° C with shaking, and then the synthesis of recombinant proteins was induced for 4 h, supplementing the medium of 1 mM IPTG culture. Recombinant proteins were partially purified using PME extraction, according to the protocol described above.

INDUSTRIAL APPLICATION

The present invention is applicable in the pharmaceutical industry, particularly in the manufacture of vaccines against E. coli producing shigatoxin and other pathogenic E.coli with which the present formulation could also protect.

Claims

1. Pharmaceutical composition useful for use as a vaccine against shigatoxin-producing E. coli bacteria, CHARACTERIZED because it comprises at least one antigen or at least one immunogenic Outer Membrane Protein (EMP) or immunogenic protein associated and conserved in Escherichia coli strains producing shigatoxins (STEC), in order to avoid cross-reactivity with Escherichia coli strains that are part of the commensal or beneficial microbiota.

2. Pharmaceutical composition according to claim 1, CHARACTERIZED in that the at least one immunogenic PME is obtained from STEC strains selected from STEC 026:H11, O103 and 0157:H7, or from a strain negative for enterocyte sloughing locus (LEE). ).

3. Pharmaceutical composition according to claim 2, CHARACTERIZED because the LEE-negative strain is 0113:H11.

4. Pharmaceutical composition according to claim 2, CHARACTERIZED because the immunogenic PMEs associated and conserved in STEC are flagellins (FliC), belonging to STEC strains.

5. Pharmaceutical composition according to claim 2, CHARACTERIZED because the immunogenic PMEs associated and conserved in STEC are porins.

6. Pharmaceutical composition according to claim 2, CHARACTERIZED in that the immunogenic PMEs associated and conserved in STEC are proteins associated with at least one STEC serotype but absent in Escherichia coli HS.

7. Pharmaceutical composition according to claim 5, CHARACTERIZED in that the immunogenic porin PMEs are selected from OmpC, OmpF and OmpA.

8. Pharmaceutical composition according to claim 7, CHARACTERIZED because the immunogenic PMEs porins OmpC, OmpF and OmpA show immunodominance for HUS sera and weak seroreactivity for control sera.

9. Pharmaceutical composition according to claim 2, CHARACTERIZED because the immunogenic PME is L-asparaginase II which is seroreactive for HUS sera.

0. Pharmaceutical composition according to claim 2, CHARACTERIZED in that the immunogenic PME is selected from EF-Tu, Ag43, Cah, OmpT, Hek and NmpC, all associated with one or more STEC serotypes and absent in E. coli HS .

11. Pharmaceutical composition according to claim 1, CHARACTERIZED in that the at least one immunogenic PME has an amino acid sequence with a percentage of identity of at least 80% with respect to the sequences selected from SEQ ID NO:1, SEQ ID NO :3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, or SEQ ID O:11.

12. Pharmaceutical composition according to claim 1, CHARACTERIZED in that the at least one immunogenic PME has a nucleotide sequence with a percentage of identity of at least 80% with respect to the sequences selected from SEQ ID NO:2, SEQ ID NO :4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, or SEQ ID NO:12.

13. Pharmaceutical composition according to any of the preceding claims, CHARACTERIZED because it also comprises at least one pharmaceutically acceptable adjuvant, at least one pharmaceutically acceptable excipient, and at least one preservative.

14. Pharmaceutical composition according to claim 13, CHARACTERIZED in that the adjuvants can be organic adjuvants, such as oil-based adjuvants, virosomal adjuvants; as well as inorganic adjuvants, such as aluminum salts, among others.

15. Pharmaceutical composition according to any of claims 1 to 12, CHARACTERIZED because it comprises at least about 1 g/ml up to about 500 pg/ml of at least one of the immunogenic PMEs.

16. Pharmaceutical composition according to any of claims 1 to 12, CHARACTERIZED because it comprises at least about 2 μg ml up to about 1,000 g/ml of at least two of the immunogenic PMEs.

17. Pharmaceutical composition according to any of claims 1 to 12, CHARACTERIZED because it comprises at least about 3 pg/ml up to about 1,500 g/ml of at least three of the immunogenic PMEs.

8. Pharmaceutical composition according to any of claims 1 to 12, CHARACTERIZED because it comprises at least about 4 pg/ml up to about 2,000 pg/ml of at least four of the immunogenic PMEs.