INFECTION

DOMAIN OF THE INVENTION

The present invention concerns the medical field and in particular vaccination field. Specifically, the present invention is related to a live-attenuated strain of Pseudomonas for its use as a vaccine, and for use in a method for preventive immunizing and for treating Pseudomonas infection, in particular in patients suffering from cystic fibrosis.

BACKGROUND

Pseudomonas aeruginosa is purveyor of an important morbidity and mortality especially in cystic fibrosis patients, and its eradication is difficult because of a huge phenotypic adaptability and thus an increase in resistance to antibiotics.

Cystic fibrosis (CF) is an inherited disease due to a defect in the CFTR gene encoding a membranous chloride channel. In the lungs of CF patients, primary defenses against bacteria are disturbed and patients rapidly suffer from infections with different bacteria. Pseudomonas aeruginosa (P. aeruginosa) is by far the most significant pathogen in CF adults and, it appears that pulmonary infections with this pathogen occur much earlier than believed previously in children (1-4). P. aeruginosa is a bacterium with a large genome particularly well adapted to colonize lungs of CF patients, firstly via the expression of molecular factors in response to host defenses (5-9), and secondly by its capacity to develop antimicrobial resistance mechanisms. The average prevalence of P. aeruginosa infections in these patients is 57.8 % and increases with age (4), and the acquisition of this microorganism is associated with a clinical poor outcome (6-9).

Curative treatments such as antibiotics are not sufficient to eradicate P. aeruginosa. Indeed, this microorganism is particularly resistant to the current antibiotic arsenal; it displays intrinsic multidrug resistance and has a tremendous capacity to acquire further resistance mechanisms (10,1 1 ). Moreover, during chronic infections this organism can sometimes adopt a mucoid phenotype and is also thought to adopt a biofilm-like mode of growth, resulting in protection from host immune system and antibiotic attack, with oxygen limitation and low bacterial metabolic activity (12). Thereby, the best would be to develop a vaccination strategy, as curative but also prophylactic therapy, that would limit chronic colonization of CF lungs by P. aeruginosa and by this way acute pulmonary infection, and maintain an optimal composition of pulmonary microbiota. But after 40 years of research and clinical trials up to phase 3, no biotechnology company has met the challenge of achieving a safe, immunogenic and effective vaccine.

Some literatures reviewed several studies conducted to date and attempted to dissect the necessary elements for a good immunization coverage against P. aeruginosa (13-15). It appears that only a vaccine which would cause a broad immune response (humoral and cellular) would be protective and could help to neutralize or eliminate this microorganism.

A number of recombinant vaccines have already been tested and some elements stand out as important to elicit a good immune response. Those are lipopolysaccharide (LPS), alginate, flagellum, type 3 secretion system (T3SS) with PopB and PcrV, and outer membrane proteins OprF and Oprl. But these recombinant vaccines, despite numerous clinical trials, failed to be protective for CF patients probably due to the phenotypic plasticity of P. aeruginosa. However, a recent Cochrane Database (16) comparing several trials on P. aeruginosa vaccines to placebo or no intervention concluded that vaccines against P. aeruginosa cannot currently be recommended in patients with CF.

After the failure of several recombinant vaccines which solicit only humoral response, live-attenuated vaccines are the focus of attention as they elicit a broad immune response (humoral and cellular) indispensable to fight against its pathogen (17). For example, P. aeruginosa mutants having a deletion of the aroA gene, which is required for the synthesis of aromatic amino acids, have proven to be both highly attenuated and immunogenic in animal models (18,19). These live-attenuated P. aeruginosa vaccine strains confer a T cell-mediated immune response which protect rodents during infectious challenge, regardless the associated LPS serotype and the production of neutralizing antibodies (22,23). Indeed, IL-17 cytokine produced in large amounts by the T cells is found in great quantity in the bronchoalveolar lavage and participated in the massive recruitment of neutrophils in the airways and thus in the antibacterial response (20). Moreover, the depletion of IL-17 before the challenge of the immunized mice or the absence of IL-17 receptor (IL-17R) proves that IL- 17, and therefore the cellular immune response (Th17 pathway), is necessary to obtain a good protection of the vaccine in a mouse model (20). Several studies have shown an important role of IL-17 in innate and adaptive antibacterial defenses, particularly during Klebsiella pneumoniae (21 ,22), Bacteroides frag His (23) and Escherichia coli (24) lung infections. Some proteins have been identified in P. aeruginosa to cause a high secretion of IL-17 such as PopB, FpvA, FptA, OprL and PilQ (25). The purified PopB protein which belongs to the translocon of the T3SS (highly conserved element) generates a strong Th17 response which contributes to the increasing clearance of P. aeruginosa in lung and spleen after challenge (25).

