Lipopolysaccharide (LPS)-deficient Acinetobacter baumannii polyvalent vaccine

Abstract

The present invention relates to compositions comprising LPS-deficient inactivated cells of the genus Acinetobacter and / or outer membrane vesicles forming same, and their use for the manufacture of a medicament, preferably a vaccine, for the prevention of diseases caused by K. pneumoniae, P. aeruginosa, E. coli, and / or A. pleuropneumoniae, and optionally A. baumannii.

Description

[Technical field]

[0001] The present invention relates to compositions comprising inactivated cells of the genus Acinetobacter, which are unable to synthesize lipid A, the core component of LPS, and capable of expressing heterologous outer membrane proteins from other bacteria, and / or outer membrane vesicles derived from the same cells, as well as their use for the manufacture of a medicament, preferably a vaccine, for the prevention of diseases caused by the bacteria from which the heterologous outer membrane protein is originally derived, and optionally by Acinetobacter baumannii. [Background technology]

[0002] Management of carbapenem-resistant infections is often based on antibiotics including polymyxins, tigecyclines, aminoglycosides, and combinations thereof. However, in a recent review, we found that carbapenem, aminoglycoside, polymyxin, and tigecycline co-resistant (CAPT-resistant) Gram-negative bacteria (GNB) are increasingly reported worldwide. Therefore, there is a need for further treatment options combined with rapid diagnostic methods. The most comprehensive study of the impact of antimicrobial resistance (AMR) showed that in 2019 alone, 5 million people died worldwide from causes related to infections caused by AMR pathogens. Seven bacterial species account for 73% of deaths worldwide: the gram-negative bacteria Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, and Escherichia coli, and the gram-positive bacteria Staphylococcus aureus and Streptococcus pneumoniae. There are only two vaccines available against Streptococcus pneumoniae, one of these six global causes of death. In this invention, we specifically focus on technologies that can deliver drug substances for use as vaccines against bacterial infections, particularly against gram-negative bacteria including Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, and Escherichia coli. This technique can be applied to any Gram-negative bacterium, such as Actinobacillus pleuropneumoniae, the causative pathogen of porcine pleuropneumonia.The present invention is based on the fact that, as shown above, by using as a vaccine inactivated cells derived from a lipid A mutant of Acinetobacter baumannii (A. baumannii) unable to synthesize LPS, infections caused by A. baumannii and not only by certain clones but also by diverse clones can be prevented, since the immunity raised by lipid A (LPS) null inactivated cells is focused against multiple highly conserved epitopes of the outer membrane proteins (OMPs) of the outer membrane of A. baumannii. The present invention also relies on the fact that OMPs are immunogenic bacterial components that trigger an innate immune response in humans and animals to prevent opportunistic infections. It also relies on the fact that by focusing immunity against the outer membrane protein (OMP) antigens, which are highly conserved proteins, the vaccine can provide universal immunity against all circulating clones or variants of bacterial pathogens, overcoming the typical challenges of bacterial vaccines that use LPS or capsular polysaccharide antigens as active ingredients. Antigens from LPS or capsular polysaccharides are highly diverse among clones or variants of the same bacterial species, and therefore vaccines, including conjugate vaccines, whose active components are based on immunogenic antigens derived from LPS or capsular polysaccharides, can only cover a limited number of variants. Furthermore, inactivated lipid A null mutant cells of A. baumannii used as vaccines raise immunity against OMP proteins that are adapted to their native conformation in the outer membrane of the cells, which is distinct from the immune response obtained using recombinant OMP proteins as vaccines. And finally, antigen presentation of multiple OMPs in their native conformation as part of lipid A null mutant cells of A. baumannii is enhanced over the presentation of a specific protein or peptide in the form of a recombinant protein derived from the OMP as a vaccine. With all this background, the technology that led to the present invention aimed to provide a method for producing lipid A null mutant cells of A. baumannii that can present in their membrane not only immunogenic OMPs from A. baumannii, but also immunogenic OMPs from other Gram-negative bacterial pathogens.In this way, the aforementioned advantages of using an inactivated lipid A null mutant of A. baumannii as a vaccine against A. baumannii can be applied to the fight against other Gram-negative bacterial pathogens. [Brief description of the drawings]

[0003] [Figure 1] This figure shows that AcinetoVax, a vaccine based on inactivated whole cells of an LPS-deficient A. baumannii strain, characterized by a complete inactivation of lpxC as taught in EP 2942389, plus the adjuvant aluminum hydroxide, protects against infection with various A. baumannii clinical isolates. Mice were infected with the indicated A. baumannii strains (ATCC19606, Ab-154, and Ab-113-16) 7 days after the second immunization with AcinetoVax (day 21) and survival was monitored for 7 days. ATCC19606 is a urinary infection clinical isolate used as a reference strain in sepsis and pneumonia models of infection in mice. Ab-154 is a carbapenem-susceptible clinical isolate from the outbreak at the Virgen del Rocio Hospital in Seville in 2002. Ab-113-16 is a pan-resistant clinical isolate from a patient who died as a result of the 2002 outbreak. [Diagram 2] Figure 1. Humoral responses enhanced by VXD001 (AcinetoVax) (A) only 7 days after the first dose. Analysis of IgG subtypes showed both IgG1 and IgG2 subtypes, with a partial bias towards IgG1. In B, splenocyte release of the Th1 cytokine IFN-γ (associated with CD8+ T cells) is seen by mice vaccinated with low (yellow bars) or high (grey bars) doses of VXD001 and stimulated ex-vivo with vaccine antigen. A significant response was seen after the first dose, which was only weaker and was boosted by a second injection on day 14. The same pattern was observed by analysis of IL-4 (Th1) and IL-17 (Th17) cytokine release. [Diagram 3]Analysis of LPS null derivatives from cells of the drug substance KapaVax. The left panel shows the results of PCR amplification of the genomic region containing the LPS synthesis gene lpxC. The expected length of the wild-type product is 1.5 Kb. Clones 4 and 5 are colistin-resistant mutants selected after plating cells of KapaVax2 on plates supplemented with colistin. The lpxC genomic region was then sequenced and the insertion of a 1 Kb ISAba1 transposable element at lpxC was found to be responsible for the inactivation of lpxC and the loss of LPS. The right panel shows the results of quantification of LPS (endotoxin) by chromogenic LAL assay (Pierce™ Chromogenic Endotoxin Quantification Kit, Thermo Fisher Scientific) in cultures of the parental strain A. baumannii Ab283, the LPS null derivative previously obtained from Ab283, and the two LPS null clones of KapaVax2. KapaVax or KapaVax2 are cells of the A. baumannii LPS null strain Ab283 expressing the heterologous OMPs OmpA and OmpK36 from K. pneumoniae, and the heterologous OMP OprF and the fusion protein OprI::PcrV from P. aeruginosa. [Figure 4]Analysis of LPS null derivatives by cells of the drug substance K-Vax and P-Vax. The figure on the top left shows the result of PCR amplification of the genomic region containing the LPS synthesis gene lpxC. The expected length of the wild-type product is 1.5 Kb. The figure shows PCR products obtained from DNA of several colistin-resistant derivatives of K-Vax cells, three different clones (clones 4, 20, 42) of A. baumannii Ab283 cells expressing the K. pneumoniae OMP proteins OmpA and OmpK36 in the outer membrane. The mutants showing the products marked with arrows were sequenced and found to have an ISAbA1 insertion in the lpxC gene. The insertion inactivated lpxC and therefore the cells were unable to synthesize LPS. On the top right are plates containing cells of the four detected LPS null mutants, showing growth on Mueller-Hilton medium and no growth on agar-MacConkey medium. The loss of LPS is paralleled by the inability to grow on agar-MacConkey medium. The figure on the bottom left shows the result of PCR amplification of the genomic region containing the LPS synthesis genes lpxC and lpxD of P-Vax cells, i.e. A. baumannii Ab283 cells expressing the P. aeruginosa OMP protein OprF and the fusion OprI::PcrV in the outer membrane. The expected length of the wild-type product is 1.5 Kb. The figure shows PCR products obtained from the DNA of several colistin-resistant derivatives of P-Vax cells, i.e. A. baumannii Ab283 cells expressing the P. aeruginosa OMP protein OprF and the fusion OprI::PcrV in the outer membrane. The mutants showing the products marked by arrows were sequenced and ISAbA1 insertions in the lpxC(c48) and lpxD(d69) genes were found. The insertions inactivated lpxC or lpxD and therefore these P-Vax cells were unable to synthesize LPS. At the bottom right, the plate shows growth of the parental Ab283 LPS+ but not the LPS null Ab283 derivative or the LPS null mutants of P-Vax cells c48 and d69 on agar-MacConkey. Loss of LPS is paralleled by an inability to grow on agar-MacConkey.Additionally, the results of quantification of LPS (endotoxin) by chromogenic LAL assay (Pierce™ Chromogenic Endotoxin Quantification Kit, Thermo Fisher Scientific) in cultures of several strains are shown. The only strain showing endotoxin levels above the detection level of the kit was the parental strain A. baumannii Ab283 LPS+. The results showed the absence of endotoxin (LPS) in LPS-null Ab283 derivatives or LPS-null mutants of K-Vax cells (clones 15 and 25) and P-Vax cells c48 and d69. [Diagram 5]Figure 1 shows an example of a confirmation PCR of the integration of an expression construct into the A. baumannii chromosome after a second recombination event. A: On the left are the PCR products obtained by using specific primers for the amplification of the genomic region indicated above the gel. The genomic region shown is the final expression construct after a second recombination event in A. baumannii Ab283 cells transformed with the plasmid pVXD50::K-Vax, integrated at the cysI locus. The integration resulted in the construction of K-Vax cells, i.e., A. baumannii Ab283 cells expressing the K. pneumoniae OMP proteins OmpA and OmpK36. As shown, the PCR products marked with arrows showed the appropriate size expected for the final integration of the expression construct in some clones (clones 20, 4, 42, and 44). In the gel shown on the right, the results of some confirmation PCR products due to the amplification of internal sequences in the expression construct, generated in K-Vax clones 4, 42, 44, and 20 cells, respectively, are shown. B: PCR products obtained by using specific primers for amplification of the genomic region indicated above the gel. The indicated genomic region is the final expression construct after a second recombination event in A. baumannii Ab283 cells transformed with plasmid pVXD50::Eco1-Vax, integrated at the cysI locus. The integration led to the construction of Eco1-Vax cells, i.e., A. baumannii Ab283 cells expressing the E. coli OMP proteins OmpA and OmpX. PCR products of four clones of Eco1-Vax cells showed the appropriate size expected for the final integration of the expression construct, confirming the construction of Eco1-Vax cells. [Figure 6]Figure 1 shows an example of confirmation of expression and location of heterologous OMP antigens in the drug substance K-Vax. Top panel: Surface exposure of Kp-Ompk36 and Kp-OmpA. Western blot with membrane samples obtained from DS3(K-Vax) or Ab283 LPS-vaccine batches. DS3(K-Vax) cells are A. baumannii LPS null Ab283 cells expressing the K. pneumoniae OMPs OmpA and OmpK36. Mouse polyclonal serum against either Kp-Ompk36 or Kp-OmpA was used as primary antibody. Treatment with proteinase K is indicated in each sample as +K (treated) or -K (untreated). The protocol is described in the Examples. Bottom panel: Expression analysis of DS3(K-Vax) by ELISA in vaccine batches. ELISA plates were coated with either inactivated DS3 cells or carrier cells Ab-283 LPS- after treatment with proteinase K (+) or left untreated (-). Primary antibody detection was performed with polyclonal sera raised against recombinant forms of Kp-OmpA, Kp-Ompk36, or Ab-Omp22. The average signal (error bars CI 95%) from three technical repeats is shown. [Figure 7]Figure 1 shows an example of confirmation of expression and location of heterologous OMP antigens in the drug substance P-Vax. Top left: surface exposure of Pa-OprF. Western blot with membrane samples obtained from DS4(P-Vax), Ab283 LPS-vaccine batch or wild-type P. aeruginosa strain PA14 cells. DS4(P-Vax) cells are A. baumannii LPS null Ab283 cells expressing the P. aeruginosa OMP OprF and the fusion protein OprI::PcrV. Cultures were washed after growth, resuspended in PBS and treated with 0.5 mg / ml proteinase K for 1 h at 37°C. Western blot with monoclonal antibody Hy221-n5 against Pa-OprF. Treatment with proteinase K is indicated in each sample as +K (treated) or -K (untreated). Protocol is described in the Examples. Top right: surface exposure of the Pa-OprI::PcrV fusion. Western blot with membrane samples obtained from DS4(P-Vax), Ab283 LPS-vaccine batch or wild-type P. aeruginosa strain PA14 cells. Cultures were washed after growth, resuspended in PBS and treated with 0.5 mg / ml proteinase K for 1 h at 37 °C. Western blot with monoclonal antibody Hy243-n20 against Pa-OprI. Treatment with proteinase K is indicated +K (treated) or -K (untreated) in each sample. The protocol is described in the Examples. Bottom: Detection of P. aeruginosa OprF, OprI and PcrV in two clones of DS4(P-Vax) measured by ELISA using polyclonal sera against recombinant forms of Pa-OprF, Pa-OprI and Pa-PcrV, respectively. Bar graphs represent the mean of three replicates and error bars indicate standard deviation. ELISA plates were coated with either wild-type cells of P. aeruginosa strain PA14, carrier cells Ab283 LPS-, or clones c48 and d69 of DS4opt (P-Vax). Statistical differences compared to Ab283 LPS- by one-way ANOVA multiple comparisons are indicated with asterisks (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). [Figure 8]Figure 1: Example of confirmation of expression and location of heterologous OMP antigens in drug substance KapaVax. Surface exposure of heterologous antigens (top and middle panels). Western blot with membrane samples obtained from KapaVax or Ab283 LPS-vaccine batches. KapaVax cells are A. baumannii LPS null Ab283 cells expressing K. pneumoniae OMPs OmpA and OmpK36, and P. aeruginosa OMP OprF and the fusion protein OprI::PcrV. Cultures were washed after growth, resuspended in PBS and treated with 0.5 mg / ml proteinase K for 1 h at 37°C. Western blot with polyclonal antisera against recombinant proteins Kp-OmpA, Kp-OmpK36, Pa-OprF, Pa-OprI and Pa-PcrV. Note that both anti-Pa-OprI and anti-Pa-PcrV recognize the same fusion protein OprI::PcrV expressed in KapaVax2. Treatment with proteinase K is indicated in each sample with + (treated) or - (untreated). Mb: outer membrane extract, S: extract supernatant. The protocol is described in the Examples. Bottom panel: Detection of heterologous OMP antigens measured by ELISA using polyclonal antisera against recombinant proteins of each antigen. Bar graphs represent the mean of three replicates, error bars indicate standard deviation. Significant differences in pairwise comparison two-tailed T-tests are indicated with asterisks. Anti-PaOprI and anti-PcrV sera target the same OprI::PcrV fusion protein. ELISA plates were coated with either KapaVax2@cysI vaccine cells (light grey bars) or carrier cells Ab283 LPS- (dark grey bars). [Figure 9]Figure 1 shows an example of expression of heterologous antigens by E. coli and A. pleuropneumoniae in LPS-null A. baumannii cells. Top panel: Example of differential expression of OMP between LPS-null A. baumannii carrier cells and cells expressing E. coli antigens. The upper arrow indicates a band present in the drug substance Eco2-Vax described in Example 1. This band corresponds to the antigen FyuA (native protein) with an expected molecular weight of 73.3 kDa. The lower arrow indicates a band present in the drug substance Eco1-Vax described in Example 1, but not in the carrier cells LPS-null A. baumannii LPS- (Ab LPS-) or the candidate Eco2-Vax. This band corresponds to the expected size of E. coli OmpA (38 kDa, native protein). Bottom panel: Example of differential expression of OMP between LPS-null A. baumannii carrier cells and cells expressing OMP antigens of A. pleuropneumoniae (APP). The arrow indicates a band present in the drug substance Appe3-Vax described in Example 1, which expresses APP OmpA (APP OmpA). This band corresponds to the antigen OmpA (native protein) with a predicted molecular weight of 37.5 kDa. This band is not present in the control lane of A. baumannii carrier cells LPS- (Ab LPS-). [Figure 10] Figure 1. Survival plots of a mouse sepsis model with K. pneumoniae infection. Animals were immunized with KapaVax2 (N=18), carrier Ab283 LPS-cells (N=8) or vehicle only (N=25) for two doses (109 inactivated cells / dose) every other week. IP challenge (day 7 after the second immunization) with Klebsiella pneumoniae ATCC43816 strain. Plots combine data from three independent experiments with challenge doses of 8.5×103 cfu, 6.7×103 cfu, and 6.7×103 cfu, respectively, corresponding to the minimum lethal dose (MLD). Survival was monitored for 7–12 days. Statistical differences were determined by log-rank (Mantel-Cox) test (ns denotes non-significant). K. pneumoniae ATCC 43816 is a highly virulent, hyperencapsulated clinical isolate that causes acute pneumonia and systemic spread in humans and mice. [Figure 11]Figure 1. Survival plots of a sepsis model due to P. aeruginosa infection. Animals were immunized with two doses (109 inactivated cells / dose) given every other week with the vaccine candidate KapaVax2 or vehicle alone. IP challenge with P. aeruginosa strain PA14 (4.4 x 106 cfu, equivalent to MLD) was performed 7 days after the second immunization. Survival was monitored for 7 days. N / group = 8. Statistical differences in survival curves were determined by the log-rank (Mantel-Cox) test. P. aeruginosa PA14 is a virulent wound infection clinical isolate. [Figure 12] Figure 1 shows a plot of survival rate in a mouse sepsis model with K. pneumoniae infection. Animals were immunized with DS3 (K-Vax) (N=10, dose of K-Vax 108 inactivated cells / dose), carrier Ab283 LPS-cells (N=8, dose of 109 inactivated cells / dose) or vehicle only (N=25) for two doses every other week. IP challenge (day 7 after the second immunization) with Klebsiella pneumoniae ATCC43816 strain. The challenge dose was equivalent to 5 times the minimum lethal dose (5 × MLD). Survival rates were monitored for 7–12 days. Statistical differences were determined by log-rank (Mantel-Cox) test. K. pneumoniae ATCC43816 is a highly virulent, highly encapsulated clinical isolate that causes acute pneumonia and systemic spread in humans and mice. [Figure 13] Figure 1. Survival plots of a sepsis model due to P. aeruginosa infection. Animals were immunized with two doses (109 inactivated cells / dose) given every other week with the vaccine candidate DS4 (P-Vax), the carrier Ab283 LPS-cells, or vehicle alone. IP challenge with P. aeruginosa strain PA14 (1.3x106 cfu, 1x MLD) was performed 7 days after the second immunization. Survival was monitored for 7 days. N / group=10. Statistical differences in survival curves were determined by the log-rank (Mantel-Cox) test. P. aeruginosa PA14 is a virulent wound infection clinical isolate. [Figure 14]Figure 1 shows a plot of survival rate in a sepsis model with A. baumannii. Animals (N=8 / group) were immunized with two doses of KapaVax2 (109 inactivated cells / dose combined with 0.83 mg aluminum hydroxide / dose as adjuvant), AcinetoVax (109 inactivated cells / dose combined with 0.83 mg aluminum hydroxide / dose as adjuvant) or vehicle (adjuvant only) every other week. IP challenge with 4.8x106 cells (1xMLD) of A. baumannii ATCC19606 on day 7 after the second immunization. Survival rate was monitored for 7 days. A. baumannii ATCC19606 is a urinary infection clinical isolate used as a reference strain in sepsis and pneumonia models of infection in mice. [Figure 15] Figure 2: IgG levels against K. pneumoniae cells. ELISA recognition of K. pneumoniae ATCC43816 cells by antisera from animals immunized with two doses of KapaVax (N=10) or vehicle only (N=10), sampled on day 21. Statistical analysis by two-tailed Mann-Whitney test (ns, p>0.05, *p<0.05, **p<0.005, ***p<0.001). The red dashed line indicates the limit of detection. [Figure 16] Figure 1: IgG levels against K. pneumoniae antigens. ELISA recognition of recombinant proteins Kp-OmpA and Kp-OmpK36 by antisera from animals immunized with KapaVax (N=4) or vehicle only (N=4) sampled on day 21 (day 7 after the second immunization). Statistical analysis by unpaired two-tailed T-test (ns p>0.05, *p<0.05, **p<0.005, ***p<0.001). The red dashed line indicates the limit of detection. [Figure 17] Figure 1: IgG levels against P. aeruginosa cells. ELISA recognition of P. aeruginosa PA14 cells by antisera from animals immunized with KapaVax (N=5) or vehicle only (N=5) sampled on day 21 (day 7 after second immunization). Statistical significance: ns p>0.05, *p<0.05, **p<0.005, ***p<0.001. The red dashed line indicates the limit of detection. [Figure 18] Figure 1: IgG levels against P. aeruginosa antigens. ELISA recognition of recombinant proteins by antisera from animals immunized with two doses of KapaVax (N=4) or vehicle only (N=4) sampled on day 21 (day 7 after second immunization). Statistical significance: ns p>0.05, *p<0.05, **p<0.005, ***p<0.001. The red dashed line indicates the limit of detection. [Figure 19] Figure 2: IgG levels against A. baumannii cells. ELISA recognition of A. baumannii ATCC19606 cells by antisera from animals immunized with two doses of KapaVax2 (N=9) or vehicle only (N=9), sampled on day 21 (7 days after the second immunization). Boxes indicate IQR, horizontal lines and crosses indicate median and mean, respectively. Error bars extend to CI95%. Statistical analysis by unpaired two-tailed Mann-Whitney test (ns p>0.05, *p<0.05, **p<0.005, ***p<0.001). Red dashed line indicates limit of detection. [Figure 20] Figure 1 shows detection of ELISA IgG titers (1:dilution) of 15 clinical isolates of K. pneumoniae (9 / 15 from low- and middle-income countries (LMICs)) and P. aeruginosa (8 / 15 LMICs) by antisera from KapaVax2 (LPS-deficient A. baumannii cells edited for expression of K. pneumoniae OmpA and OpmK36 and P. aeruginosa OprF and the fusion protein OprI::PcrV) and AcinetoVax (LPS-deficient A. baumannii cells). [Figure 21]Detection of ELISA IgG titers (right panel, 1: dilution) of 15 clinical isolates of A. baumannii (10 / 15 from low- and middle-income countries (LMICs)) by antisera from KapaVax2 (LPS-deficient A. baumannii cells edited for expression of OmpA and OpmK36 from K. pneumoniae and OprF from P. aeruginosa and the fusion protein OprI::PcrV) and AcinetoVax (LPS-deficient A. baumannii cells). The left panel shows information about the collection of A. baumannii clinical isolates used, specifically the country of origin of the clinical isolates and the international clonotype of A. baumannii to which they belong. [Figure 22] Figure 2: Expression of A. baumannii antigens in KapaVax and AcinetoVax vaccine batches: Total lysate preparation of vaccine batch (2x1010 cells / ml). Samples were measured on gels with a 4% to 16% acrylamide gradient. The panels show the signals obtained with monoclonal antibodies raised against OmpA (left) and Omp22 (right). [Figure 23] Figure 1. Surface exposure of A. baumannii Omp22 on the OM of KapaVax and carrier cells Ab283 LPS-. Cultures were washed after growth, resuspended in PBS and treated with 0.5 mg / ml proteinase K for 1 h at 37°C. Western blot with a monoclonal antibody raised against Ab-Omp22. Treatment with proteinase K is indicated by + (treated) or - (untreated) in each sample. Mb: outer membrane extract, S: extract supernatant. Protocol described in the Examples. [Figure 24]Top panel: Coomassie staining of A. baumannii OMV purified fractions (left) and Western blot for detection of A. baumannii Omp22 in OMV purified fractions (right). Omp22 is detected with monoclonal antibodies in LPS-null A. baumannii Ab283 carrier cells (top) and K-Vax (DS3) cells (LPS-null A. baumannii Ab283 expressing the OMP antigens K. pneumoniae OmpA and OmpK36, bottom). Bottom panel: Each sample from all fractions of the purification protocol for OMV (F1-F10, see examples) is analyzed by WB from either A. baumannii LPS-carrier cells (-) or DS3 candidates expressing the K. pneumoniae heterologous antigens OmpA and OmpK36 (D). Polyclonal antisera obtained by immunizing C57BL / 6J females with recombinant proteins OmpA or OmpK36 are used as primary antibodies. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0004] The following detailed description discloses specific and / or preferred variations of individual features of the invention. The invention also contemplates embodiments made by combining two or more of the specific and / or preferred variations described for two or more features of the invention as particularly preferred embodiments. Unless expressly stated otherwise, the term "comprising" is used in the context of this document to indicate that further members may optionally be present in addition to the members of the list introduced by "comprising".