All these data suggest the interest to explore the development of a vaccine candidate which involves multiple immune effectors, and not only opsonic antibodies production against LPS O antigen or other antigens of the bacterium.

For the P. aeruginosa-based vaccine, type III secretion system (T3SS) has been demonstrated by many laboratories to be a major virulence factor of this bacterium. The T3SS apparatus is like an injectisome dedicated to the injection of toxins inside host cells (29). Recently, it has been shown that this injectisome could be hijack to inject proteins of interest, and this could help in the development of a novel antigen delivery tool (30).

In 2013, Le Gouellec et al. have applied the KBMA attenuation strategy on P. aeruginosa to develop a microsyringe for antigen delivery based on the T3SS of this bacterium (30). They have shown that the KBMA has the potential to deliver antigens to human antigen-presenting cells in vitro via T3SS with considerable attenuated cytotoxicity as compared with the wild- type vector (31 ). In a mouse model of cancer, this KBMA strain which cannot replicate in its host, efficiently disseminates into lymphoid organs and delivers its heterologous antigens (31 ). The attenuated strain effectively induces a cellular immune response against the cancerous cells while lowering the systemic inflammatory response.

The inventors have now surprisingly shown that the use of a live-attenuated Pseudomonas strain is able to elicit a safe, immunogenic and effective response against Pseudomonas infection. Therefore, they showed that the live-attenuated Pseudomonas strain developed in the prior art as a vehicle or tool for delivering a specific protein of interest, and in particular tumoral antigens, is an efficient vaccine against Pseudomonas infection when used as an empty vehicle, viz. with no specific protein of interest to be delivered. Furthermore, they demonstrated the ability to rationally design this vaccine against Pseudomonas infection by overexpression of beneficial effectors in order to obtain the broadest immune response as possible and/or by modification to improve safety of such a vaccine based on killed but metabolically active attenuation method. The present invention thus provides an innovative and safer live-attenuated Pseudomonas vaccine. SUMMARY OF THE INVENTION

The present invention is related to a live-attenuated strain of Pseudomonas for use in a method for preventive immunizing a patient prone to suffer from a Pseudomonas infection or for treating a patient suffering from a Pseudomonas infection, said live-attenuated strain comprising deletion of the genes ExoS, ExoT.

Preferably, this live-attenuated strain of Pseudomonas for use in a method for preventive immunizing a patient prone to suffer from a Pseudomonas infection or for treating a patient suffering from a Pseudomonas infection, further expresses the gene ExsA encoding the activator of the Pseudomonas aeruginosa type III secretion system, and the sequence encoding the 54 first amino acids of the ExoS toxin (exoS54). In an advantageous embodiment of the invention, the live-attenuated Pseudomonas strain to be used according to the invention is furthermore treated with the KBMA process to become 'killed but metabolically active'.

In a preferred embodiment, the live-attenuated Pseudomonas strain to be used according to the invention may further express beneficial effectors so as to obtain the broadest immune response as possible, and in particular express at least one of the following proteins: proteins of the main virulence factor T3SS, such as PopB, PopD, PscJ, Pscl and PcrV; proteins of the flagella, such as FliC; and porin such as OprF and Oprl.

The invention is also related to a vaccine comprising at least the live-attenuated strain of Pseudomonas as defined above and a pharmaceutically acceptable carrier or adjuvant.

The present invention is also related to a vaccination strategy characterized by multi- position injections of the live-attenuated strain of Pseudomonas, to the organism. In particular, the present invention is thus related to a method of inducing an immune response in a patient prone to suffer from a Pseudomonas infection or in a patient suffering from a Pseudomonas infection, comprising administering to said patient by injection in multiple positions an effective amount of the above-described vaccine.

Finally, the invention is also related to the use of the live-attenuated strain of Pseudomonas as defined above for preparing a vaccine. DETAILLED DESCRIPTION OF THE INVENTION

All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety. However, publications mentioned herein are cited for the purpose of describing and disclosing the protocols, reagents and vectors which are reported in the publications and which might be used in connection with the invention. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

Furthermore, the practice of the present invention employs, unless otherwise indicated, conventional microbiological and molecular biological techniques within the skill of the art. Such techniques are well known to the skilled worker, and are explained fully in the literature. See, for example, Sambrook et al., 2001 . Conventional immunological techniques are explained in Current protocol in Immunology, Coligan, John Wiley & Sons (2005)

It must be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to "a strain" includes a plurality of such strains, and so forth. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any materials and methods similar or equivalent to those described herein can be used to practice or test the present invention, the preferred materials and methods are now described.

As used herein, the following terms may be used for interpretation of the claims and specification.