[0005] The present invention relates to a. A lipopolysaccharide (LPS)-deficient A. baumannii strain characterized by partial or complete inactivation of one or various cellular nucleic acid molecules encoding endogenous LPS biosynthetic genes, wherein the lipopolysaccharide (LPS)-deficient A. baumannii strain is characterized by partial or complete inactivation of genes selected from the group consisting of lpxA, lpxB, lpxC, lpxD, lpxK, lpxL and / or lpxM, and wherein the lipopolysaccharide (LPS)-deficient A. baumannii strain expresses one or more heterologous antigens, preferably outer membrane protein (OMP) antigens, having targeting sites in the outer membrane, and there is surface exposure of such heterologous antigens, or b. Outer membrane vesicles (OMVs) derived from the lipopolysaccharide (LPS)-deficient A. baumannii strains described in the above paragraphs; Regarding.

[0006] Inactivated LPS-null A. baumannii cells can be efficiently used as a vaccine to prevent infection with A. baumannii. The efficient antigen presentation of this immunogen is shown by the enhanced rapid humoral response and prominent T cell-mediated response in mice. Since the most variable antigenic component of the bacterial cell wall, i.e. LPS, is absent from the immunogen, the immune response is directed against conserved outer membrane proteins, which are present in multiple copies and, importantly, in their native conformation. This results in a highly universal immune response, allowing the same vaccine product to neutralize infection with diverse variants of A. baumannii. The properties of the immune response enhanced by inactivated LPS-null whole cells of A. baumannii may possibly overcome classical challenges of vaccination against bacterial pathogens, such as the lack of immunogenicity or the excessive specificity of vaccines based on recombinant proteins or defined saccharide antigens with LPS or capsular polysaccharides.

[0007] The aim of this invention is to exploit the immunogenic properties of LPS-null cells of A. baumannii and extend their applicability to combat other bacterial pathogens, in particular Gram-negative bacteria, including but not limited to Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, and Escherichia coli. This technology can be applied to any Gram-negative bacteria, for example Actinobacillus pleuropneumoniae, the causative agent of porcine pleuropneumonia.

[0008] The present invention teaches how to construct A. baumannii LPS-null cells that express A. baumannii and other heterologous bacterial species OMPs on the outer membrane in the same cell. The authors of the present invention demonstrate that expression of other bacterial cell OMP proteins in A. baumannii can be achieved. The properties of these OMP proteins are known in the art (Koebnik et al., 2000, Smithers et al. 2021). The outer membrane protects Gram-negative bacteria from harsh environments. At the same time, embedded proteins perform several roles that are very important for bacterial cells, such as solute and protein translocation and signal transduction. One type of OMP considered in the present invention is the integral OMP protein (Koebnik et al. 2000). Unlike membrane proteins from all other sources, integral OMP proteins do not consist of transmembrane α-helices, but instead fold into an antiparallel β-barrel. They include OmpA membrane domains, OmpX proteins, phospholipase A, general porins, substrate-specific porins, and iron siderophore transporters (Koebnik et al. 2000). The second type of OMP protein considered in the present invention is bacterial lipoproteins (Juncker et al. 2003, Smithers et al. 2021). Bacterial lipoproteins are a class of lipid post-translationally / translationally modified outer membrane-anchored proteins that often perform various essential functions (Smithers et al. 2021). A hallmark of all lipoproteins is an N-terminal signal sequence followed by a cysteine. So far, several hundred putative lipoproteins have been annotated in Gram-negative bacteria (Juncker et al. 2003). The presence of a transmembrane domain for anchoring to the outer membrane of Gram-negative bacteria makes the transmembrane subunit of pili also a type of protein considered in the present invention (Zhang et al. 2000; Antenuci et al. 2020).

[0009] Since expression of heterologous OMP proteins of any family, including those described in Koebnik et al., 2000 (endogenous OMPs) or Smithers et al. 2021 (bacterial lipoproteins), is intended to be carried out in A. baumannii cells and the fate of the expressed OMP protein is intended to be the outer membrane of A. baumannii, in the present invention, the heterologous OMP protein includes a signal sequence of the OMP protein that can be processed by the A. baumannii cells to promote the location of the expressed protein in the outer membrane of A. baumannii. Preferably, the signal sequence is derived from any OMP of A. baumannii, most preferably from the OmpA protein of A. baumannii. It should be noted that the OmpA protein of A. baumannii consists of SEQ ID NO:1.

[0010] The present invention also encompasses any signal sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the full / length sequence of SEQ ID NO:1 and / or known signal sequences of OMP proteins from A. baumannii, provided that these sequences can be processed by A. baumannii cells to promote location of the expressed protein in the outer membrane of A. baumannii.

[0011] The term "sequence identity" or "percent identity" in the context of antigens, peptides or proteins referred to throughout the present invention refers to two or more sequences or subsequences that are the same ("identical") or have a specified percentage of amino acid residues that are identical ("percent identity"), when compared and aligned for maximum correspondence with a second molecule, as measured using a sequence comparison algorithm (e.g., by BLAST alignment, or any other algorithm known to one of skill in the art), or alternatively, by visual inspection.

[0012] Thus, the expression of heterologous antigens in the outer membrane of the lipopolysaccharide (LPS)-deficient A. baumannii strains of the present invention includes three unique properties. First, the post-translational processing of the heterologous OMP protein must be induced by a signal sequence derived from the OMP protein defined above, which can be processed by A. baumannii to induce the location of the expressed protein in the outer membrane of A. baumannii. Second, the heterologous OMP protein must contain an OMP transmembrane domain, either a typical transmembrane domain of an endogenous OMP protein as described in Koebnik et al. 2000, or an OMP transmembrane domain by a bacterial lipoprotein as described in Juncker et al. 2003, which allows the insertion of the expressed protein in the outer membrane of A. baumannii. And third, the heterologous OMP protein must contain an immunogenic domain. It is noted that sera from humans infected with bacterial pathogens recognize OMP proteins primarily on crude protein extracts of bacterial cells, and show the presence of significant titers of serum antibodies raised against bacterial OMP proteins.

[0013] It should be noted that the immunogenic domains of the heterologous OMP proteins expressed in A. baumannii are those contained in the wild-type OMPs of the heterologous pathogen. In the examples herein, the construction of various drug substances based on A. baumannii inactivated cells is shown. In particular, the examples show the expression of full-length proteins of endogenous OMPs of K. pneumoniae (OmpA and OmpK36), P. aeruginosa (OprF), E. coli (OmpA, OmpX, FuyA, HmA, IutA) or A. pleuropneumoniae (OmpA, OmpW, TbpA, and ApfA), i.e., the native full-length OMP sequences including the native transmembrane domains and the immunogenic domains.

[0014] Furthermore, it should be noted that the immunogenic domain of a heterologous OMP protein expressed in A. baumannii can be engineered to remove unnecessary sequences or to add another immunogenic domain, either from the pathogen's OMP protein or from any other protein, provided such removal or addition does not prevent correct post-translational processing and incorporation of the expressed OMP protein into the outer membrane of A. baumannii. In the examples herein, the immunogenic domain of the P. aeruginosa type 3 secretion apparatus protein PcrV was fused to the P. aeruginosa outer membrane lipoprotein OprI.

[0015] It should be noted that the OMP proteins expressed in A. baumannii shown in the examples belong not only to different bacterial pathogens but also to different OMP types, including several integral OMP families as described in Koebnik et al. 2000, including proteins of the small β-barrel membrane anchor family OmpA (OmpA in either K. pneumoniae, E. coli or A. pleuropneumoniae) or OmpX (OmpX in E. coli), classical trimeric porins such as OmpK36 in K. pneumoniae, non-specific porins such as OprF in P. aeruginosa, or substrate-specific porins such as TbpA in A. pleuropneumoniae, or TonB-dependent iron receptors such as FuyA, Hma and IutA in E. coli. It should also be noted that the OMP protein that can be directed to the outer membrane of A. baumannii cells by the methods of the invention can be an outer membrane lipoprotein such as OprI of P. aeruginosa, or the membrane-anchored type IV pilus subunit ApfA of A. pleuropneumoniae, as shown in the Examples.

[0016] Furthermore, the authors of the present invention have demonstrated that inoculation of a subject in need thereof, preferably a human subject, with LPS-deficient whole cells of A. baumannii expressing one or more copies of an antigenic outer membrane heterologous protein by one or more microorganisms, preferably inactivated, results in immunization not only against A. baumannii infections, but also against infections caused by any microorganism that naturally expresses one or more copies of the antigenic outer membrane protein, demonstrating the utility of these cells as a prophylactic multivalent vaccine.

[0017] It is noted that the immunodominant proteins of the vaccine consisting of inactivated LPS-null cells of A. baumannii are the A. baumannii OMP proteins OmpA and Omp22, which are the most abundant endogenous OMP proteins on the outer membrane of these cells. It is also noted and shown in the examples that these proteins remain the most abundant proteins detected on the outer membrane of A. baumannii LPS-null cells expressing outer membrane OMP proteins of other bacterial pathogens. Importantly, it is noted and shown in the examples that the presence of A. baumannii antigens in LPS-null A. baumannii cells expressing OMP proteins of different heterologous bacterial pathogens not only enhances protective immunity against infections caused by A. baumannii, but also enhances partial cross-protective immunity against infections caused by heterologous pathogens. The authors showed, for example, that a drug substance consisting of inactivated LPS-null A. baumannii cells expressing the K. pneumoniae OMP proteins OmpA and OmpK36 used as a vaccine was able to neutralize infection caused by the highly virulent, highly encapsulated strain K. pneumoniae ATCC 43816 (see Figures 10 and 12), and that removal of the heterologous K. pneumoniae OMP proteins OmpA and OmpK36 significantly reduced protection against infection caused by K. pneumoniae ATCC 43816, although the partial cross-protective protection enhanced by the A. baumannii antigens was still present (see Figures 10 and 12). It has also been shown in the Examples that a drug substance consisting of inactivated LPS-null cells of A. baumannii expressing the P. aeruginosa OMP protein OprF and the fusion OprI::PcrV used as a vaccine is able to neutralize infection caused by the highly virulent strain P. aeruginosa PA14 (see Figure 13), and that removal of the heterologous P. aeruginosa OMP protein OprF and the fusion OprI::PcrV significantly reduces protection against infection caused by P. aeruginosa PA14 (see Figure 13), although the partial cross-protective protection enhanced by the A. baumannii antigen is still present.