In the claims which follow and in the preceding description of the invention, except where the context requires otherwise due to express language or necessary implication, the word "comprise" or variations such as "comprises" or "comprising" is used in an inclusive sense, i.e. to specify the presence of the stated features but not to preclude the presence or addition of further features in various embodiments of the invention.

The term "Pseudomonas" designates a genus of gammaproteobacteria belonging to the family Pseudomonadaceae containing 191 validly described species. All species and strains of Pseudomonas are Gram-negative rods, and are classified as strict aerobes. Among them, Pseudomonas aeruginosa is a highly relevant opportunistic human pathogen.

Gram-negative bacteria use different types of secretion systems for their own purposes. In particular, the type III secretion system (T3SS) is involved in the cytotoxicity of Pseudomonas strains.

The present invention is related to a live-attenuated strain of Pseudomonas for use in a method for preventive immunizing a patient prone to suffer from a Pseudomonas infection or for treating a patient suffering from a Pseudomonas infection, wherein said live-attenuated strain comprising deletion of the genes ExoS, ExoT.

Advantageously, the live-attenuated strain of Pseudomonas for use in a method for preventive immunizing a patient prone to suffer from a Pseudomonas infection or for treating a patient suffering from a Pseudomonas infection further expresses the gene ExsA encoding the activator of the Pseudomonas aeruginosa type III secretion systemand the sequence encoding the 54 first amino acids of the ExoS toxin (exoS54).

In one embodiment, the live-attenuated strain of Pseudomonas to be used as described above, comprises deletion of the genes ExoS, ExoT, expresses the gene ExsA encoding the activator of the Pseudomonas aeruginosa type III secretion system and the sequence encoding the 54 first amino acids of the ExoS toxin (exoS54), and further expresses Orf1

(also named SpcS) the specific chaperone of ExoS.

Indeed, the inventors demonstrated that a specific live-attenuated Pseudomonas, and in particular a specific live-attenuated Pseudomonas aeruginosa "killed but metabolically active", elicited a high and broad humoral immune response in mice against several antigens of particular interest such as OprF porin and PcrV protein, a component of the type 3 secretion system, the major virulence factor of Pseudomonas aeruginosa. They also demonstrated that while stimulating humoral immunity, this specific live-attenuated Pseudomonas aeruginosa elicited also several pathways of cellular immunity, especially Th1 , Th2 and especially Th17, known as necessary to eradicate this bacterium. Moreover, they demonstrated that the vaccine strain according to the present invention is safe and has a protective efficacy in mice during a pulmonary infectious challenge.

The live-attenuated Pseudomonas strain to be used according to the present invention comprises at least deletion of the genes encoding the two major T3S toxin exoenzymes - ExoS and ExoT. In particular, such a live-attenuated Pseudomonas strain may be obtained starting from the initial strain CHA (wild-type, P. aeruginosa mucoid strain as described in

(Toussaint, B., I. Delic-Attree and P. M. Vignais (1993). "Pseudomonas aeruginosa contains an IHF-like protein that binds to the algD promoter." Biochem Biophys Res Commun 196(1 ):

416-421 ).

Advantageously the live-attenuated Pseudomonas strain to be used according to the invention is a strain which is already attenuated by deletion of these two genes such as CHA- OST as described by Epaulard O, Derouazi M, Margerit C, Marlu R, Filopon D, Polack B, et al. Optimization of a type III secretion system-based Pseudomonas aeruginosa live vector for antigen delivery. Clin Vaccine Immunol 2008 Feb;15(2):308-13. For the aspect of the present invention where the live-attenuated Pseudomonas strain further exhibit other features with relation to ExsA and the sequence encoding the 54 first amino acids of the ExoS toxin (exoS54), optionally with expression of Orf1 (SpcS) the chaperone protein of ExoS, a strain which is already attenuated by deletion of the two genes ExoS and ExoT such as CHA-OST as described by Epaulard et al. 2008 (supra) may be modified to expression those other features, and in particular transformed with a plasmid containing all the other necessary sequences as defined above, as for example with the plasmid pEiS54 (GeneBank JQ733380, version 1 of March 14, 2012) for exhibiting the other requested features.

Whatever the aspect of the invention, the live-attenuated strain of Pseudomonas to be used is advantageously furthermore treated to become 'killed but metabolically active'.

The process to obtain a "killed but metabolically active" bacteria can be summarized as follow:

• deleting the gene uvrAB encoding exonucleotidase A and B subunit; any technique known by the man skilled in the art can be used to obtain this deletion; and

• A photochemical inactivation with UVA is performed in presence of psoralen.