[0018] The ability of the technology to deliver multi-pathogen vaccine candidates is another aspect of the present invention. The authors have shown, for example, that it is possible to construct a drug substance consisting of inactivated LPS-null cells of A. baumannii expressing in the same cells the OMP proteins OmpA and OmpK36 of K. pneumoniae and the OMP protein OprF and the fusion OprI::PcrV of P. aeruginosa. When used as a vaccine, the immunity raised by such a drug substance is able to neutralize infection caused by the highly virulent and highly encapsulated strain K. pneumoniae ATCC43816 (see FIG. 10) and also by the highly virulent strain P. aeruginosa PA14 (see FIG. 11). Moreover, the immunity raised by the drug substance, which still contains antigens of A. baumannii, as do all drug substances of the present invention, provided protection against lethal sepsis caused by A. baumannii ATCC19606 (see FIG. 14).

[0019] It is also noted and shown in the Examples that expression of heterologous OMP antigens in LPS-null A. baumannii cells does not prevent the formation of typical outer membrane vesicles derived from Gram-negative bacteria (see FIG. 24).

[0020] Thus, a first aspect of the present invention provides a method for producing a method for treating a pulmonary circulation comprising the steps of: a. a lipopolysaccharide (LPS)-deficient A. baumannii strain characterized by a partial or complete inactivation of one or various cellular nucleic acid molecules encoding endogenous LPS biosynthetic genes, wherein the lipopolysaccharide (LPS)-deficient A. baumannii strain is characterized by a partial or complete inactivation of a gene selected from the group consisting of lpxA, lpxB, lpxC, lpxD, lpxK, lpxL and / or lpxM, and wherein the lipopolysaccharide (LPS)-deficient A. baumannii strain expresses one or more copies of one or more microbial antigenic outer membrane heterologous proteins having targeting sites in the outer membrane; and / or b. Outer membrane vesicles (OMVs) derived from the lipopolysaccharide (LPS)-deficient A. baumannii strains described in the above paragraphs; Refers to...

[0021] "Lipopolysaccharide (LPS) or lipooligosaccharide" is understood to be a component found in the outer membrane of gram-negative bacteria. The term LPS is often used interchangeably with "endotoxin" due to the history of its discovery. LPS consists of a polysaccharide chain and another lipid known as lipid A, which is responsible for endotoxic activity. The polysaccharide chain varies between different bacteria and determines the serotype. Endotoxins are approximately 10 kDa in size, but can form large aggregates up to 1000 kDa. Humans are capable of producing antibodies against LPS, but generally these antibodies are only able to protect against specific serotypes of bacteria. Endotoxins are responsible for many of the clinical signs of infections caused by gram-negative bacteria such as Neisseria meningitidis and A. baumannii.

[0022] By "cells of A. baumannii" in the present invention is understood such cells belonging to the bacterial domain, phylum Proteobacteria, class Gammaproteobacteria, order Pseudomonadales, family Moraxellaceae, genus Acinetobacter, species Acinetobacter baumannii. Acinetobacter species are obligate aerobic non-fermenting and non-motile rods that are oxidase negative and appear in pairs under microscopic observation. They are widespread in nature and are important in soils, contributing to their mineralization.

[0023] The lipopolysaccharide (LPS)-deficient A. baumannii strains of the present invention are preferably whole inactivated cells, and whole "inactivated cells" in the present invention are understood to be cells that are not capable of replicating but preserve their immunogenicity. The cells of the present invention are inactivated before inoculation in order to prevent their replication in the host and therefore to prevent infections resulting from their administration. Inactivation of the cells of the present invention can be performed using various methods known in the art, such as, but not limited to, adsorption, heat, ultraviolet light, ionizing radiation, ultrasound, phenol, formol, formaldehyde, crystal violet, glyceraldehyde, ethylene oxide, propiolactone, ethyleneamine, bromoethyleneamine or formalin. In a preferred embodiment, the cells of the present invention are heat inactivated. In another preferred embodiment, the cells of the present invention are of the Acinetobacter baumannii species and are heat inactivated.

[0024] In a preferred embodiment of this aspect of the invention, the loss of LPS can be achieved by partial or complete inactivation of one or various cellular molecules of the endogenous genes for the LPS subunits, in particular the nucleic acids encoding LPS lpxA, lpxB and / or lpxC, resulting in a complete loss of LPS, as taught in EP 2942389. The Acinetobacter species, in particular the A. baumannii lpxA, lpxB and / or lpxC sequences. In a preferred embodiment of the invention, the endogenous genes for LPS are selected from lpxA, lpxB and / or lpxC, or any combination of these genes.

[0025] Preferably, one or more mutations in endogenous LPS biosynthesis genes of the lipopolysaccharide (LPS)-deficient A. baumannii strain of the first aspect of the invention are obtained by selecting colistin-resistant mutants on plates supplemented with the antibiotic colistin and screening the selected colistin-resistant mutants for mutations in the LPS synthesis genes. In this sense, the authors of the present invention show in the examples that several diverse strains of A. baumannii can become colistin-resistant by different mutations in the LPS synthesis genes that lead to LPS loss (see Table 1).

[0026] [Table 1]

[0027] Table 1. Examples of LPS null derivatives from some parental A. baumannii strains obtained by the authors. The table shows examples of LPS null mutants obtained by the authors from three different unrelated clinical isolates of A. baumannii, specifically an old clinical isolate currently available at ATCC as strain ATCC19606 and used as a reference A. baumannii strain in experiments with other animal models, as well as clinical isolates named IB001 and Ab283 that caused an outbreak in 2002 at the Virgen del Rocio Hospital in Seville. All LPS null mutants were constructed by selecting colistin-resistant mutants on plates supplemented with colistin, except for K1, which was obtained by direct mutagenesis of the lpxA gene. The table shows a description of the mutations found or generated in the LPS synthesis genes, the minimum inhibitory concentration of colistin, and the amount of endotoxin (LPS) measured in cell cultures of each strain by a chromogenic LAL assay for the quantification of bacterial endotoxin.

[0028] In a preferred embodiment of the first aspect of the invention, the sequences of the LPS synthesis genes are inactivated, for example by construction of a suicide vector containing any of the genes lpxA, lpxB, lpxC, lpxD, lpxK, lpxL and / or lpxM or a combination thereof, or by interfering with a marker gene for selection, transforming target cells with the vector and screening for positive cells that become negative for LPS expression. In yet another preferred embodiment of the invention, Acinetobacter cells lacking LPS are obtained by deletion and / or insertion of one or various nucleotides in the nucleic acid sequences encoding the genes involved in the biosynthesis of LPS and / or in the sequences controlling their expression. Deletions and / or insertions can be generated by homologous recombination, by insertion of transposons or by other suitable methods known in the art.

[0029] In another preferred embodiment of the first aspect of the invention, the cell of the invention is preferably an A. baumannii cell, although such cells may potentially be other Acinetobacter species, such as Acinetobacter baylyi, A. beijerinckii, A. bereziniae, A. boissieri, A. bouvetii, A. brisouii, A. calcoaceticus, A. gerneri, A. guillouiae, A. grimontii, A. gyllenbergii, A. haemolyticus, A. indicus, A. indicus, A. johnsonii, A. junii, A. Iwoffii, A. nectaris, A. nosocomialis, A. parvus, A. pittii, A. puyangensis, A. radioresistens, A. rudis, A. schindleri, A. soli, A. tandoii, A. tjernbergiae, A. towneri, A. arthingii, A. In the present invention, Acinetobacter is understood to refer to the kingdom Bacteria, the phylum Proteobacteria, the class Gammaproteobacteria, the order Pseudomonadales, and the family Moraxellaceae.

[0030] On the other hand, as already indicated earlier in this specification, three specific properties are required for the expression and exposure of a heterologous antigen at the outer membrane of A. baumannii cells according to the first aspect of the invention. First, the post-translational processing of the heterologous OMP protein must be induced by a signal sequence derived from the OMP protein, which can be processed by A. baumannii to induce the location of the expressed protein at the outer membrane of A. baumannii. Second, the heterologous OMP protein must contain an OMP transmembrane domain characteristic of endogenous OMP proteins as described in Koebnik et al. 2000, or a typical signal sequence of bacterial lipoproteins as described in Juncker et al. 2003, which allows the insertion of the expressed protein at the outer membrane of A. baumannii. And third, the heterologous OMP protein must contain an immunogenic domain.

[0031] Thus, one or more heterologous antigens (or heterologous OMP proteins) are characterized in that they contain a signal sequence at their N-terminus derived from an OMP protein that is processed by A. baumannii to direct the location of the expressed protein to the outer membrane of A. baumannii. Furthermore, one or more heterologous antigens are characterized in that they contain a typical transmembrane domain from an endogenous OMP protein as described in Koebnik et al. 2000 or an OMP transmembrane domain from a bacterial lipoprotein as described in Juncker et al. 2003, which allows insertion of the expressed protein at the outer membrane of A. baumannii. And thirdly, one or more heterologous antigens are characterized in that they contain an immunogenic domain.

[0032] Thus, in another preferred embodiment of the first aspect of the invention, the one or more heterologous antigens (or heterologous OMP proteins) having a targeting site in the outer membrane are characterised in that they comprise: 1. At the N-terminus of the heterologous antigen (or heterologous OMP protein), a signal sequence from an OMP protein that is processed by A. baumannii to direct the location of the expressed protein to the outer membrane of A. baumannii; 2. An integral OMP protein or an OMP transmembrane domain unique to a bacterial lipoprotein that allows insertion of the expressed protein at the outer membrane of A. baumannii, and 3. Immunogenic domains.

[0033] Preferably, the heterologous antigen (or heterologous OMP protein) is comprised of a signal sequence at the N-terminus of the protein linked directly, or optionally via a linker, to the N-terminal amino acid of a portion of the protein that includes the transmembrane and immunogenic domains.

[0034] Preferably, as mentioned above, in the present invention, the heterologous OMP protein includes a signal sequence of an OMP protein that can be processed by A. baumannii cells to promote the location of the expressed protein in the outer membrane of A. baumannii. Preferably, the signal sequence is derived from any OMP of A. baumannii, most preferably from the OmpA protein of A. baumannii. The present invention also encompasses any signal sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the full / length sequence of SEQ ID NO:1 from the OmpA protein of A. baumannii, provided that the resulting sequence is capable of being processed by A. baumannii cells to promote the location of the expressed protein in the outer membrane of A. baumannii.

[0035] Preferably, the immunogenic domain of the heterologous OMP protein expressed in A. baumannii is such as that contained in the wild-type OMP of the heterologous pathogen. In the examples herein, the construction of various drug substances based on A. baumannii inactivated cells is shown. In particular, the examples show the expression of full-length proteins of endogenous OMPs of K. pneumoniae (OmpA and OmpK36), P. aeruginosa (OprF), E. coli (OmpA, OmpX, FuyA, HmA, IutA) or A. pleuropneumoniae (OmpA, OmpW, TbpA, and ApfA), i.e., the native full-length OMP sequences, including the native transmembrane domain and the immunogenic domain. All of these immunogenic domains are expressly included in the present invention. Also preferably, it should be noted that the immunogenic domain of the heterologous OMP protein expressed in A. baumannii may be engineered to remove unnecessary sequences or to add another immunogenic domain, either from the pathogen's OMP protein or from any other protein, provided such removal or addition does not prevent correct post-translational processing and integration of the expressed OMP protein into the outer membrane of A. baumannii. In the examples herein, the immunogenic domain of the P. aeruginosa type 3 secretion apparatus protein PcrV was fused to the P. aeruginosa outer membrane lipoprotein OprI.

[0036] In another preferred embodiment of the first aspect of the invention, the heterologous OMP proteins expressed in A. baumannii shown in the examples belong not only to different bacterial pathogens but also to different OMP types, including several endogenous OMP families as described in Koebnik et al. 2000, including proteins of the small β-barrel membrane anchor family OmpA (OmpA of either K. pneumoniae, E. coli or A. pleuropneumoniae) or OmpX (OmpX of E. coli), classical trimeric porins such as OmpK36 of K. pneumoniae, non-specific porins such as OprF of P. aeruginosa, or substrate-specific porins such as TbpA of A. pleuropneumoniae, TonB-dependent iron receptors such as FuyA, Hma and IutA of E. coli. It should also be noted that the heterologous OMP protein that can be directed to the outer membrane of an A. baumannii cell by the methods of the invention can be an outer membrane lipoprotein such as OprI of P. aeruginosa, or the membrane-anchored type IV pilus subunit ApfA of A. pleuropneumoniae, as shown in the Examples.

[0037] Furthermore, as already indicated, the authors of the present invention have demonstrated that inoculation of a subject in need thereof, preferably a human subject, with whole LPS-deficient A. baumannii cells expressing one or more copies of the antigenic outer membrane heterologous protein defined above by one or more microorganisms, preferably inactivated, results in immunization not only against A. baumannii infections, but also against infections caused by any microorganism which naturally expresses one or more copies of the antigenic outer membrane protein, demonstrating the practical utility of these cells as a prophylactic and even therapeutic multivalent vaccine.

[0038] On the other hand, as reflected above, A. baumannii strains lacking lipopolysaccharide (LPS) must express one or more copies of one or more microbial antigenic outer membrane heterologous proteins that have targeting sites in their outer membrane. Such expression must be performed so that such heterologous antigens are surface exposed. To that end, the vaccine candidates of the present invention can be constructed by genome editing of A. baumannii using any allelic exchange technique for recombinant strain production, including one or more recombination steps for the insertion of an expression construct at a specific locus into the A. baumannii genome. The expression construct to be inserted can be carried into the cell by a linear piece of DNA or a plasmid. In particular, a construct encoding one or more copies of an antigenic outer membrane heterologous protein can be inserted into any suitable locus of the A. baumannii strain before, simultaneously or after the cell is made lacking LPS, as taught, for example, in EP 2942389. Suitable loci are any loci on the A. baumannii chromosome that can integrate an insert by recombination, such as cysI, trpE, lpxA, lpxC, lpxD, lpxB, lpxK, lpxL, lpxM, and / or Tn5 / Tn7 sites. It should be noted that Acinetobacter baumannii cells can be transformed with a vector, preferably a suicide vector, that contains sequences to facilitate recombination at a desired target locus into the A. baumannii genome, and such loci are any loci of the A. baumannii genome that contain sequences suitable to undergo recombination as described above.

[0039] In this sense, in a preferred embodiment of the invention, the strain is modified to express one or more heterologous antigens (or heterologous OMP proteins) having a target site in the outer membrane by the insertion of an expression construct encoding one or more heterologous antigens. More preferably, the Acinetobacter baumannii cells can be preferably transformed with a vector, characterized in that the vector comprises sequences for promoting recombination flanking the expression construct, which in turn comprises at least one or more transcription promoter sequences, one or more ORFs encoding heterologous proteins for A. baumannii, and one or more transcription termination sequences. The promoter sequences can be any known sequence in the art that is capable of promoting transcription in A. baumannii cells. These promoter sequences are routinely used in bacteriological research. In a preferred embodiment of the invention, the promoter sequence used is a promoter sequence located upstream of the A. baumannii ORF encoding the outer membrane protein OmpA. The expression construct may contain several ORFs in series, each flanked by a promoter and an end, a transcription termination sequence upstream and downstream of the ORF, respectively, thus constructed to allow independent expression of each ORF controlled by a specific promoter. Alternatively, the expression construct may have one or several ORFs in an operon-like structure with polycistronic expression controlled by a common promoter. The ORFs of the expression vectors code for outer membrane proteins or peptides selected for their immunogenic properties, derived from known human or animal pathogens different from A. baumannii, whose expression in A. baumannii cells is intended to enhance the immune response against the pathogen different from A. baumannii in humans or animals vaccinated with A. baumannii cells.Since expression of a heterologous outer membrane protein, including any family of proteins described in Koebnik et al., 2000 (intrinsic OMPs) or Smithers et al. 2021 (bacterial lipoproteins), is intended to be carried out in A. baumannii cells and the fate of the expressed OMP protein is intended to be the outer membrane of A. baumannii, in the present invention, the heterologous OMP protein includes a signal sequence of the OMP protein that can be processed by the A. baumannii cells to promote location of the expressed protein in the outer membrane of A. baumannii. Preferably, the signal sequence is derived from any OMP of A. baumannii, most preferably from the OmpA protein of A. baumannii, including any signal sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the full / length sequence of SEQ ID NO: 1 from the OmpA protein of A. baumannii and capable of being processed by the A. baumannii cell to promote location of the expressed protein in the outer membrane of A. baumannii. Preferably, the ORF of the expression vector encodes a heterologous antigen (or heterologous OMP protein) as defined above, characterized in that it comprises: 1. At the N-terminus of the heterologous antigen (or heterologous OMP protein), a signal sequence from an OMP protein that is processed by A. baumannii to direct the location of the expressed protein to the outer membrane of A. baumannii; 2. An integral OMP protein or an OMP transmembrane domain unique to a bacterial lipoprotein that allows insertion of the expressed protein at the outer membrane of A. baumannii, and 3. Immunogenic domains.

[0040] Preferably, the signal sequence is derived from any OMP of A. baumannii, most preferably from the OmpA protein of A. baumannii, including any signal sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the full / length sequence of SEQ ID NO:1 from the OmpA protein of A. baumannii and capable of being processed by the A. baumannii cell to promote location of the expressed protein in the outer membrane of A. baumannii.

[0041] Preferably, the immunogenic domain of the heterologous OMP protein expressed in A. baumannii is such as contained in the wild-type OMP of the heterologous pathogen. In the examples herein, the construction of various drug substances based on A. baumannii inactivated cells is shown. In particular, the examples show the expression of full-length proteins of endogenous OMPs of K. pneumoniae (OmpA and OmpK36), P. aeruginosa (OprF), E. coli (OmpA, OmpX, FuyA, Hma, IutA) or A. pleuropneumoniae (OmpA, OmpW, TbpA, and ApfA), i.e., full-length native OMP sequences including native transmembrane and immunogenic domains. All of these immunogenic domains are expressly included in the present invention as potentially encoded by the ORFs of the expression construct. Also preferably, it should be noted that the immunogenic domain of the heterologous OMP protein encoded by the ORF of the expression construct may be engineered to remove unnecessary sequences or to add another immunogenic domain, either from the pathogen's OMP protein or from any other protein, provided such removal or addition does not interfere with correct post-translational processing and integration of the expressed OMP protein into the outer membrane of A. baumannii. In the examples herein, the immunogenic domain of the P. aeruginosa type 3 secretion apparatus protein PcrV was fused to the P. aeruginosa outer membrane lipoprotein OprI.