Toxicity and optionally secretion via the TTSS system by the thus treated strain may be assessed. For more details on the KBMA process, see Brockstedt et al., 2005 (Brockstedt DG, Bahjat KS, Giedlin MA, Liu W, Leong M, Luckett W, Gao Y, Schnupf P, Kapadia D, Castro G, Lim JY, Sampson-Johannes A, Herskovits AA, Stassinopoulos A, Bouwer HG, Hearst JE, Portnoy DA, Cook DN, Dubensky TW Jr. Killed but metabolically active microbes: a new vaccine paradigm for eliciting effector T-cell responses and protective immunity. Nat Med. 2005 Aug; 1 1 (8):853-60) and Wollowitz, 2001 (Wollowitz S. Fundamentals of the psoralen-based Helinx technology for inactivation of infectious pathogens and leukocytes in platelets and plasma. Semin Hematol. 2001 Oct;38(4 Suppl 1 1 ):4-1 1 ), herein incorporated by reference.

In a preferred embodiment of the invention, a KBMA live-attenuated strain of Pseudomonas may be obtained by culturing the live-attenuated strain of Pseudomonas with deletion of the gene uvrAB encoding exonucleotidase A and B subunit and exposure to long wavelength UVA light in presence of psoralen. Preferably, the exposure is carried out in presence of a concentration of 10μΜ psoralen during 40 minutes prior to exposition to UVA irradiation at a dose of 7,2J/cm ² (about 365 nm) during 40 minutes.

In addition, the inventors demonstrated that the live-attenuated strain of Pseudomonas to be used according to the invention is an engineered life biological product which may be rationally designed by overexpressing beneficial effectors for optimization of the vaccination efficacy in order to obtain the broadest immune response as possible.

In an advantageous embodiment of the present invention, the live-attenuated strain of Pseudomonas to be used is further expressing at least one of the following proteins: proteins of the main virulence factor T3SS, such as PopB, PopD, PscJ, Pscl and PcrV; proteins of the flagella, such as FliC; and porin such as OprF and Oprl.

Also, as described above, a Pseudomonas strain which is already attenuated by deletion of the aroA gene encoding the 3-phosphoshikimate 1-carboxyvinyltransferase which is a key enzyme in aromatic amino acid synthesis and/or of the last gene encoding the enzyme which produces quorum sensing (QS) homoserine lactones 3-oxo-C12-HSL may be used as starting strain for obtaining the live-attenuated strains to be used according to the invention. As previous described, Pseudomonas strains with attenuation of its virulence while keeping a good efficiency to induce an immunogenic response when administered to an organism, are already available in the art, such as for example CHA-OST and CHA-OAL (Epaulard et al., 2008 supra).

In a preferred embodiment of the invention, the live-attenuated Pseudomonas strain belongs to the species Pseudomonas aeruginosa.

In another preferred embodiment, the present invention is related to the live- attenuated strain of Pseudomonas for use as defined above in a method for preventive immunizing or for treating a patient suffering from cystic fibrosis, since these patients are particularly disposed to pathogens and especially to Pseudomonas infection.

The invention is also related to a vaccine comprising at least the live-attenuated strain of Pseudomonas such as described above and a pharmaceutically acceptable carrier or adjuvant. The man skilled in the art knows the best adjuvant for each vaccine composition.

The present invention is also related to a method of inducing an immune response in a host comprising administering to the host by injections in multiple positions an effective amount of the vaccine such as described above, and preferably said host is a patient prone to suffer from a Pseudomonas infection or in a patient suffering from a Pseudomonas infection.

Finally, the invention is also related to the use of the live-attenuated strain of Pseudomonas such as described above, for preparing a vaccine composition.

DRAWINGS

Figure 1. Polyvalent humoral response elicited by the vaccine vector KBMA. ELISAs were performed on sera of immunized with either KBMA (n=6) or KBMA + IPTG (n=18) and unimmunized mice (n=18) collected 3 weeks after the last immunization. A) PcrV and B) OprF as coated antigens. Non-parametric unpaired Mann-Whitney t tests were used for statistical analysis. Error bars are SEM. Positive threshold of 0.26 for PcrV and 0.51 for OprF. **p<0.005, ****p<0.0001. OD: optical density.

Figure 2. Cellular response elicited by the vaccine vector KBMA.

A multiplex immunoassay was performed in duplicate to determine the expression levels of 23 different cytokines in sera samples of immunized (n=10 per group) and unimmunized mice (n=30: pool of all unimmunized mice) collected at days 1 , 7 and 21 after the last immunization. Only the cytokines that were significantly different from the control (*p< 0.05) are shown in mice immunized with KBMA + IPTG. Non-parametric unpaired Mann-Whitney t tests were used for statistical analysis. Scatter plots represent averages and bars mean with SD. *p<0.05, **p<0.005, ***p<0.0005, ****p<0.0001. D1: day 1; D7: day 7; D21: day 21 after the last immunization.

Figure 3. Cellular response elicited by the vaccine vector KBMA.