[0042] In another preferred embodiment of the first aspect of the invention, the OMP proteins encoded by the ORFs of the expression constructs shown in the examples belong not only to different bacterial pathogens but also to different OMP types, including several endogenous OMP families as described in Koebnik et al. 2000, including proteins of the small β-barrel membrane anchor family OmpA (OmpA of either K. pneumoniae, E. coli or A. pleuropneumoniae) or OmpX (OmpX of E. coli), classical trimeric porins such as OmpK36 of K. pneumoniae, non-specific porins such as OprF of P. aeruginosa, or substrate-specific porins such as TbpA of A. pleuropneumoniae, or TonB-dependent iron receptors such as FuyA, Hma and IutA of E. coli. Also, note that the heterologous OMP protein encoded by the ORF of the expression construct and targeted to the outer membrane of A. baumannii cells can be an outer membrane lipoprotein such as OprI of P. aeruginosa, or the membrane-anchored type IV pilus subunit ApfA of A. pleuropneumoniae, as shown in the Examples.

[0043] Furthermore, as described herein below, when A. baumannii cells are transformed with a vector, preferably a suicide vector, recombination between sequences at the target locus in the host and sequences within the vector results in the production of recombinant cells in which the sequences of the vector are integrated into the target locus on the A. baumannii chromosome. This recombination event can be assisted by strategies known in the art, such as inducing a double-stranded break at the insertion site by genome editing tools, such as those based on CRISPR methodology. In a preferred embodiment of the present invention, the first recombination event results in the insertion of vector sequences including a selectable marker and an expression construct. The selectable marker is used to select cells in which such a first recombination event has occurred. A second recombination event can result in recombinant cells in which only the expression construct remains integrated at the insertion site, but not the sequence encoding the selectable marker. The absence of the selectable mark can be used as a selection tool to select A. baumannii cells in which such a second recombination event has occurred. Once selected, common techniques such as PCR and DNA sequencing are used to confirm whether the integration of the vector sequences into the target locus has occurred as expected.

[0044] On the other hand, before the expression of the analyzed heterologous protein in the recombinant A. baumannii cells is deemed sufficient, simultaneously or subsequently, loss of LPS can be induced in A. baumannii cells by alternative methods such as site-directed mutagenesis of the LPS synthesis genes, resulting in partial or complete inactivation of the LPS synthesis genes, including genes selected from the group consisting of lpxA, lpxB, lpxC, lpxD, lpxK, lpxL and / or lpxM, or by selecting A. baumannii cells resistant to the antibiotic colistin and screening the cells by common genomic methods such as PCR and / or DNA sequencing for mutations resulting in partial or complete inactivation of the LPS synthesis genes, including genes selected from the group consisting of lpxA, lpxB, lpxC, lpxD, lpxK, lpxL and / or lpxM.

[0045] In a particular embodiment of the invention, when the target locus for insertion of the above recombination event is a locus comprising an ORF encoding an LPS synthesis gene comprising a gene selected from the group consisting of lpxA, lpxB, lpxC, lpxD, lpxK, lpxL and / or lpxM, and the recombination results in partial or complete inactivation of such ORF, the resulting recombinant A. baumannii cell is an LPS negative cell due to partial or complete inactivation of the LPS synthesis gene comprising a gene selected from the group consisting of lpxA, lpxB, lpxC, lpxD, lpxK, lpxL and / or lpxM.

[0046] After inserting the construct into a suitable locus, such as the cysI locus, the expression and localization of the heterologous antigen at the outer membrane can be demonstrated (as taught in the examples), and then the selection of the LPS-negative derivatives can be demonstrated, for example, by plating in colistin and selecting LPS-negative mutants. The expression and localization of each antigen at the outer membrane can be confirmed by Western blot and ELISA. Thus, Western blot analysis of whole cell lysates and outer membrane extracts with antigen-specific antibodies can be used to confirm the expression and localization of the heterologous antigen in A. baumannii cells. The recognition of the recombinant antigen on the surface of the whole cell can also be verified using antigen-specific antibodies in ELISA experiments.

[0047] In further particular embodiments of the first aspect of the invention, the heterologous antigens expressed on the outer membrane of the lipopolysaccharide (LPS)-deficient A. baumannii strain are derived from at least K. pneumoniae, P. aeruginosa, E. coli, and / or A. pleuropneumoniae.

[0048] In particular, the heterologous antigen expressed in the outer membrane of A. baumannii strains lacking lipopolysaccharide (LPS) is selected from the group consisting of Kp-OmpA (SEQ ID NO: 31 or 17) and Kp-Ompk36 (SEQ ID NO: 32 or 18), which is derived from at least K. pneumoniae and comprises any sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the full / length sequence of any of SEQ ID NO: 31, 32, 17 or 18, and when expressed or exposed in one or more copies in the outer membrane of A. baumannii as taught in the present invention by using a signal sequence that can be processed by A. baumannii cells to facilitate the location of the expressed protein in the outer membrane of A. baumannii, when inoculated into a subject in need thereof, preferably a human subject, produces an immunizing, preferably protective, immune response not only against A. baumannii infections but also against infections caused by K. pneumoniae. SEQ ID NO: 2 or 3 identified in the present invention retain the native signal peptide of K. pneumoniae. However, as taught in the present invention, the native peptide is replaced by a signal sequence of an OMP protein that can be processed by A. baumannii cells to promote the location of the expressed protein in the outer membrane of A. baumannii. Preferably, the signal sequence is from any OMP of A. baumannii, most preferably from the OmpA protein of A. baumannii (SEQ ID NO: 1). SEQ ID NO: 17 and 18 show the final sequences of Kp-OmpA and Kp-OmpK36 with the signal peptide of A. baumannii OmpA. SEQ ID NO: 31 and 32 show the final sequences of Kp-OmpA and Kp-Ompk36 without the signal peptide, i.e., the transmembrane domain and the antigen domain.

[0049] Kp-OmpA. OmpA is a highly conserved membrane porin among the Enterobacteriaceae. It is not only the homologue of one of the two major antigens identified to be involved in the protection exerted by our LPS-null A. baumannii cells in animal models, but has also been described as the protein antigen most recognized by antisera from patients with acute infections caused by K. pneumoniae (Kurupati et al. 2006). Furthermore, its use as a DNA vaccine (Kurupati et al. 2011) resulted in a protective immune response in a mouse model of sepsis. In summary, the results published by Kurupati et al. (2011) showed that intramuscular immunization of BALB / c females with four doses of 50 μl of an ompA DNA preparation increased survival at day 8 by up to 60% against challenge with a lethal dose of a K. pneumoniae clinical isolate.

[0050] Kp-OmpK36. OmpK36 is also a highly conserved membrane porin in K. pneumoniae, reported as the second most recognized protein (after OmpA) by antisera from infected patients and the best covered by sera from various clinical infections (Kurupati et al. 2006). Its use as a DNA vaccine in the above mentioned mouse sepsis model (Kurupati et al. 2011) showed an even higher level of protection than that obtained by vaccination with OmpA (Kurupati et al. 2011), specifically, the survival rate at day 8 was increased by up to 75% against 60% obtained with a vaccine expressing OmpA alone. Furthermore, when used as a single subunit vaccine, immunization with three doses of 25 μg full-length recombinant OmpK36 combined with incomplete Freund's adjuvant was able to protect mice against intraperitoneal challenge (survival rate 60%) and immune sera were able to neutralize diverse strains of K. pneumoniae (Babu et al. 2017, Hussein et al. 2018).

[0051] In another particular embodiment of the first aspect of the invention, the heterologous antigen expressed on the outer membrane of the lipopolysaccharide (LPS)-deficient Acinetobacter baumannii strain is derived from at least P. aeruginosa and is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 1109%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 149%, 150%, 151%, 152%, 153%, 154%, 155%, 156%, 157%, 158%, 159%, 160%, 161%, 162%, 163%, 164%, 165%, 166%, 167%, 168%, 169%, 170%, 171%, 172%, 173%, 174%, 175%, 176%, 177%, 178%, 179%, 180%, 181%, 182%, 183%, 184%, 185%, 186%, 187%, 188%, 189%, 1 or any sequence having 99% identity thereto, which when expressed or exposed in one or more copies in the outer membrane of A. baumannii as taught in the present invention, when inoculated into a subject in need thereof, preferably a human subject, produces an immunizing, preferably protective, immune response against A. baumannii infections as well as infections caused by P. aeruginosa. SEQ ID NOs: 4-7 retain the native signal peptide of P. aeruginosa. As taught in the present invention, the native peptide is replaced by a signal sequence of an OMP protein that can be processed by A. baumannii cells to promote the location of the expressed protein in the outer membrane of A. baumannii. Preferably, the signal sequence is derived from any OMP of A. baumannii, most preferably from the OmpA protein (SEQ ID NO: 1) of A. baumannii. SEQ ID NOs: 19-21 show the final sequences of Pa-OprF, Pa-OprF::PcrV, and Pa-OprI, respectively, with the signal peptide of A. baumannii OmpA. SEQ ID NOs: 33-35 show the final sequences of Pa-OprF, Pa-OprF::PcrV, and Pa-OprI, respectively, without the signal peptide, i.e., the transmembrane domain and the antigen domain. SEQ ID NO: 45 shows the final sequence of Pa-PcrV without the signal, i.e., the antigen domain.

[0052] Pa-OprI.OprI is a highly conserved membrane lipoprotein that, when used as an adjuvant in a protein-based vaccine against Mycobacterium tuberculosis consisting of the fusion protein N-terminal OprI-Ag85, has been demonstrated to exert immunostimulatory activity through activation of Toll-like receptors (TLR) 2 and 4, a key step in the induction of Th1-type T cells (Gartner et al. 2007). This Th1-inducing property of OprI has been previously demonstrated in a mouse model of cutaneous leishmaniasis (Cote-Sierra et al. 2002) and in a model of classical swine fever (Rau et al. 2006). The protective effect of OprI against P. aeruginosa infection was demonstrated in a mouse model of sepsis using immunodeficient Balb / c females, with LD50s up to 35-fold higher in animals immunized intraperitoneally with four doses of 50 μg purified OprI versus non-immunized animals (Finke et al. 1990 & 1991). Importantly, immunization with OprI was shown to be safe and immunogenic in healthy subjects (von Specht et al. 1996).

[0053] Pa-PcrV. PcrV is the major antigen of the needles formed by the P. aeruginosa type 3 secretion apparatus. It has been shown that anti-PcrV antibodies contribute significantly to protection against virulent P. aeruginosa infections (Sawa et al. 1999 & 2014, Moriyama et al. 2009, Milla et al. 2014) and that active vaccination with PcrV can induce protective immunity against P. aeruginosa infections (Meynet et al. 2018, Aguilera-Herce et al. 2019). The results obtained by Meynet et al. (2018) showed that immunization with 1 × 10 killed but metabolically active P. aeruginosa overexpressing PcrV significantly increased the risk of infection with P. aeruginosa. 8 CFU ~ 2 × 10 8showed that subcutaneous immunization of C57Bl / 6J females with three doses at 2-week intervals containing 1000 CFU resulted in a 58.3% day 7 survival rate compared to unvaccinated animals in an acute pulmonary infection model. Furthermore, Hamaoka et al. (2017) tested the effect of immunization with recombinant PcrV (rPcrV) in combination with three adjuvants in a mouse model of acute pneumonia. AlOH 3 Intraperitoneal immunization with three doses of 10 μg each of rPcrV combined with rPcrV resulted in an increased survival rate of 73% compared to controls immunized with an equal dose of adjuvant. Importantly, two monoclonal antibody-based therapies targeting PcrV are currently in clinical development, with one study demonstrating a significant reduction in P. aeruginosa infection rates (pneumonia) compared to placebo (Francois et al. 2012, Ali et al. 2019).

[0054] Pa-OprF. OprF is a highly conserved OmpA homolog and is the major outer membrane porin in P. aeruginosa. Previous immunization studies in experimental models have shown that OprF protects against P. aeruginosa infection. Gilleland et al. (1984) demonstrated that intraperitoneal immunization with two doses of 10 μg of purified OprF of P. aeruginosa PAO1 provided up to 67% protection in a mouse model of sepsis using CD-1 mice. Furthermore, Hassan et al. 2018 recently reported immunization of mice with recombinant full-length OprF of P. aeruginosa obtained by heterologous expression in E. coli under the control of an inducible promoter. Full-length OprF used as a subunit vaccine in a mouse model of acute pneumonia was able to protect Balb / c female mice against infection with nonmucoid and mucoid P. aeruginosa strains PAO1 and PAK, respectively. While 100% of non-immunized animals died 48-72 days after intranasal inoculation with P. aeruginosa, the survival rates at day 7 of animals immunized subcutaneously with four doses of recombinant OprF (rOprF) containing 50 µg of recombinant protein were 50% and 60% for PaO1 and PAK, respectively. Nevertheless, in the same study, combining rOprF with recombinant OprI improved survival to 100% against PAO1 challenge.

[0055] In another particular embodiment of the first aspect of the invention, the heterologous antigen expressed in the outer membrane of an Acinetobacter baumannii strain lacking lipopolysaccharide (LPS) is at least one of E. coli derived from E. coli and comprising any sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the full / length sequence of any of SEQ ID NOs: 22-26 or 36-40, including Ec-OmpA (SEQ ID NO: 36 or 22), Ec-OmpX (SEQ ID NO: 37 or 23), Ec-FuyA (SEQ ID NO: 38 or 24), Ec-Hma (SEQ ID NO: 39 or 25), Ec-Hma (SEQ ID NO: 40 or 41), Ec-Hma (SEQ ID NO: 42 or 43), Ec-Hma (SEQ ID NO: 44 or 45), Ec-Hma (SEQ ID NO: 46 or 46), Ec-Hma (SEQ ID NO: 47 or 48), Ec-Hma (SEQ ID NO: 49 or 50), Ec-Hma (SEQ ID NO: 51 or 52), Ec-Hma (SEQ ID NO: 53 or 54), Ec-Hma (SEQ ID NO: 55 or 56), Ec-Hma (SEQ ID NO: 57 or 58), Ec-Hma (SEQ ID NO: 59 or 60), Ec-Hma (SEQ ID NO: 61 or 62), Ec-Hma (SEQ ID NO: 63 or 64), Ec-Hma (SEQ ID NO: 65 or 66), Ec-Hma (SEQ ID NO: 67 or 68), Ec-Hma (SEQ ID 5) or Ec-IutA (SEQ ID NO: 40 or 26), which when expressed or exposed in one or more copies in the outer membrane of A. baumannii as taught in the present invention, when inoculated into a subject in need thereof, preferably a human subject, produces an immunizing, preferably protective, immune response against A. baumannii infections as well as infections caused by E. coli. SEQ ID NO: 8-12 retain the native signal peptide of E. coli. As taught in the present invention, the native peptide is replaced by a signal sequence of an OMP protein which can be processed by A. baumannii cells to promote the location of the expressed protein in the outer membrane of A. baumannii. Preferably, the signal sequence is derived from any OMP of A. baumannii, most preferably from the OmpA protein (SEQ ID NO: 1) of A. baumannii. SEQ ID NOs: 22 to 26 show the final sequences of Ec-OmpA, Ec-OmpX, Ec-FuyA, Ec-Hma, and Ec-IutA, respectively, having the signal peptide of A. baumannii OmpA. SEQ ID NOs: 36 to 40 show the final sequences of Ec-OmpA, Ec-OmpX, Ec-FuyA, Ec-Hma, and Ec-IutA, respectively, not including the signal peptide, i.e., the transmembrane domain and the antigen domain.

[0056] Ec-OmpA. Outer membrane protein A (OmpA) is a major protein in the outer membrane of Escherichia coli. OmpA is composed of three functional domains, including a hydrophilic extracellular mass, a β-barrel transmembrane structure, and a peptidoglycan-binding domain (Wang, 2002). Gu et al. 2018 showed that vaccination of mice with an OmpA-based vaccine induced Th1, Th2, and Th17 immune responses and conferred effective protection. Furthermore, OmpAVac-specific antibodies could mediate opsonophagocytosis and inhibit bacterial invasion, thereby conferring prophylactic protection in E. coli K1-challenged adult and neonatal mice. These results suggest that OmpA-based vaccines could be good vaccine candidates for the control of E. coli infections.

[0057] Ec-OmpX. The integral outer membrane protein X (OmpX) of Escherichia coli belongs to a family of highly conserved bacterial proteins that promote bacterial adhesion and invasion into mammalian cells. In addition, these proteins play a role in resistance to attack by the human complement system (Vogt and Schulz, 1999). Using recombinant OmpX of E. coli, Maisnier-Patin et al. (2003) reported that EcOmpX binds to and is internalized by human antigen-presenting cells.

[0058] Ec-FuyA. Yersiniabactin is a siderophore found in several enterobacterial strains, including the pathogenic bacteria Yersinia pestis, Yersinia pseudotuberculosis, and Yersinia enterocolitica, as well as the enteropathogenic Escherichia coli and Salmonella enterica. Uropathogenic E. coli possess highly efficient mechanisms of iron acquisition, one of which is the yersiniabactin system. The fyuA gene, which encodes the yersiniabactin receptor, was found to be crucial for biofilm formation in iron-deficient environments, such as human urine (Hancock et al. 2008). In a mouse model, Habibi et al. (2017) showed that vaccination with FyuA induced both IgG1 (Th2) and IgG2a (Th1) type humoral responses and reduced uropathogenic E. coli colonization in the bladder and kidneys.