A) Flow cytometry successive gating strategy for detection of helper T cells from the spleen. The singlets cells without damaged membranes and thus not permeable to the reactive dye were considered as viable cells and gated on CD3 CD4 ⁺ . B) Flow cytometry threshold strategy for detection of helper T cells effector activity by cytokine intracellular staining assay. Isotypes control antibodies were used to set-up the background. C and D) Unspecific stimulations with PMA/iono were used to analyze the frequency of helper T cells producing IL-17 defining the Th17 subtype and IFN-γ but not the IL-17 defining the Th1. We also analyzed the TNF-a production by the helper T cells. C) Shows a single example of helper T cells cytokine production by spleen cells from unimmunized mice and immunized mice. D) Summarize the data. E) Syngeneic DC ₂₄ APCs presenting KBMA antigens (KBMA) after overnight processing were used to stimulate spleen and measure the helper T cells specific responses to the KBMA vaccine. APCs not exposed to the bacteria (DC 0) were used as negative stimulating condition for background evaluation. D and E) Mean and SEM frequency of Th17, Th1 and TNF-a helper T cells is depicted for unimmunized mice and immunized mice. Unpaired t test differences between the two groups are indicated (*p<0.05, **p<0.005, ***p<0.0005).

Figure 4. No toxicity on weight for the vaccine vector KBMA.

Immunized (n=35) and unimmunized mice (n=30) were weighted weekly since the first immunization to the intranasal infectious challenge. Non parametric unpaired Mann-Whitney t test was used for statistical analysis. Error bars are mean with SD, p=0.4418. Figure 5. Protective efficacy conferred by the vaccine vector KBMA. Intranasal infectious challenge with 5 x 10 ⁶ CFU/mouse of CHA strain were performed on KBMA+IPTG immunized mice (n=12) and unimmunized mice (n=10), 3 weeks after the last immunization. Animals were then observed for survival every four hours for up to one week, animal health was recorded and mice were euthanized if necessary according to different clinical signs. Log- rank test was used for statistical analysis. ****p<0.0001.

EXAMPLES Before describing the present invention in detail, it is to be understood that this invention is not limited to particularly exemplified methods and may, of course, vary.

Bacterial strains, plasmids and media

The bacterial strains and plasmids used in this work are listed in Table 1.

Table 1

Name Relevant features

Bacterial strains

CHA Wild type, P. aeruginosa mucoid strain, CF isolate from CHU

Grenoble

OST CHA strain deleted for Exotoxins S and T (AexoS, AexoT)

OSTAB2 CHA-OST strain deleted for uvrA and uvrB genes (AuvrA::lox

AuvrB::lox), susceptible to photochemical treatment

OSTAB2 pEiS54 CHA-OSTAB2 transformed with empty plasmid pEAi-S54

(inducible exsA gene; secretion tag)

Plasmids Empty plasmid with inducible exsA gene and secretion tag pEiS54 Gift from Ina Atree (CEA, Grenoble, France)

pET15b-pcrV The photochemical sensitive strain OSTAB2 mutant was previously generated from the P. aeruginosa strain CHA-OST (35) by deleting uvrA and uvrB genes by Cre/lox-based mutagenesis (36). The strain is transformed with pEiS54 plasmid (GenBank accession number JQ733380, version 1 of March 14, 2012).

Media used for bacterial cultures were Lysogeny broth (Miller) and Pseudomonas isolation agar (PIA, Difco™, BD). Medium used for cell cultures was Iscove's Modified Dulbecco's Media (IMDM, Sigma-Aldrich). Whenever it is cited, carbenicillin (Cb) concentration was used at 300μg/mL, isopropyl-beta-D-thiogalactopyranoside (IPTG) at 0.5mM and gentamicin at 50μg/mL (Sigma-Aldrich).

Preparation of vaccinal strainFrozen bacterial stocks of OSTAB2 pEiS54 strain were grown overnight in Lysogeny broth (LB) containing Cb, at 37°C, 300 rpm. After washing the overnight culture twice with 1 mL LB, bacteria were resuspended in culture at an optical density at 600nm (OD600) of 0.2 in LB containing either Cb (condition KBMA) or Cb + IPTG (condition KBMA + IPTG) until the OD600 reached a value of 0.5. Inactivation of bacteria was then obtained using the photochemical treatment: after the addition of 10μΜ of amotosalen (a gift from Grenoble EFS, France) in the culture medium and when the OD600 reached a value of 1 , bacteria are subjected to illumination with UVA 365nm (7,2J/cm ² ) during 40 minute, as previously described (31 ). Concentrations were adjusted spectrophotometrically and confirmed retrospectively by colony-forming units (CFU) counting on PIA at 37°C.