[0059] Ec-Hma.Hma is a heme acquisition protein, a 79 kDa heme receptor in the outer membrane, that functions independently of ChuA to mediate hemin uptake by uropathogenic E. coli strains (Hagan and Mobley, 2009). Intranasal immunization of mice with the outer membrane iron receptors FyuA, Hma, IreA, and IutA conjugated to cholera toxin provided protection in the bladder or kidney under challenge with uropathogenic E. coli strains in mice (Forsyth et al. 2020).

[0060] Ec-IutA.IutA is the 75 kDa ferric aerobactin receptor of the outer membrane of E. coli. Active immunization of mice with recombinant antigens EcpA, EcpD, IutA or IroN elicited high levels of total IgG antibodies of the IgG1 / IgG2a isotype and was determined to be highly protective against E. coli infection in lethal and non-lethal septic challenge (Mellata et al. 2016).

[0061] In another particular embodiment of the first aspect of the invention, the heterologous antigen expressed in the outer membrane of the lipopolysaccharide (LPS)-deficient Acinetobacter baumannii strain is at least one of Ap-OmpA (SEQ ID NO: 27 or 41), Ap-OmpW (SEQ ID NO: 28 or 42), Ap-TbpA (SEQ ID NO: 29 or 43), or Ap-TbpA (SEQ ID NO: 30 or 44), which is derived from A. pleuropneumoniae and comprises any sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the full / length sequence of any of SEQ ID NOs: 27-30 or 41-44. By using a signal sequence selected from the group consisting of ApfA (SEQ ID NO: 30 or 44), which can be processed by A. baumannii cells to promote the location of the expressed protein in the outer membrane of A. baumannii, when expressed or exposed in one or more copies in the outer membrane of A. baumannii as taught in the present invention, when inoculated into a subject in need thereof, preferably a human subject, it generates an immunizing, preferably protective, immune response against A. baumannii infections as well as infections caused by A. pleuropneumoniae. SEQ ID NO: 13-16 retain the native signal peptide of A. pleuropneumoniae. As taught in the present invention, the native peptide is replaced by a signal sequence of an OMP protein which can be processed by A. baumannii cells to promote the location of the expressed protein in the outer membrane of A. baumannii. Preferably, the signal sequence is derived from any OMP of A. baumannii, most preferably from the OmpA protein (SEQ ID NO: 1) of A. baumannii. SEQ ID NOs: 27 to 30 show the final sequences of Ap-OmpA, Ap-OmpW, Ap-TbpA, and Ap-ApfA, respectively, having the signal peptide of A. baumannii OmpA. SEQ ID NOs: 41 to 44 show the final sequences of Ap-OmpA, Ap-OmpW, Ap-TbpA, and Ap-ApfA, respectively, not including the signal peptide, i.e., the transmembrane domain and antigen domain.

[0062] Ap-OmpA. It is the A. pleuropneumoniae homologue of the major protein in the outer membrane of other Gram-negative bacteria such as E. coli. OmpA is composed of three functional domains including a hydrophilic extracellular mass, a β-barrel transmembrane structure, and a peptidoglycan-binding domain (Wang, 2002).

[0063] Ap-OmpW. It is another endogenous OMP protein in A. pleuropneumoniae. The ompW gene is found in many bacteria, such as Escherichia coli, Aeromonas hydrophila, and Vibrio harveyi. Immune responses against the OmpW protein have been shown to provide immunity to Aeromonas challenge. In A. pleuropneumoniae, OmpW appears to regulate the phenotype during infection (Chen et al. 2022).

[0064] Ap-TbpA.TbpA (transferrin-binding protein A) is a 100 kDa outer membrane β-barrel that forms the import channel for transferrin and has been shown to be a molecular determinant of virulence and pathogenesis in A. pleuropneumoniae (Nahar et al. 2021).

[0065] Ap-ApfA. ApfA is an outer membrane protein that anchors type IV pili of A. pleuropneumoniae. Type 4 pili have been shown to increase bacterial-bacterial interactions and promote bacterial attachment and colonization, thereby facilitating the progression of bacterial infection (Zhang et al. 2000). In A. pleuropneumoniae, a conserved general secretory pathway (GSP) domain in the N-terminal part of the outer membrane protein ApfA adjacent to the transmembrane domain has been identified. ApfAs are used as outer membrane anchors that can bind potential immunogens (Antenuci et al. 2020).

[0066] SEQ ID NO:1. A. baumannii OmpA signal peptide MKLSRIALATMLVAAPLAAANA Nucleotide sequence of SEQ ID NO:1 Atgaaattgagtcgtattgcacttgctactatgcttgttgctgctccattagctgctgctaatgct SEQ ID NO:2. K. pneumoniae OmpA TIFF2024540918000002.tif26170 * In bold is the K. pneumoniae native signal peptide. Nucleotide sequence of SEQ ID NO:2 TIFF2024540918000003.tif67170 SEQ ID NO:3. K. pneumoniae OmpK36 TIFF2024540918000004.tif26170 * In bold is the K. pneumoniae native signal peptide. Nucleotide sequence of SEQ ID NO:3 TIFF2024540918000005.tif72170 SEQ ID NO: 4. P. aeruginosa OprF TIFF2024540918000006.tif26170 * Bold indicates P. aeruginosa native signal peptide Nucleotide sequence of SEQ ID NO:4 TIFF2024540918000007.tif72170 SEQ ID NO:5. P. aeruginosa OprI::PcrV fusion protein TIFF2024540918000008.tif26170 * Bold indicates P. aeruginosa native signal peptide * The linker of the fusion protein is underlined. Nucleotide sequence of SEQ ID NO:5 TIFF2024540918000009.tif72170 SEQ ID NO:6. P. aeruginosa OprI TIFF2024540918000010.tif10170 * Bold indicates P. aeruginosa native signal peptide Nucleotide sequence of SEQ ID NO:6 TIFF2024540918000011.tif21170 SEQ ID NO:7. P. aeruginosa PcrV TIFF2024540918000012.tif20170 * Bold indicates P. aeruginosa native signal peptide Nucleotide sequence of SEQ ID NO:7 TIFF2024540918000013.tif61170 SEQ ID NO:8. E. coli OmpA TIFF2024540918000014.tif26170 * The bold letters indicate the native E. coli signal peptide. Nucleotide sequence of SEQ ID NO:8 TIFF2024540918000015.tif72170 SEQ ID NO: 9. E. coli OmpX TIFF2024540918000016.tif15170 * The bold letters indicate the native E. coli signal peptide. Nucleotide sequence of SEQ ID NO:9 TIFF2024540918000017.tif36170 SEQ ID NO:10. E. coli FyuA TIFF2024540918000018.tif46170 * The bold letters indicate the native E. coli signal peptide. Nucleotide sequence of SEQ ID NO:10 TIFF2024540918000019.tif133170 SEQ ID NO:11. E. coli Hma TIFF2024540918000020.tif46170 * The bold letters indicate the native E. coli signal peptide. Nucleotide sequence of SEQ ID NO:11 TIFF2024540918000021.tif138170 SEQ ID NO:12. E. coli IutA TIFF2024540918000022.tif51170 * The bold letters indicate the native E. coli signal peptide. Nucleotide sequence of SEQ ID NO:12 TIFF2024540918000023.tif144170 SEQ ID NO: 13. A. pleuropneumoniae OmpA TIFF2024540918000024.tif26170 * Bold indicates A. pleuropneumoniae native signal peptide Nucleotide sequence of SEQ ID NO:13 TIFF2024540918000025.tif72170 SEQ ID NO: 14. A. pleuropneumoniae OmpW TIFF2024540918000026.tif15170 * Bold indicates A. pleuropneumoniae native signal peptide Nucleotide sequence of SEQ ID NO:14 TIFF2024540918000027.tif46170 SEQ ID NO:15. A. pleuropneumoniae TbpA TIFF2024540918000028.tif61170 * Bold indicates A. pleuropneumoniae native signal peptide Nucleotide sequence of SEQ ID NO:15 TIFF2024540918000029.tif185170 SEQ ID NO: 16. A. pleuropneumoniae ApfA TIFF2024540918000030.tif10170 * Bold indicates A. pleuropneumoniae native signal peptide Nucleotide sequence of SEQ ID NO: 16 TIFF2024540918000031.tif31170 SEQ ID NO: 17. K. pneumoniae OmpA TIFF2024540918000032.tif26170 * Bold indicates A. baumannii signal peptide Nucleotide sequence of SEQ ID NO:17 TIFF2024540918000033.tif72170 SEQ ID NO: 18. K. pneumoniae OmpK36 TIFF2024540918000034.tif26170 * Bold indicates A. baumannii signal peptide Nucleotide sequence of SEQ ID NO:18 TIFF2024540918000035.tif72170 SEQ ID NO: 19. P. aeruginosa OprF TIFF2024540918000036.tif26170 * Bold indicates A. baumannii signal peptide Nucleotide sequence of SEQ ID NO:19 TIFF2024540918000037.tif72170 SEQ ID NO:20. P. aeruginosa OprI::PcrV fusion protein TIFF2024540918000038.tif26170 * Bold indicates A. baumannii signal peptide * The linkers are underlined Nucleotide sequence of SEQ ID NO:20 TIFF2024540918000039.tif77170 SEQ ID NO:21. P. aeruginosa OprI TIFF2024540918000040.tif10170 * Bold indicates A. baumannii signal peptide Nucleotide sequence of SEQ ID NO:21 TIFF2024540918000041.tif20170 SEQ ID NO:22. E. coli OmpA TIFF2024540918000042.tif26170 * Bold indicates A. baumannii signal peptide Nucleotide sequence of SEQ ID NO:22 TIFF2024540918000043.tif72170 SEQ ID NO:23. E. coli OmpX TIFF2024540918000044.tif15170 * Bold indicates A. baumannii signal peptide Nucleotide sequence of SEQ ID NO:23 TIFF2024540918000045.tif36170 SEQ ID NO:24. E. coli FyuA TIFF2024540918000046.tif46170 * Bold indicates A. baumannii signal peptide Nucleotide sequence of SEQ ID NO:24 TIFF2024540918000047.tif133170 SEQ ID NO:25. E. coli Hma TIFF2024540918000048.tif46170 * Bold indicates A. baumannii signal peptide Nucleotide sequence of SEQ ID NO:25 TIFF2024540918000049.tif138170 SEQ ID NO:26. E. coli IutA TIFF2024540918000050.tif51170 * Bold indicates A. baumannii signal peptide Nucleotide sequence of SEQ ID NO:26 TIFF2024540918000051.tif144170 SEQ ID NO:27. A. pleuropneumoniae OmpA TIFF2024540918000052.tif26170 * Bold indicates A. baumannii signal peptide Nucleotide sequence of SEQ ID NO:27 TIFF2024540918000053.tif72170 SEQ ID NO: 28. A. pleuropneumoniae OmpW TIFF2024540918000054.tif15170 * Bold indicates A. baumannii signal peptide Nucleotide sequence of SEQ ID NO:28 TIFF2024540918000055.tif46170 SEQ ID NO:29. A. pleuropneumoniae TbpA TIFF2024540918000056.tif61170 * Bold indicates A. baumannii signal peptide Nucleotide sequence of SEQ ID NO:29 TIFF2024540918000057.tif185170 SEQ ID NO:30. A. pleuropneumoniae ApfA TIFF2024540918000058.tif10170 * Bold indicates A. baumannii signal peptide Nucleotide sequence of SEQ ID NO:30 TIFF2024540918000059.tif31170 SEQ ID NO:31. K. pneumoniae OmpA GGKLGWSQYHDTGFYGNGFQNNNGPTRNDQLGAGAFGGYQVNPYLGFEMGYDWLGRMAYKGSVDNGAFKAQGVQLTAKLGYPITDDLDIYTRLGGMVWRADSKGNYASTGVSRSEHDTGVSPVFAGGVEWAVTRDIATRLEYQWVNNIGDAGTVGTRPDNGML SLGVSYRFGQEDAAPVVAPAPAPAPEVATKHFTLKSDVLFNFNKATLKPEGQQALDQLYTQLSNMDPKDGSAVVLGYTDRIGSEAYNQQLSEKRAQSVVDYLVAKGIPAGKISARGMGESNPVTGNTCDNVKARAALIDCLAPDRRVEIEVKGYKEVVTQPQA Nucleotide sequence of SEQ ID NO:31 ggtggtaaactgggttggtcccagtatcacgacaccggtttctacggtaacggtttccagaacaacaacggtccgacccgtaacgatcagcttggtgctggtgcgttcggtggttaccaggttaacccgtacctcggtttcgaaatgggttatgactggctgggccgtatggcatataaaggcagcgttgacaacggtgctttcaaagctcagggcgttcagctgaccgctaaactgggttacccgatcactgacgatctggacatctacacccgtctgggcggcatggtttggcgcgctgactccaaaggcaactacgcttctaccggcgtttcccgtagcgaacacgacactggcgtttccccagtatttgctggcggcgtagagtgggctgttactcgtgacatcgctacccgtctggaataccagtgggttaacaacatcggcgacgcgggcactgtgggtacccgtcctgataacggcatgctgagcctgggcgtttcctaccgcttcggtcaggaagatgctgcaccggttgttgctccggctccggctccggctccggaagtggctaccaagcacttcaccctgaagtctgacgttctgttcaacttcaacaaagctaccctgaaaccggaaggtcagcaggctctggatcagctgtacactcagctgagcaacatggaCccgaaagacggttccgctgttgttctgggctacaccgaccgcatcggttccgaagcttacaaccagcagctgtctgagaaacgtgctcagtccgttgttgactacctggttgctaaaggcatcccggctggcaaaatctccgctcgcggcatgggtgaatccaacccggttactggcaacacctgtgacaacgtgaaagctcgcgctgccctgatcgattgcctggctccggatcgtcgtgtagagatcgaagttaaaggctacaaagaagttgtaactcagccgcaggcttaa SEQ ID NO:32. K. pneumoniae OmpK36 AEIYNKDGNKLDLYGKIDGLHYFSDDKSVDGDQTYMRVGVKGETQINDQLTGYGQWEYNVQANNTESSSDQAWTRLAFAGLKFGDAGSFDYGRNYGVVYDVTSWTDVLPEFGGDTYGSDNFLQSRANGVATYRNSDFFGLVDGLNFALQYQGKNGSVSGEGALSPTNNGRTAL KQNGDGYGTSLTYDIYDGISAGFAYSNSKRLGDQNSKLALGRGDNAETYTGGLKYDANNIYLATQYTQTYNATRAGSLGFANKAQNFEVVAQYQFDFGLRPSVAYLQSKGKDLEGYGDQDILKYVDVGATYYFNKNMSTYVDYKINLLDDNSFTHNAGISTDDVVALGLVYQF Nucleotide sequence of SEQ ID NO:32 SEQ ID NO:33. P. aeruginosa OprF QGQNSVEIEAFGKRYFTDSVRNMKNADLYGGSIGYFLTDDVELALSYGEYHDVRGTYETGNKKVHGNLTSLDAIYHFGTPGVGLRPYVSAGLAHQNITNINSDSQGRQQMTMANIGAGLKYYFTENFFAKASLDGQYGLEKRDNGHQGEWMAGLGVGFNFGGS KAAPAPEPVADVCSDSDNDGVCDNVDKCPDTPANVTVDANGCPAVAEVVRVQLDVKFDFDKSKVKENSYADIKNLADFMKQYPSTSTTVEGHTDSVGTDAYNQKLSERRANAVRDVLVNEYGVEGGRVNAVGYGESRPVADNATAEGRAINRRVEAEVEAEAK Nucleotide sequence of SEQ ID NO:33 cagggccagaactcggtagagatcgaagccttcggcaagcgctacttcaccgacagcgttcgcaacatgaagaacgcggacctgtacggcggctcgatcggttacttcctgaccgacgacgtcgagctggcgctgtcctacggtgagtaccatgacgttcgtggcacctacgaaaccggcaacaagaaggtccacggcaacctgacctccctggacgccatctaccacttcggtaccccgggcgtaggtctgcgtccgtacgtgtcggctggtctggctcaccagaacatcaccaacatcaacagcgacagccaaggccgtcagcagatgaccatggccaacatcggcgctggtctgaagtactacttcaccgagaacttcttcgccaaggccagcctcgacggccagtacggtctggagaagcgtgacaacggtcaccagggcgagtggatggctggcctgggcgtcggcttcaacttcggtggttcgaaagccgctccggctccggaaccggttgccgacgtttgctccgactccgacaacgacggcgtttgcgacaacgtcgacaagtgcccggataccccggccaacgtcaccgttgacgccaacggctgcccggctgtcgccgaagtcgtacgcgtacagctggacgtgaagttcgacttcgacaagtccaaggtcaaagagaacagctacgctgacatcaagaacctggctgacttcatgaagcagtacccgtccacttccaccaccgttgaaggtcacaccgactccgtcggcaccgacgcttacaaccagaagctgtccgagcgtcgtgccaacgccgttcgtgacgtactggtcaacgagtacggtgtagaaggtggtcgcgtgaacgctgttggttacggcgagtcccgcccggttgccgacaacgccaccgctgaaggccgcgctatcaaccgtcgcgttgaagccgaagtagaagctgaagccaagtaa SEQ ID NO:34. P. aeruginosa OprI::PcrV fusion protein SHSKETEARLTATEDAAARAQARADEAYRKADEALGAAQKAQQTADEANERALRMLEKASRK GGGGSGGGGSGGGGS SAAPASAEQEELLALLRSERIVLAHAGQPLSEAQVLKALAWLLAANPSAPPGQGLEVLREVLQARRQPGAQWDLRELVVSAYFSLHGRLDEDVIGVYKDVLQTQDGKRKALLDELKALTAELKVYSVIQSQINAALS AKQGIRIDAGGIDLVDPTLYGYAVGDPRWKDSPEYALLSNLDTFSGKLSIKDFLSGSPKQSGELKGLSDEYPFEKDNNPVGNFATTVSDRSRPLNDKVNEKTTLLNDTSSRYNSAVEALNRFIQKYDSVLRDILSAI Nucleotide sequence of SEQ ID NO:34 agccactccaaagaaaccgaagctcgtctgaccgctaccgaagacgcagctgctcgtgctcaggctcgcgctgacgaagcctatcgcaaggctgacgaagctctgggcgctgctcagaaagctcagcagactgctgacgaggctaacgagcgtgccctgcgcatgctggaaaaagccagccgcaag ggcggcggcggcagcggcggcggcggcagcggcggcggcggcagctcggcggcgcctgccagtgccgagcaggaggaactgctggccctgttgcgcagcgagcggatcgtgctggcccacgccggccagccgctgagcgaggcgcaagtgctcaaggcgctcgcctggttgctcgcggccaatccgtccgcgcctccggggcagggcctcgaggtactccgcgaagtcctgcaggcacgtcggcagcccggtgcgcagtgggatctgcgcgagttcctggtgtcggcctatttcagcctgcacgggcgtctcgacgaggatgtcatcggtgtctacaaggatgtcctgcagacccaggacggcaagcgcaaggcgctgctcgacgagctgaaggcgctgaccgcggagttgaaggtctacagcgtgatccagtcgcagatcaacgccgcgctgtcggccaagcagggcatcaggatcgacgctggcggtatcgatctggtcgaccccacgctatatggctatgccgtcggcgatcccaggtggaaggacagccccgagtatgcgctgctgagcaatctggataccttcagcggcaagctgtcgatcaaggattttctcagcggctcgccgaagcagagcggggaactcaagggcctcagcgatgagtaccccttcgagaaggacaacaacccggtcggcaatttcgccaccacggtgagcgaccgctcgcgtccgctgaacgacaaggtcaacgagaagaccaccctgctcaacgacaccagctcccgctacaactcggcggtcgaggcgctcaaccgcttcatccagaaatacgacagcgtcctgcgcgacattctcagcgcgatctag SEQ ID NO: 35. P. aeruginosa OprI SHSKETEARLTATEDAAARAQARADEAYRKADEALGAAQKAQQTADEANERALRMLEKASRK The nucleotide sequence of SEQ ID NO: 35 agccactccaaagaaaccgaagctcgtctgaccgctaccgaagacgcagctgctcgtgctcaggctcgcgctgacgaagcctatcgcaaggctgacgaagctctgggcgctgctcagaaagctcagcagactgctgacgaggctaacgagcgtgccctgcgcatgctggaaaaagccagccgcaatag SEQ ID NO: 36. E. coli OmpA APKDNTWYTGAKLGWSQYHDTGFIPNNGPTHENQLGAGAFGGYQVNPYVGFEMGYDWLGRMPYKGDNINGAYKAQGVQLTAKLGYPITDDLDIYTRLGGMVWRADTKANVPGGASFKDHDTGVSPVFAGGVEYAITPEIATRLEYQWTNNIGDAHTIGTRPDNG MLSLGVSYRFGQGEVAPVVAPAPAPAPEVQTKHFTLKSDVLFTFNKATLKPEGQAALDQLYSQLSNLDPKDGSVVVLGYTDRIGSDAYNQALSERRAQSVVDYLISKGIPADKISARGMGESNPVTGNTCDNVKQRAALIDCLAPDRRVEIEVKGIKDVVTQPQA Nucleotide sequence of SEQ ID NO:36 gctccgaaagataacacctggtacactggtgctaaactgggctggtcccagtaccatgacactggttttattcctaacaatggtccgacccacgaaaaccaactgggtgcaggtgcttttggtggttaccaggttaacccgtatgttggctttgaaatgggttacgactggttaggtcgtatgccgtacaaaggcgacaacatcaacggcgcatacaaagctcagggcgttcagctgaccgctaaactgggttacccaatcactgacgatctggacatctacactcgtctgggtggtatggtatggcgtgcagacaccaaggctaacgtacctggtggcgcatcctttaaagaccacgacaccggcgtttctccggtcttcgctggcggtgttgagtatgcgatcactcctgaaatcgctacccgtctggaataccagtggaccaacaacatcggtgacgcacacaccatcggcactcgtccggacaacggcatgctgagcctgggtgtttcctaccgtttcggtcagggcgaagtagctccagtagttgctccggctccagctccggcaccggaagtacagaccaagcacttcactctgaagtctgacgttctgttcaccttcaacaaagcaaccctgaaaccggaaggtcaggctgctctggatcagctgtacagccagctgagcaacctggatccgaaagacggttccgtagttgttctgggttacactgaccgcatcggttctgacgcttataaccaggctctgtccgagcgtcgtgctcagtccgttgttgattacctgatctctaaaggtatcccggcagacaaaatctccgcacgtggtatgggcgaatccaacccggttactggcaacacctgtgacaacgtgaaacagcgtgctgcactgatcgattgcctggctccggatcgtcgcgtagagatcgaagttaaaggcatcaaagacgttgtaactcagccgcaggcttaa SEQ ID NO: 37. E. coli OmpX ATSTVTGGYAQSDAQGQMNKMGGFNLKYRYEEDNSPLGVIGSFTYTEKSRTASSGDYNKNQYYGITAGPAYRINDWASIYGVVGVGYGKFQTTEYPTYKHDTSDYGFSYGAGLQFNPMENVALDFSYEQSRIRSVDVGTWIAGVGYRF Nucleotide sequence of SEQ ID NO:37 gcgacttctaccgtaactggcggttacgcacaaagcgacgctcagggccaaatgaacaaaatgggcggtttcaacctgaaataccgctatgaagaagacaacagcccgctg ggtgtgatcggttctttcacttacaccgagaaaagccgtactgcaagctctggtgactacaacaaaaaccagtactacggcatcactgctggtccggcttaccgcattaacg actgggcaagcatctacggtgtagtgggtgtgggttatggtaaattccagaccactgaatacccgacctacaaacacgacaccagcgactacggtttctcctacggtgctgg tttgcagttcaacccgatggaaaacgttgctctggacttctcttacgagcagagccgtattcgtagcgttgacgtaggcacctggattgccggtgttggttaccgcttctaa SEQ ID NO:38. E. coli FyuA NAQTSQQDESTLVVTASKQSSRSASANNVSSTVVSAPELSDAGVTASDKLPRVLPGLNIENSGNMLFSTISLRGVSSAQDFYNPAVTLYVDGVPQLSTNTIQALTDVQSVELLRGPQGTLYGKSAQGGIINIVTQQPDSTPRGYIEGGVSSRDSYRSKFNLSG PIQDGLLYGSVTLLRQVDDGDMINPATGSDDLGGTRASIGNVKLRLAPDDQPWEMGFAASRECTRATQDAYVGWNDIKGRKLSISDGSPDPYMRCTDSQTLSGKYTTDDWVFNLISAWQQQHYSRTFPSGSLIVNMPQRWNQDVQELRAATLGDARTVDMVF GLYRQNTREKLNSAYDMPTMPYLSSTGYTTAETLAAYSDLTWHLTDRFDIGGGVRFSHDKSSTQYHGSMLGNPFGDQGKSNDDQVLGQLSAGYMLTDDWRVYTRVAQGYKPSGYNIVPTAGLDAKPFVAEKSINYELGTRYETADVTLQAATFYTHTKDMQLY SGPVGMQTLSNAGKADATGVELEAKWRFAPGWSWDINGNVIRSEFTNDSELYHGNRVPFVPRYGAGSSVNGVIDTRYGALMPRLAVNLVGPHYFDGDNQLRQGTYATLDSSLGWQATERMNISVYVDNLFDRRYRTYGYMNGSSAVAQVNMGRTVGINTRIDFF Nucleotide sequence of SEQ ID NO:38 SEQ ID NO: 39. E. coli Hma TSSETKISNEETLVVTTNRSASNLWESPATIQVIDQQTLQNSTNASIADNLQDIPGVEITDNSLAGRKQIRIRGEASSRVLILIDGQEVTYQRAGDNYGVGLLIDESALERVEVVKGPYSVLYGSQAIGGIVNFITKKGGDKLASGVVKAVYNSATAGWEESIAVQGSIGG FDYRINGSYSDQGNRDTPDGRLPNTNYRNNSQGVWLGYNSGNHRFGLSLDRYRLATQTYYEDPDGSYEAFSVKIPKLEREKVGVFYDTDVDGDYLKKIHFDAYEQTIQRQFANEVKTTQPVPSPMIQALTVHNKTDTHDKQYTQAVTLQSHFSLPANNELVTGAQYKQDRVS QRSGGMTSSKSLTGFINKETRTRSYYESEQSTVSLFAQNDWRFADHWTWTMGVRQYWLSSKLTRGDGVSYTAGIISDTSLARESASDHEMVTSTSLRYSGFDNLELRAAFAQGYVFPTLSQLFMQTSAGGSVTYGNPDLKAEHSNNFELGARYNGNQWLIDSAVYYSEAKDY IASLICDGSIVCNGNTNSSRSSYYYYDNIDRAKTWGLEISAEYNGWVFSPYISGNLIRRQYETSTLKTTNTGEPAINGRIGLKHTLVMGQANIISDVFIRAASSAKDDSNGTETNVPGWATLNFAVNTEFGNEDQYRINLALNNLTDKRYRTAHETIPAAGFNAAIGFVWNF Nucleotide sequence of SEQ ID NO:39 SEQ ID NO: 40. E. coli IutA QQNDDNEIIVSASRSNRTVAEMAQTTWVIENAELEQQIQGGKELKDALAQLIPGLDVSSQSRTNYGMNMRGRPLVVLIDGVRLNSSRSDSRQLDSVDPFNIDHIEVISGATALYGGGSTGGLINIVTKKGQPETMMEFEAGTKSGFNSSKDHDERIAGAVSGGNDHISGRLSVAYQK FGGWFDGNGDATLLDNTQTGLQHSNRLDIMGTGTLNIDESRQLQLITQYYKSQGDDNYGLNLGKGFSAISGSSTPYVSKGLNSDRIPGTERHLISLQYSDSDFLRQELVGQVYYRDESLRFYPFPTVNANKQATAFSSSQQDTDQYGMKLTLNSQLMDGWQITWGLDAEHERFTSNQ MFFDLAQASASGGLNNHKIYTTGRYPSYDITNLAAFLQSSYDINDIFTVSGGVRYQYTENRVDDFIDYTQQQKIAAGKAISADAIPGGSVDYDNFLFNAGLLMHITERQQAWFNFSQGVALPPDGKYYGRGIYGAAVNGHLPLTKSVNVSDSKLEGVKVDSYELGWRFTGDNLRTQI AAYYSLSNKSVERNKDLTISVKDDRRRIYGVEGAVDYLIPDTDWSTGVNFNVLKTESKVNGQWQKYDVKESSPSKATAYINWAPEPWSLRVQSTTSFDVSDAEGNDINGYTTVDFISSWQLPVGTLSFSVENLFDRDYTTVWGQRAPLYYSPGYGPASLYDYKGRGRTFGLNYSVLF Nucleotide sequence of SEQ ID NO:40 SEQ ID NO: 41. A. pleuropneumoniae OmpA AAPQQNTFYAGAKAGWASFHDGIEQLDSAKNTDRGTKYGINRNSVTYGVFGGYQILNQDKLGLAAELGYDYFGRVRGSEKPNGKADKKTFRHAAHGATIALKPSYEVLPDLDVYGKVGIALVNNTYKTFNAAQEKVKTRRFQSSLILGAGVEYAILPELAARVEYQWLNNAGK ASYSTLNRMGATDYRSDISSVSAGLSYRFGQGAVPVAAPAVETKNFAFSSDVLFAFGKSNLKPAAATALDAMQTEINNAGLSNAAIQVNGYTDRIGKEASNLKLSQRRAETVANYIVSKGALAANVTAVGYGEANPVTGATCDKVKGRKALIACLAPDRRVEVQVQGTKEVTM Nucleotide sequence of SEQ ID NO:41 SEQ ID NO: 42. A. pleuropneumoniae OmpW AHQAGDVIFRAGAIGVIANSSSDYQTGADVNLDVNNNIQLGLTGTYMLSDNLGLELLAATPFSHKITGKLGATDLGEVAKVKHLPSLYLQYYFFDSNATVRPYVGAGLNYTRFFSAESLKPQLVQNLRVKKHSVAPIANLGVDVKLTDNLSFNAAAWYTRIKTTADYDVPGLGHVSTPITLDPVVLFSGISYKF Nucleotide sequence of SEQ ID NO:42 gcacatcaagcgggcgatgtgattttccgtgccggtgcgatcggtgtgattgcaaattcaagttcggattatcaaaccggggcggacgtaaacttagatgtaaataataatattcagcttggtttaaccggtacctatatgttaagt gataatttaggtcttgaattattagcggcaacaccgtttttctcacaaaatcaccggtaagttaggtgcaacagatttaggcgaagtggcaaaagtaaaacatcttccgccgagcctttacttacaatattatttctttgattctaat gcgacagttcgtccatacgttggtgccggtttaaactatactcgctttttcagtgctgaaagtttaaaaccgcaattagtacaaaacttacgtgttaaaaaacattccgtcgcaccgattgcgaatttaggtgttgatgtgaaatta acggataatctatcattcaatgcggcagcttggtacacacgtattaaaactactgccgattatgatgttccgggattaggtcatgtaagtacaccgattactttagatcctgttgtattattctcaggtattagctacaaattctaa SEQ ID NO: 43. A. pleuropneumoniae TbpA AEQAVQLNDVYVTGTKKKAHKKENEVTGLGKVVKTPDTLSKEQVLGIRDLTRYDPGISVVEQGRGATTGYSIRGVDRNRVGLALDGLPQIQSYVSQYSRSSSGAINEIEYENLR SIQISKGASSSEFGSGSLGGSVQFRTKEVSDIIKPGQSWGLDTKSAYSSKNQQWLNSLAFAGTHNGFDALVIYTHRDGKETKAHKNAESRSQNITRVGVETNELDTSNRYTATT NNQHTYGWFLIKDECPTLDCTPKQMARVTKDTPSFRSYPEYTPEEKQAYENQKHITERLNAQDYTGEYRALPDPLKYKSDSWLVKLGYTFSPKHYVAGTYEHSKQRYDTRDMTY TAYWQPSDLLRTGRNWYPMNNAKGLYRDNALDGVAIDYFTEDGVKSSKGLRWAKARFIDEWHTRDRLGALYRYTNQDGNRLIDRLSLSFDQQKINLSTRLRENNCSEYPTIDKNC RATLDKLWSSTKNEQSSYEEKHDTIQLSLDKTVQTGLGKHQLNMLLGSDRFNSTLKRHEILSEFSVGSWGLVRDIGYRNGSYNNPYVYELKDQAIYSKNECDYSGTIAGRADCA TSKIKGHNHYIALRDNFAITKYLDIGLGYRFDKHKFRSTHRWANQGDYKNSAWNIGIVAKPTSFLSLSYRASSGFRVPSFQELFGLRYDGAMKGSSDAYQKTEKLSPEKSLNQE VAATFKGDFGVVEVSYFKNDYKQLIAPAERMHQTQSMINYFNVQDIKLDGINLIGKLDWNGVFDKIPEGIYTTLAYSKMRVKEVKNYQGYMNIRSPLLDTIQPARYVVGVGYDQ PDEKWGVNLTMTHSSSGKNPNELRGNEQVGFANYERTATKKRTRSWHTFDLTGYITPWKHTTVRAGVYNLMNYRYTTWESVRQSSLNAIHQHTNVKDYARYAAPGRNYVVSFEMKF Nucleotide sequence of SEQ ID NO:43 SEQ ID NO: 44. A. pleuropneumoniae ApfA FTLIELMIVIAIIAILATVAIPSYNSYNSYTQKAALSELLAASASYKTDVEICIYNTGDSKNCSGGQNGVRKMTELRQAKYLNAITVEGGTITVTGKGNLQEYGYTMTPIHNGSTISWETKCKGEDLSLFPANFCAIN Nucleotide sequence of SEQ ID NO:44 tttactttaattgaattgatgatcgtgattgcgattattgccattttagctacggttgcaattccgtcatataacagttatacccaaaaagcggcgctttcg gagctattggcggcatcggcttcttataaaacggatgtcgagatctgcatatataacaccggagattctaaaaactgtagcggcggtcaaaacggtgtcaga aaaatgacggagcttagacaggctaaatatttaaatgccattacggtggaaggcggaacgattacggtaacggggaaggggaatctgcaggaatacggttatacgatgacaccgattcataacggtagcactatttcttgggaaacgaaatgtaaaggggaggacttaagtttatttccggcaaatttctgtgcgataaattag SEQ ID NO: 45. P. aeruginosa PcrV AAPASAEQEELLALLRSERIVLAHAGQPLSEAQVLKALAWLLAANPSAPPGQGLEVLREVLQARRQPGAQWDLRELVVSAYFSLHGRLDEDVIGVYKDVLQTQDGKRKALLDELKALTAELKVYSVIQSQINAALS AKQGIRIDAGGIDLVDPTLYGYAVGDPRWKDSPEYALLSNLDTFSGKLSIKDFLSGSPKQSGELKGLSDEYPFEKDNNPVGNFATTVSDRSRPLNDKVNEKTTLLNDTSSRYNSAVEALNRFIQKYDSVLRDILSAI Nucleotide sequence of SEQ ID NO:45 tcggcggcgcctgccagtgccgagcaggaggaactgctggccctgttgcgcagcgagcggatcgtgctggcccacgccggccagccgctgagcgaggcgcaagtgctcaaggcgctcgcctggttgctcgcggccaatccgtccgcgcctccggggcagggcctcgaggtactccgcgaagtcctgcaggcacgtcggcagcccggtgcgcagtgggatctgcgcgagttcctggtgtcggcctatttcagcctgcacgggcgtctcgacgaggatgtcatcggtgtctacaaggatgtcctgcagacccaggacggcaagcgcaaggcgctgctcgacgagctcaaggcgctgaccgcggagttgaaggtctacagcgtgatccagtcgcagatcaacgccgcgctgtcggccaagcagggcatcaggatcgacgctggcggtatcgatctggtcgaccccacgctatatggctatgccgtcggcgatcccaggtggaaggacagccccgagtatgcgctgctgagcaatctggataccttcagcggcaagctgtcgatcaaggattttctcagcggctcgccgaagcagagcggggaactcaagggcctcagcgatgagtaccccttcgagaaggacaacaacccggtcggcaatttcgccaccacggtgagcgaccgctcgcgtccgctgaacgacaaggtcaacgagaagaccaccctgctcaacgacaccagctcccgctacaactcggcggtcgaggcgctcaaccgcttcatccagaaatacgacagcgtcctgcgcgacattctcagcgcgatctag

[0067] In another embodiment of the first aspect of the invention, the heterologous antigen expressed on the outer membrane of the A. baumannii strain lacking lipopolysaccharide (LPS) is at least derived from K. pneumoniae and selected from the group consisting of Kp-OmpA and / or Kp-OmpK36, and / or any identical sequences thereof as taught in the present invention, and / or derived from P. aeruginosa and selected from the group consisting of Pa-OprF and / or Pa-OprI, and / or the fusion protein Pa-OprI::PcrV, and / or any identical sequences thereof as taught in the present invention, and / or derived from E. coli and selected from the group consisting of Ec- The A. baumannii strain is selected from the group consisting of OmpA and / or Ec-OmpX and / or Ec-FuyA and / or Ec-Hma and / or Ec-IutA, and / or any identical sequences thereof as taught in the present invention, and / or is derived from A. pleuropneumoniae and is selected from the list of Ap-OmpA and / or Ap-OmpW and / or Ap-TbpA and / or Ap-ApfA, and / or any identical sequences thereof as taught in the present invention, preferably the A. baumannii strain lacking lipopolysaccharide (LPS) further comprises expression of the A. baumannii antigens Ab-OmpA and Ab-Omp22.