Immunization of mice

We used female C57BI/6J mice as they are known susceptible to chronic bronchopulmonary P. aeruginosa infection and able to produce high antibody levels and Th2 response (37,38). Mice were purchased at an age of 6-8 weeks from Janvier SA (Le Genest-Saint-lsle, France) and were kept under specific pathogen-free conditions in the PHTA animal facility at the University Grenoble Alpes (Grenoble, France). All mice were anesthetized (isoflurane) and inoculated three times by placing 100μΙ of the vaccine preparation subcutaneously (SC) in the right flank with either KBMA or KBMA + IPTG. Escalating doses of 1 x 10 ⁸ CFU/mouse, 2 x 10 ⁸ CFU/mouse and 2 x 10 ⁸ CFU/mouse were administered at fifteen days' interval.

Evaluation of the humoral immune response

Anti-PcrV and anti-OprF specific antibodies were assessed by enzyme linked immunosorbent assay (ELISA) using Nunc MaxiSorp® flat bottomed 96-well plates (Dutscher, France) coated overnight at 4°C with 5μg/mL of recombinant proteins as antigens in 0.01 M phosphate buffered saline (PBS), pH 7.4. For that, we have produced and purified the recombinant PcrV protein (from the clone BL21 (DE3)pET15b-PcrV, a gift from Grenoble CEA, France); and the OprF porin in a proteoliposome (39). The plates were blocked with 2% BSA for 1 hour. Serial sera dilutions were added to each well and incubated for 1 hour. Following three washes with PBS containing 0.5% Tween 20, a peroxidase-conjugated sheep anti-mouse IgG (Sigma- Aldrich) diluted 1 : 1 ,000 in PBS was added and incubated for 1 hour. Then, 100μΙ_ of 3,3', 5,5'- tetramethylbenzidine (TMB) solution was added to each well, incubated for about 15 minutes in darkness, and then an equal volume of stopping solution (1 N H ₂ S0 ₄ ) was added. Optical density at 450nm was measured with a microplate reader (TriStar Berthold Technologies). For the interpretation of the results, a positive threshold corresponding to the upper limit of the negative controls was fixed (sum of the average of the negative controls plus three times the standard deviation). Evaluation of the cellular immune response

A multiplex immunoassay was used to determine the expression levels of 23 different cytokines in plasma samples (Bio-Plex Pro™ Mouse Cytokine Standard 23-Plex, Bio-Rad) of KBMA + IPTG immunized and unimmunized mice collected at days 1 , 7 and 21 after the last immunization. The experiment was performed in duplicate according to the detailed instructions provided. Plate was read on Bio-Plex MAGPIX™ Multiplex Reader and analyzed with the software Luminex Xponent.

Thereafter, the spleen cells were harvested for ex vivo analysis and evaluated for effector activity by an intracellular staining assay using syngeneic DC _{2 4} cell line (a gift from APcure, Grenoble) as professional antigen presenting cells (APCs) prepared using the procedure described hereinafter. The DC _{2 4} APCs were exposed to KBMA during 1 hour, washed and then cultured for overnight antigen processing in RPMI 1640 medium supplemented with antibiotics (50 μg/mL tobramycin and 200 μg/mL gentamicin) and 10 % FCS. Then the APCs were counted and used to stimulate spleen cells at a ratio of 1 DC ₂₄ to 10 spleen cells. APCs not exposed to the bacteria were used as control to measure the background responses by helper T cells. The spleen cells and targeted APCs were cocultured for 6 hours in the presence of 1 μg/mL brefeldin A protein transport inhibitor (BioLegend). Next, the cells were labeled with the Live/Dead FixableDead Cell Stain Kit (Invitrogen) and surface-labeled with antibodies specific for CD4 (RM4-5, BioLegend) and CD3 (145-2C1 1 , BD Biosciences), and then labeled intracellular^ with antibodies specific for mouse IFN-γ (XMG1.2), IL-17 (T TC1 1 - 18H10.1 ) and TNFa (MP6-XT22) (all from BioLegend) using cytofix/cytoperm and perm/wash reagents from BD Biosciences. Unspecific stimulation with 20 ng/mL phorbol 12-myristate 13- acetate (PMA) + 1 μg/mL ionomycin (iono) were used to measure the total helper T cells producing IL ₁₇ defining the Th ₁₇ subtype and IFN-γ but not the IL17 defining the Th-ι subtype and the TNF-a production. Samples were acquired on a FACSCanto II (BD Biosciences) and analyzed using Diva dedicated software.

Safety and toxicity assay

Immunized and unimmunized mice were weighed weekly since the day of the first vaccination until the challenge experiment to assess the impact of the vaccination on their growth. The vaccine preparation ready to use was frozen at -80°C in LB + Cb + 20% glycerol. In vitro cellular injection test was then performed to assess these storage conditions (40).

Efficacy assay

Frozen bacterial stocks of CHA strain were grown overnight in LB without additional antibiotic, at 37°C, 300 rpm. After washing the overnight culture with 1 mL LB, bacteria were inoculated in LB at an OD600 of 0.2 and grown until an OD600 of 1.5-1 .8 at 37°C, 300 rpm. Bacteria were resuspended in PBS. Concentrations were adjusted spectrophotometrically and confirmed after overnight growth on PIA at 37°C. An acute pulmonary infection was performed on mice by placing 20μί of this bacterial suspension on each nostril (40μΙ/ηιου8β; 5 x 10 ⁶ CFU/mouse) three weeks after the last immunization. Animals were then observed for survival every four hours for up to 96 hours. Animal health was recorded and mice were euthanized if necessary according to different clinical signs based on the mouse grimace scale recently published (41 ). Statistical analysis

Statistical analysis on mice data were generated by the Graph Pad Prism 7 for Windows (Graph Pad Software, La Jolla California USA, www.graphpad.com). Tests undertaken were t test non parametric unpaired analysis (Mann-Whitney) and Log-rank test. Example 1 : the live-attenuated Pseudomonas strain vaccine vector is able to elicit a polyvalent humoral immune response with regard to the pathogen P. aeruginosa.

KBMA + IPTG elicited a significant anti-PcrV antibody titer (p=0.001 1 ) compare to control (Fig. 1A), while KBMA did not (p=0.9591 ). KBMA and KBMA + IPTG elicited both a significant anti-OprF antibody titer (p=0.0015 and p<0.0001 respectively) compare to control (Fig. 1 B). ExsA, the main activator factor of the T3SS on which is located the PcrV protein, is contained in the plasmid which transforms the vaccine strain, and is under the control of an IPTG- inducible promoter (ptaq). Thus, it is demonstrated that the addition of IPTG during the bacterial growth allows generating a specific anti-PcrV antibodies rate significantly higher. These data suggest that the addition of IPTG boosts the number of T3SS to the bacterial surface, enabling to generate a stronger humoral response against this important virulence factor and therefore against the protein PcrV, while it has no impact for the outer membrane OprF porin. As whole bacteria has been used as vaccine vector, the presentation of the different bacterial antigens to host immune cells is as natural as possible, identical to that occurs during a P. aeruginosa infection, which allows mice eliciting antigens-specific antibodies as natural as possible able to recognize the bacterium P. aeruginosa in its entirety. Furthermore, whole proteins and not just epitopes have been used to highlight antibodies, and even proteins included in a liposome as in the outer membrane of P. aeruginosa for the OprF porin, in order to detect at best specific antibodies elicited by this whole bacterium as vaccine vector. Thus, these coating antigens are in their most natural conformation. The average rates of anti-PcrV and anti-OprF antibodies obtained in mice were consequent, and these antibodies are only functional antibodies as they recognize conformational epitopes of PcrV and OprF naturally available. So it is demonstrated here that KBMA P. aeruginosa vaccine vector allows a relevant humoral immune response with regard to the pathogen P. aeruginosa.

Example 2: the live-attenuated Pseudomonas strain vaccine vector is able to elicit with regard to the pathogen P. aeruginosa.

A multiplex immunoassay screening for cytokines and chemokines was used to identify the different pathways of cellular response involved in mice immunized with KBMA P. aeruginosa vaccine vector. Cytokine and chemokine kinetics were also measured. Among the molecules analyzed, levels of IL-12p70, IL-17, GM-CSF, G-CSF, IL-5, IL-10, IL-13, TNFa, KC, ΜΙΡ-1 β and MCP-1 were significantly higher (p<0.05) at days 1 and 7 in KBMA+IPTG immunized mice than in unimmunized animals (Fig. 2). Indeed, at day 1 after the last immunization, levels of G-CSF and ΜΙΡ-1 β were significantly higher (pO.0001 ) in KBMA+IPTG immunized mice than in unimmunized mice; levels of IL-17, TNFa and KC were significantly higher (p<0.0005) in KBMA+IPTG immunized mice than in unimmunized mice; levels of IL-13 and MCP-1 were significantly higher (p<0.005) in KBMA+IPTG immunized mice than in unimmunized mice; levels of IL-12p70, GM-CSF and IL-10 were significantly higher (p<0.05) in KBMA+IPTG immunized mice than in unimmunized mice. At day 7 after the last immunization, levels of IL-17 were significantly higher (p<0.0001 ) in KBMA+IPTG immunized mice than in unimmunized mice; levels of TNFa and ΜΙΡ-1 β were significantly higher (p<0.0005) in KBMA+IPTG immunized mice than in unimmunized mice; levels of IL-12p70, G-CSF, INFy, IL-13, IL-1 a and MCP-1 were significantly higher (p<0.005) in KBMA+IPTG immunized mice than in unimmunized mice; levels of GM-CSF, IL-5, IL-10, and MIP-1 a were significantly higher (p<0.05) in KBMA+IPTG immunized mice than in unimmunized mice. At day 21 , all cytokines and chemokines levels were back down at the same levels than in unimmunized mice. Therefore, it is demonstrated here that KBMA P. aeruginosa vaccine vector elicited several pathways of cellular response, such as Th17 (IL-17), Th1 (INFy and IL- 12) and Th2 (IL-5, IL-10, IL-13). These immunity pathways, and particularly Th17 pathway, have been shown as necessary to eradicate P. aeruginosa (21 ,22).