[0068] In another embodiment of the first aspect of the invention, the lipopolysaccharide (LPS)-deficient A. baumannii strain is characterized by partial or complete inactivation of a gene selected from the group consisting of lpxA, lpxC, and lpxD.

[0069] A second aspect of the present invention refers to a composition comprising a lipopolysaccharide (LPS)-deficient A. baumannii strain expressing one or more copies of one or more microbial antigenic outer membrane heterologous proteins in its outer membrane, as defined in the first aspect of the invention or any of its preferred embodiments, hereinafter Composition of the Invention.

[0070] Preferably, the composition of the invention is a pharmaceutical composition, optionally comprising an acceptable pharmaceutical vehicle, carrier and / or excipient. Even more preferably, the composition of the invention is a vaccine formulation, optionally comprising an adjuvant. Preferably, the composition or vaccine formulation comprises about 10 microbial cells expressing one or more copies of an antigenic outer membrane heterologous protein by one or more microorganisms in their outer membrane, as defined in the first aspect of the invention or any of its preferred embodiments. 6 pieces ~ about 10 12 A. baumannii strain lacking about 10 lipopolysaccharides (LPS), preferably about 10 9 Also preferably, the adjuvant, when present in the vaccine formulation, is preferably Al(OH) 3 More preferably, the lipopolysaccharide (LPS)-deficient A. baumannii strain present in the composition, preferably in the vaccine formulation, is inactivated. Notwithstanding the above, the administration to obtain an effective therapeutic amount depends on various factors, such as the age, weight, sex, resistance of the mammal. As used herein, any therapeutic amount should be effective and therefore refer to the amount of inactivated Acinetobacter cells that produces the desired effect, and is generally determined by the desired therapeutic effect.

[0071] The term "excipient" refers to a substance that serves to activate or assist the preparation of a medicament to provide absorption of the components of the composition of the medicament of the present invention, stabilization of said components, a more palatable consistency or flavor, etc. Excipients can maintain the components of the excipient together with the function of protecting the medicament, such as isolating it from the atmosphere and / or humidity, for example in the case of starch, sugar, cellulose, sweeteners, colorants, filling the pill, capsule or any other form of presentation, facilitating the dissolution of the components and their absorption in the intestine, for example in the case of dicalcium phosphate, without excluding other types of excipients described in this paragraph.

[0072] Vehicles, also referred to as excipients, are substances used to dilute any of the components of the present invention to the desired volume or weight in a medicament. Pharmaceutically acceptable vehicles are inert substances or of similar action to any of the elements of the present invention. The function of a vehicle is to facilitate the incorporation of other elements, allow good dosing and administration, and give consistency and form to the medicament. When the form of presentation is liquid, the pharma-ceutically acceptable vehicle is a diluent.

[0073] The adjuvants and pharma- ceutically acceptable vehicles that can be used in the composition of the present invention are such vehicles known to those skilled in the art.In the present invention, the term "adjuvant" refers to any agent that does not contain and does not have antigenic activity itself, and can be used to stimulate the immune system and increase the response to vaccines.There are many adjuvants, such as, but not limited to, aluminum phosphate, aluminum hydroxide, toll-like receptor agonist, cytokine, squaline, Freund's incomplete and complete adjuvants.In a preferred form of this aspect of the present invention, the adjuvant is selected from the list consisting of aluminum phosphate, aluminum hydroxide, toll-like receptor agonist, cytokine, squaline, saponin, Freund's incomplete and complete adjuvants.

[0074] In the present invention, the term "vaccine" refers to an antigenic preparation used to induce an immune response against a disease. Vaccines are made from antigens that, when introduced into the host, induce an immune response through the production of antibodies and create an immunological memory that results in temporary or permanent immunity.

[0075] The manufacture of the vaccine formulation of the present invention can be accomplished through, but is not limited to, a two-stage fermentation process followed by heat inactivation. The vaccine formulation shall preferably be in the form of a sterile freeze-dried preparation of the vaccine. Once the excipients and adjuvants are blended, freeze-drying shall be applied to the final product. Vaccine formulations based on LPS-null A. baumannii cell technology are preferably not filtered or autoclaved to obtain sterility. The cells are usually inactivated in the final step of drug substance manufacturing after fermentation and before final product blending. The inactivation method shall preferably be based on a mild heat inactivation protocol (75°C, 30 min) to preserve the potency of the whole cell multi-antigen active components.

[0076] The third aspect refers to a lipopolysaccharide (LPS)-deficient A. baumannii strain expressing one or more copies of one or more microbial antigenic outer membrane heterologous proteins in its outer membrane, as defined in the first aspect of the invention or any of its preferred embodiments, for use as a medicament or for use in therapy, more preferably as a vaccine to induce an immune response against a disease caused by any microorganism that naturally expresses one or more copies of the antigenic outer membrane protein.

[0077] The term "medicine" as used herein refers to any substance used in the prevention, mitigation, treatment or cure of disease in humans or animals.

[0078] In a fourth aspect, the lipopolysaccharide (LPS)-deficient A. baumannii strain of the first aspect of the invention or any of its preferred embodiments, or the composition of the second aspect of the invention or any of its preferred embodiments, is used to deliver a bacterial outer membrane antigen to a subject in need thereof, preferably a human subject, in order to immunize said subject against any infectious disease which contains, harbors or expresses said antigen. In a preferred embodiment, the lipopolysaccharide (LPS)-deficient A. baumannii strain of the first aspect of the invention or any of its preferred embodiments, or the composition of the second aspect of the invention or any of its preferred embodiments, is used to deliver a bacterial outer membrane antigen to induce an immune response against K. pneumoniae, P. aeruginosa, E. coli, and / or A. pleuropneumoniae and optionally A. baumannii in a subject in need thereof. In a preferred embodiment, the lipopolysaccharide (LPS)-deficient A. baumannii strain of the first aspect of the invention or any of its preferred embodiments, or the composition of the second aspect of the invention or any of its preferred embodiments, is used to deliver bacterial outer membrane antigens for the prevention, amelioration or treatment of infections caused by K. pneumoniae, P. aeruginosa, E. coli, and / or A. pleuropneumoniae and optionally A. baumannii.Preferably, for any of these purposes, the heterologous antigens expressed on the outer membrane of the A. baumannii strain lacking lipopolysaccharide (LPS) are derived from at least any of those presented throughout the present invention for K. pneumoniae, P. aeruginosa, E. coli, and A. pleuropneumoniae, for example those selected from the group consisting of Kp-OmpA and / or Kp-OmpK36 for K. pneumoniae, and Pa-OprF and / or Pa-OprI, and / or the fusion protein Pa-OprI::PcrV for P. aeruginosa. and for E. coli selected from the group consisting of Ec-OmpA and / or Ec-OmpX and / or Ec-FuyA and / or Ec-Hma and / or Ec-IutA, and for A. pleuropneumoniae selected from the list of Ap-OmpA and / or Ap-OmpW and / or Ap-TbpA and / or Ap-ApfA, preferably the A. baumannii strain lacking lipopolysaccharide (LPS) further comprises expression of the A. baumannii antigens Ab-OmpA and / or Ab-Omp22.

[0079] The medicaments and compositions of the invention can be used alone or in combination with other medicaments or active ingredients or compositions for the treatment of diseases caused by organisms of the Acinetobacter genus.

[0080] As used herein, the term "active ingredient" refers to any component that potentially provides pharmacological activity or other various effects in the diagnosis, cure, mitigation, treatment or prevention of disease, or affects the structure or function of the human or animal body. The term includes such components that exist in similar and modified forms that facilitate chemical changes in the production of drugs and provide a specific activity or effect.

[0081] By "infections caused by K. pneumoniae, P. aeruginosa, E. coli, and / or A. pleuropneumoniae and optionally A. baumannii" is understood such diseases in which the causative pathogen of the lesion is either K. pneumoniae, P. aeruginosa, E. coli, and / or A. pleuropneumoniae and / or A. baumannii. Acinetobacter causes a variety of lesions, such as, but not limited to, bacteremia, meningitis, urinary tract infections, skin and soft tissue infections, surgical site infections and pneumonia. For these reasons, the disease caused by organisms of the Acinetobacter genus, which is one of the more preferred forms, is selected from the group consisting of bacteremia, meningitis, urinary tract infections, skin and soft tissue infections, surgical site infections and pneumonia.

[0082] A fifth aspect of the present invention refers to an antibody or an active fragment thereof obtainable by immunization of a mammal with a composition of the first or second aspect of the present invention, preferably said antibody or active fragment consisting of a composition which is preferably a pharmaceutical composition, said pharmaceutical composition being particularly used as a therapy for the treatment of infections caused by K. pneumoniae, P. aeruginosa, E. coli and / or A. pleuropneumoniae and optionally A. baumannii.

[0083] Method for preparing LPS-null Acinetobacter cells capable of presenting heterologous outer membrane antigens according to the present invention This particular section constitutes a non-limiting example of a method for preparing an A. baumannii strain according to the first aspect of the invention or any of its preferred embodiments.

[0084] First, A. baumannii cells are transformed with a vector, preferably a suicide vector, that contains a sequence to facilitate recombination at a desired target locus into the A. baumannii genome, such locus being any locus in the A. baumannii genome that contains a sequence suitable for undergoing recombination. For example, a suitable locus for integration of an insert by recombination may be cysI, trpE, lpxA, lpxC, lpxD, lpxB, lpxK, lpxL, lpxM, and / or Tn5 / Tn7 sites. In a preferred embodiment of the invention, the suitable locus is cysI. The cysI locus contains the coding gene cysIORF, which contains a 1644 nt ORF that codes for a 547 amino acid protein essential for the biosynthesis of the amino acid cysteine. In another preferred embodiment, the suitable locus is lpxA, which contains an ORF that codes for the acyltransferase LpxA, the first enzyme in the lipopolysaccharide biosynthetic pathway.

[0085] The vector sequence for promoting recombination is flanked by an expression construct that includes at least one or more transcription promoter sequences, one or more ORFs encoding heterologous OMP proteins for A. baumannii, and one or more transcription termination sequences. The promoter sequence can be any known sequence in the art that can promote transcription in A. baumannii cells. These promoter sequences are routinely used in bacteriological research. In a preferred embodiment of the present invention, the promoter sequence used is a promoter sequence located upstream of the A. baumannii ORF encoding the outer membrane protein OmpA. The expression construct can contain several ORFs in series, each flanked by a promoter and a termination, transcription termination sequence upstream and downstream of the ORF, respectively, thus constructing to allow independent expression of each ORF controlled by a specific promoter. Alternatively, the expression construct can have one or several ORFs in an operon-like structure with polycistronic expression controlled by a common promoter. The ORFs of the expression vectors code for OMP proteins or peptides selected for their immunogenic properties, derived from known human or animal pathogens different from A. baumannii, whose expression in A. baumannii cells is intended to enhance the immune response against the pathogen different from A. baumannii in humans or animals vaccinated with A. baumannii cells. The technology of the present invention is based on the use of a signal sequence in the upstream part of each ORF for post-translational processing of the encoded protein, in particular for promoting the location of the encoded protein at the outer membrane of A. baumannii cells. The nature of signal sequences capable of promoting the location of expressed proteins at the outer membrane of A. baumannii cells is known in the art. In a preferred embodiment of the present invention, the signal sequence of the outer membrane protein OmpA of A. baumannii is used to promote the location of the encoded protein at the outer membrane of A. baumannii.It is also known in the art that any OMP protein having a transmembrane domain, even by any other bacterial cell different from A. baumannii, when expressed as a fusion protein containing an A. baumannii signal sequence at its N-terminus, will be processed by A. baumannii cells for integration into the outer membrane of the A. baumannii cell. Thus, the techniques developed in the present invention can be applied to any OMP protein of bacterial cells, particularly Gram-negative bacterial cells, having a known OMP transmembrane domain, regardless of the nature of the remaining domain of the OMP protein selected for expression in A. baumannii cells.

[0086] Therefore, the heterologous OMP protein selected for expression in A. baumannii cells as described above must be an OMP protein from any known bacterial pathogen that has an OMP transmembrane domain for insertion into the bacterial outer membrane. The properties of these OMP proteins are known in the art. The outer membrane protects Gram-negative bacteria from the harsh environment. At the same time, the embedded proteins perform a number of roles that are very important for the bacterial cell, such as solute and protein translocation, and signal transduction. Unlike membrane proteins from all other sources, integral OMP proteins do not consist of transmembrane α-helices, but instead fold into antiparallel β-barrels. They include OmpA membrane domains, OmpX proteins, phospholipase A, general porins, substrate-specific porins, and iron siderophore transporters. It is also known in the art that the location of these OMP proteins at the outer surface of bacterial pathogens confers immunogenic properties to them. It is known in the art that sera from humans infected with bacterial pathogens recognize primarily OMP proteins on crude protein extracts of bacterial cells and demonstrate the presence of significant titers of serum antibodies raised against bacterial OMP proteins.

[0087] In certain embodiments of the invention, the sequence of a heterologous OMP protein derived from a pathogen other than A. baumannii can be modified by inserting a peptide of the same or another pathogen with the intent of adding another antigenic sequence to a domain of the OMP protein, such that expression and final location in the outer membrane of A. baumannii as taught in the present invention is not hindered by the insertion of the other antigenic sequence in the final fusion protein based on the original OMP.

[0088] In a preferred embodiment of the invention, the heterologous OMP selected for expression in A. baumannii cells is derived from the most fastidious Gram-negative bacteria, in particular K. pneumoniae, P. aeruginosa and / or E. coli. In another preferred embodiment of the invention, the heterologous OMP is selected from Actinobacillus pleuropneumoniae, the causative agent of porcine pleuropneumonia. In another preferred embodiment of the invention, the OMP protein derived from K. pneumoniae is the K. pneumoniae OMP protein OmpA and / or OmpK36. In another preferred embodiment of the invention, the OMP protein derived from P. aeruginosa is the OMP protein OprF, and / or a fusion of the P. aeruginosa OMP protein OprI with the P. aeruginosa type 3 secretion apparatus protein PcrV. In yet another preferred embodiment of the invention, the OMP protein derived from E. coli is the outer membrane receptors FuyA, HmA, and IutA involved in iron acquisition, or the OMPs OmpA and OmpX. And in another preferred embodiment of the invention, the OMP proteins from Actinobacillus pleuropneumoniae are OMPs OmpA and OmpW, transferrin binding protein A, TpbA, and / or type IV pilus subunit protein ApfA.

[0089] The transcription termination sequence of the expression construct may be any transcription termination sequence capable of allowing transcription termination in A. baumannii known in the art.

[0090] Second, when A. baumannii cells are transformed with a vector, preferably a suicide vector, recombination between a specific selected sequence of the target locus in the host and a homologous sequence in the vector results in the production of a recombinant cell in which the sequence of the vector is integrated into the target locus on the A. baumannii chromosome. This recombination event can be assisted by strategies known in the art, such as inducing a double-stranded break at the insertion site by genome editing tools, such as those based on CRISPR methodology. In a preferred embodiment of the present invention, the first recombination event results in the insertion of vector sequences, including a selectable marker and an expression construct. The selectable marker is used to select cells in which such a first recombination event has occurred. A second recombination event can result in recombinant cells in which only the expression construct remains integrated into the insertion site, but not the sequence encoding the selectable marker. The absence of the selectable marker can be used as a selection tool to select A. baumannii cells in which such a second recombination event has occurred. Once selected, common techniques such as PCR and DNA sequencing are used to confirm whether the integration of the vector sequences into the target locus has occurred as expected.