These results demonstrated that KBMA P. aeruginosa vaccine vector allows an early production of IL-17 (high rates at days 1 and 7, p<0.0005 and p<0.0001 respectively) ; such a response is already known to play a protective role during acute pulmonary P. aeruginosa infection in mice (42).

Furthermore, a rationally design of the KBMA P. aeruginosa vaccine vector may be carried out by overexpressing beneficial effectors of Th17 pathway such as PopB protein, in order to increase this essential Th17 immune response and eradicate the pathogen. Indeed, it has been shown that PopB-immunized mice were protected from lethal pneumonia in an antibody-independent IL-17-dependent manner; and that PopB elicited an important Th17 response and also an enhanced clearance of P. aeruginosa from lung and spleen after challenge (25). Thus, including PopB to KBMA P. aeruginosa vaccine vector is a way to improve the effectiveness of the vaccine.

Thereafter, helper T cells cytokine production in spleen cells was analyzed by flow cytometry using cytokine intracellular staining assay. It is demonstrated that KBMA immunized mice produced a significantly higher rate of Th1 and Th17 helper T cells than unimmunized mice (p<0.05 and p<0.0005 respectively), but not significant for TNF a helper T cells (Fig. 3D). Unimmunized mice spleen cells co-cultured with either APCs not exposed to the bacteria or presenting KBMA antigens were not able to allow the activation of the different cellular pathways (Fig. 3E). Immunized mice spleen cells co-cultured with APCs presenting KBMA antigens elicited a significantly higher rate of Th17 and TNF a helper T cells than unimmunized mice (p<0.005 and p<0.0005 respectively), while co-cultured with APCs not exposed to the bacteria were not able to allow the activation of the different cellular pathways (Fig. 3E). These data show that KBMA P. aeruginosa vaccine vector is able to elicit a specific cellular immune response with several pathways such as Th17, Th1 and TNF a. Th17 pathway is particularly interesting as already shown as necessary to eradicate P. aeruginosa (21 ,22). Example 3: safety of use and easy storage

To assess the toxicity in mice of the KBMA P. aeruginosa vaccine vector, the weight was followed since the first immunization to the intranasal infectious challenge. There was no significant difference (Fig. 4) in term of weight loss between KBMA+IPTG immunized mice and unimmunized mice (p=0.4418), although there is a slowdown in weight gain from day 21 corresponding to the establishment of immunity. Thus, this general toxicity test in mice revealed normal weight gains following 3 injections of KBMA P. aeruginosa vaccine. Therefore, it is demonstrated that KBMA P. aeruginosa vaccine vector is not toxic in mice.

Example 4: efficacy

Three weeks after the last immunization, intranasal infectious challenges were performed on KBMA+IPTG immunized mice and unimmunized mice. With a dose of 5 x 10 ⁶ CHU/mouse, all unimmunized mice were dead within the first 48 hours (Fig. 5), whereas the first death in immunized mice occurred at the 48 ^th hour after the infectious challenge. At the 150 ^th hour, 58.333% of immunized mice were still alive and in good health. Therefore, it is demonstrated that KBMA P. aeruginosa vaccine vector allows a good protective efficacy in mice, with a very significant difference compare to unimmunized mice (p<0.0001 ).

Conclusions

It is demonstrated here that KBMA P. aeruginosa vaccine vector is immunogenic as it elicits a broad immune response, humoral with good amount of specific antibodies particularly against PcrV protein by the adjunction of IPTG, and cellular with the activation of several pathways such as Th1 , Th2 and notably Th17 pathway known as necessary to eradicate P. aeruginosa. It is demonstrated that this live-attenuated vaccine, which cannot replicate in its host, is very promising in term of safety of use in mice and has a protective efficacy in mice during a pulmonary infectious challenge. Furthermore, the ability to rationally design this engineered life biological product by overexpressing beneficial effectors for vaccination efficacy in order to obtain the broadest immune response as possible renders the live-attenuated Pseudomonas strain of the invention strongly attractive. REFERENCES

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