[0091] Third, the expression of the proteins encoded by the expression constructs in recombinant A. baumannii cells can be analyzed by basic proteomic techniques such as Western blot or ELISA. The location of the encoded proteins at the outer membrane of recombinant A. baumannii cells can be analyzed by common proteomic methods previously described in the art, such as those described in the Examples.

[0092] Fourth, when the expression of the analyzed heterologous protein in the recombinant A. baumannii cells is deemed sufficient, the loss of LPS is induced in the A. baumannii cells. All steps carried out up to this point, i.e., transformation of A. baumannii cells with the expression construct and integration of the expression construct into the chromosome, were carried out in wild-type A. baumannii cells. It is known in the art that any strain, clone or mutant of A. baumannii can be transformed by the techniques described in the present invention so far. Furthermore, it is known in the art that it is possible to obtain viable A. baumannii mutants that are unable to synthesize lipid A, i.e., the core component of LPS. In a preferred embodiment of the present invention, A. baumannii cells are grown on plates with the antibiotic colistin to select for colistin-resistant mutants. It is known in the art that the loss of LPS renders A. baumannii cells colistin-resistant. Since other mutations that do not affect LPS synthesis may also result in colistin resistance, once colistin-resistant mutants are isolated, specific PCR reactions and DNA sequencing are used to look for mutations in LPS synthesis genes, including genes selected from the group consisting of lpxA, lpxB, lpxC, lpxD, lpxK, lpxL and / or lpxM. Once a mutation in an LPS synthesis gene is identified, the absence of LPS can be confirmed by techniques known in the art, such as a quantitative chromogenic LAL-based assay to detect the presence of endotoxin, i.e., LPS, in bacterial cultures, or the lack of detection by anti-LPS antibodies of crude extracts by bacterial cells. As taught in the examples, loss of LPS can be induced and LPS-null A. baumannii mutants can be obtained regardless of the A. baumannii strain, clone or variant used. As taught in the examples, various A. baumannii strains have been used in our laboratory to induce loss of LPS, and various mutations in LPS synthesis genes have been found to be involved in loss of LPS.

[0093] In addition to the above methods, loss of LPS can be induced by alternative methods such as site-directed mutagenesis of LPS synthesis genes, resulting in partial or complete inactivation of the LPS synthesis genes, including genes selected from the group consisting of lpxA, lpxB, lpxC, lpxD, lpxK, lpxL and / or lpxM.

[0094] In a particular embodiment of the invention, when the target locus for insertion of the above recombination event is a locus comprising an ORF encoding an LPS synthesis gene comprising a gene selected from the group consisting of lpxA, lpxB, lpxC, lpxD, lpxK, lpxL and / or lpxM, and the recombination results in partial or complete inactivation of such ORF, the resulting recombinant A. baumannii cell is an LPS negative cell due to partial or complete inactivation of the LPS synthesis gene comprising a gene selected from the group consisting of lpxA, lpxB, lpxC, lpxD, lpxK, lpxL and / or lpxM.

[0095] An "infection" in the present invention is understood to be a lesion resulting from the invasion or colonization of any host tissue by any organism of the genus Acinetobacter, preferably A. baumannii, or any other bacterial species targeted by the vaccine candidate, including the bacterial species K. pneumoniae, P. aeruginosa, E. coli and A. pleuropneumoniae.

[0096] The term "antigen" in the present invention refers to a molecule (generally a protein or polysaccharide) capable of inducing the formation of an antibody. There are many different types of molecules that can act as antigens, including proteins, peptides, polysaccharides, and rarely other molecules such as nucleic acids.

[0097] The following examples are illustrative only and are not intended to limit the scope of the invention. EXAMPLES

[0098] Abbreviation cysI: a gene encoding sulfite reductase involved in the biosynthesis of the amino acid L-cysteine. trpE: a gene encoding anthranilate synthase (subunit I) involved in the biosynthesis of the amino acid tryptophan. lpxA: a gene encoding acyl-[acyl carrier protein]-UDP-N-acetylglucosamine O-acyltransferase involved in the biosynthesis of lipid A, a phosphorylated glycolipid that anchors lipopolysaccharide to the outer membrane of the cell. lpxC: a gene encoding DP-3-O-acyl-N-acetylglucosamine deacetylase, which catalyzes the hydrolysis of UDP-3-O-myristoyl-N-acetylglucosamine, a committed step in lipid A biosynthesis, to form UDP-3-O-myristoylglucosamine and acetate. lpxD: a gene encoding DP-3-O-acylglucosamine N-acyltransferase involved in the biosynthesis of lipid A, which catalyzes the N-acylation of UDP-3-O-acylglucosamine using 3-hydroxyacyl-ACP as the acyl donor. lpxB: a gene encoding lipid A disaccharide synthase involved in the condensation of UDP-2,3-diacylglucosamine with 2,3-diacylglucosamine-1-phosphate to form lipid A disaccharide, the precursor of lipid A. lpxK: a gene encoding a tetraacyldisaccharide 4'-kinase that transfers the gamma phosphate of ATP to the 4' position of a tetraacyldisaccharide 1-phosphate intermediate (designated DS-1-P) to form tetraacyldisaccharide 1,4'-bis-phosphate (lipid IVA). This protein is involved in step 6 of the subpathway that synthesizes lipid IV(A) from (3R)-3-hydroxytetradecanoyl-[acyl carrier protein] and UDP-N-acetyl-α-D-glucosamine. lpxL: Lauroyl-acyl carrier protein (ACP) to Kdo 2 - Catalyzes the transfer of lauric acid to lipid IV(A) by Kdo 2 -A gene encoding lipid A biosynthesis lauroyltransferase, which forms (lauroyl)-lipid IV(A). lpxM: a gene encoding lipid A biosynthesis myristoyltransferase involved in step 4 of the subpathway that synthesizes KDO(2)-lipid A from CMP-3-deoxy-D-manno-octulosonic acid and lipid IV(A). Tn5 / Tn7 site: The region of the A. baumannii genome that is preferably inserted by the transposons Tn5 and Tn7.

[0099] Example 1. A method for the selection of LPS null mutants of A. baumannii that can be applied to any A. baumannii strain and results in various selected mutations in the LPS synthesis genes. As shown by Moffatt et al. 2010, Garcia-Quintanilla et al. 2014, Pulido et al. 2018, and Carretero-Ledesma et al. 2018, colistin-resistant mutants of a given strain of A. baumannii can be isolated by directly plating these parent strains on Mueller-Hinton agar containing 10 mcg / ml colistin sulfate, and colistin-resistant colonies can be identified after one round of selection (10 8 1 to 10 per piece 9(at a frequency of 1 in 1000 individuals). As also shown by Moffat et al. 2010, Garcia-Quintanilla et al. 2014, Pulido et al. 2018, and Carretero-Ledesma et al. 2018, the colistin-resistant phenotype of selected colonies is frequently acquired by loss of LPS due to a mutation in one of the LPS synthesis genes. Table 1 shows examples of colistin-resistant mutants selected by the present authors by using the colistin plating method described above. In Table 1, examples of LPS-null colistin-resistant derivatives of three different unrelated clinical isolates of A. baumannii, specifically an old clinical isolate currently available in ATCC as strain ATCC19606 and used as a reference A. baumannii strain in experiments with other animal models, and clinical isolates named IB001 and Ab283, which caused an outbreak in the Virgen del Rocio Hospital in Seville in 2002, are shown. Various LPS null derivatives are shown, indicating the specific mutations found in the LPS biosynthetic genes.

[0100] Furthermore, Table 1 shows one LPS null derivative of Ab283 obtained by direct mutagenesis of the LPS synthesis gene lpxA. This derivative was named K1 or K1-Vax. To construct K1-Vax, Acinetobacter baumannii Ab283 cells were transformed with a suicide vector (pVXD-40::K1-Vax) containing sequences to promote recombination at the lpxA target locus into the A. baumannii genome. The pVXD-40::K1-Vax plasmid was constructed by cloning an expression construct flanked by homologous regions (2000 bp upstream and 2000 bp downstream of the lpxA gene) adjacent to the Ab283 lpxA gene. The suicide plasmid (a non-replicating plasmid in A. baumannii) carries a tetracycline resistance cassette inducing resistance to tetracycline as a selection marker, and the gene sacB as a counter selection marker. The pVXD-40::K1-Vax suicide plasmid was introduced into Ab283 cells by electroporation, conjugation or chemical transformation followed by two-step allelic exchange. The first recombination event resulted in the insertion of the vector when recombination occurred between the upstream region cloned into the plasmid and the upstream region of the Ab283 genome. Successfully recombined cells with the plasmid integrated into the genome were selected on tetracycline agar plates. Common techniques such as PCR and DNA sequencing were used to confirm whether integration of the plasmid into the target locus occurred as expected. The second recombination event was induced by growing the selected tetracycline-resistant Ab283 cells in the presence of sucrose, resulting in recombinant cells in which the vector backbone upstream and downstream of the homologous region was removed and only the expression construct remained in the recombinant Aab283 cells. Such Ab283 cells in which the second recombination event had occurred were obtained by selecting recombinant Ab283 cells that were sucrose-resistant and tetracycline-sensitive. Once selected, common techniques such as PCR and DNA sequencing were used to verify whether integration of the expression construct into the target locus occurred as expected. The second recombination also resulted in the complete inactivation of lpxA, which is essential for the biosynthesis of LPS in A. baumannii.Thus, the second recombination directly led to recombinant LPS-null Ab283 cells.

[0101] Example 2. Construction and validation of LPS-null A. baumannii strains expressing OMP proteins from other bacterial species Drug substances based on LPS-null A. baumannii cells expressing OMP proteins of other bacterial species to be used as vaccine candidates against infections by different bacterial pathogens can be constructed by genome editing of A. baumannii using allelic exchange techniques for recombinant strain production. In summary, we selected A. baumannii carrier strains. In particular, we screened pan-susceptible clinical isolates of A. baumannii that can survive without LPS as taught in EP 2942389. The final strain selected in the screening process was named Ab283. Insertion of the expression construct into the A. baumannii genome can be assessed by recombination by using various strategies known in the art, such as suicide plasmids or linear DNA fragments. For selection of LPS-negative cells, we plated the strains on colistin plates (Ab283 was grown in the presence of colistin and selection of colistin-resistant mutants was performed) and selected colistin-resistant mutants. We then screened by PCR for large insertions or deletions in the lpx gene.

[0102] Preparation of the first drug substance, KapaVax2, by insertion of an expression construct at the cysI locus. For construction of KapaVax2 at the cysI locus, the construct was inserted at the cysI locus in the Ab283 wild-type strain and expression and localization of the heterologous antigen at the outer membrane was demonstrated, followed by plating on colistin to select for an LPS-negative mutant carrying a 1 Kb ISAba insertion in the lpxC ORF, followed by selection of an LPS-negative derivative (see Fig. 3 ).

[0103] This candidate KapaVax2 contains both the selected K. pneumoniae antigens (OmpA and OmpK36) and three P. aeruginosa antigens (OprF, OprI and PcrV). Each antigen was expressed from an operon-like construct under the control of the A. baumannii OmpA promoter. PcrV was fused to the N-terminus of OprI as a fusion protein. The expression and location of each antigen in the outer membrane was confirmed by Western blot and ELISA (see Figure 8). Western blot analysis of whole cell lysates and outer membrane extracts with antigen-specific antibodies was also used to confirm the expression and localization of the heterologous antigens in A. baumannii cells.

[0104] Prior to LPS null mutant selection, A. baumannii cells were transformed with a suicide vector (pVXD-50::KapaVax2) containing sequences to promote recombination at the cysI target locus into the A. baumannii genome for integration of the expression construct at the cysI target locus. This procedure included the design of a suicide plasmid containing the expression construct flanked by homologous regions adjacent to the target gene, as well as a selection marker and a counterselection marker. The ORFs contained in the expression construct include sequences derived from the outer membrane proteins OmpA and OmpK36 of K. pneumoniae, as well as the outer membrane proteins OprF and OprI of P. aeruginosa, and the protein PcrV of the type 3 secretion apparatus of P. aeruginosa. The oprI and pcrV coding sequences were combined into one coding sequence to create a novel chimeric gene, oprI::pcrV. The four coding sequences were expressed from an operon-like construct under the control of the A. baumannii OmpA promoter. The expression construct contained the signal sequence of the A. baumannii outer membrane protein OmpA upstream of each coding sequence to facilitate localization of each antigen in the outer membrane of A. baumannii. The expression construct also contained the transcription termination sequence of the A. baumannii protein OmpA.

[0105] The pVXD-50::KapaVax2 plasmid was constructed by cloning an expression construct flanked by homologous regions adjacent to the Ab283 cysI gene (600 bp upstream and 600 bp downstream of the cysI gene). The suicide plasmid (a non-replicating plasmid in A. baumannii) carries the kanamycin resistance gene, which induces resistance to kanamycin as a selection marker, and the gene sacB as a counter selection marker.

[0106] The pVXD-50::KapaVax2 suicide plasmid was introduced into Ab283 cells by electroporation, conjugation or chemical transformation followed by two-step allelic exchange. The first recombination event resulted in the insertion of the vector when recombination occurred between the upstream region cloned into the plasmid and the upstream region of the Ab283 genome. Successfully recombined cells with the plasmid integrated into the genome were selected on kanamycin agar plates. Common techniques such as PCR and DNA sequencing were used to confirm whether integration of the plasmid into the target locus occurred as expected.

[0107] A second recombination event was induced by growing the selected kanamycin-resistant Ab283 cells in the presence of sucrose, resulting in recombinant cells in which the vector backbone upstream and downstream of the homologous region was removed and only the expression construct remained in the recombinant Ab283 cells. Such Ab283 cells in which a second recombination event had occurred were obtained by selecting recombinant Ab283 cells that were sucrose-resistant and kanamycin-sensitive. Once selected, common techniques such as PCR and DNA sequencing were used to confirm whether integration of the expression construct into the target locus had occurred as expected.

[0108] Preparation of a second drug substance, K-Vax, by insertion of an expression construct at the cysI locus. For construction of K-Vax at the cysI locus, the construct was inserted at the cysI locus in the Ab283 wild-type strain and expression and localization of the heterologous antigen at the outer membrane was demonstrated followed by plating on colistin to select for an LPS-negative mutant carrying a 1 Kb ISAba insertion in the lpxC ORF, followed by selection of an LPS-negative derivative (see Fig. 4 ).

[0109] This candidate K-Vax contains selected K. pneumoniae antigens (OmpA and OmpK36), each expressed from an operon-like construct under the control of the A. baumannii OmpA promoter. Expression and localization of each antigen at the outer membrane was confirmed by Western blot and ELISA (see FIG. 6). Western blot analysis of whole cell lysates and outer membrane extracts with antigen-specific antibodies was also used to confirm the expression and localization of heterologous antigens in A. baumannii cells.

[0110] Prior to LPS null mutant selection, A. baumannii cells were transformed with a suicide vector (pVXD-50::K-Vax) containing sequences to facilitate recombination at the cysI target locus into the A. baumannii genome for integration of the expression construct at the cysI locus of A. baumannii Ab283. This procedure included the design of a suicide plasmid containing the expression construct flanked by homologous regions adjacent to the target gene, as well as a selection marker and a counter selection marker. The ORFs contained in the expression construct included sequences from the K. pneumoniae outer membrane proteins OmpA and OmpK36. Both coding sequences were expressed from an operon-like construct under the control of the A. baumannii OmpA promoter. The expression construct contained the signal sequence of the A. baumannii outer membrane protein OmpA upstream of each coding sequence to facilitate location of each antigen in the outer membrane of A. baumannii. The expression construct also contains the transcription termination sequence of the A. baumannii protein OmpA.

[0111] The pVXD-50::K-Vax plasmid was constructed by cloning an expression construct flanked by homologous regions adjacent to the Ab283 cysI gene (600 bp upstream and 600 bp downstream of the cysI gene). The suicide plasmid (a non-replicating plasmid in A. baumannii) carries the kanamycin resistance gene, which induces resistance to kanamycin as a selection marker, and the gene sacB as a counter selection marker.

[0112] The pVXD-50::K-Vax suicide plasmid was introduced into Ab283 cells by electroporation, conjugation or chemical transformation followed by two-step allelic exchange. The first recombination event resulted in the insertion of the vector when recombination occurred between the upstream region cloned into the plasmid and the upstream region of the Ab283 genome. Successfully recombined cells with the plasmid integrated into the genome were selected on kanamycin agar plates. Common techniques such as PCR and DNA sequencing were used to confirm whether integration of the plasmid into the target locus occurred as expected.

[0113] A second recombination event was induced by growing the selected kanamycin-resistant Ab283 cells in the presence of sucrose, resulting in recombinant cells in which the vector backbone upstream and downstream of the homologous region was removed and only the expression construct remained in the recombinant Ab283 cells. Such Ab283 cells in which a second recombination event had occurred were obtained by selecting recombinant Ab283 cells that were sucrose-resistant and kanamycin-sensitive. Once selected, common techniques such as PCR and DNA sequencing were used to confirm whether integration of the expression construct into the target locus had occurred as expected (see Figure 5).

[0114] Preparation of a third drug substance, P-Vax, by insertion of an expression construct at the cysI locus. For construction of P-Vax at the cysI locus, the construct was inserted at the cysI locus in the Ab283 wild-type strain and expression and localization of the heterologous antigen at the outer membrane was demonstrated followed by plating on colistin to select for an LPS-negative mutant carrying a 1 Kb ISAba insertion in the lpxD ORF, followed by selection of an LPS-negative derivative (see Figure 4).

[0115] This candidate P-Vax contains three P. aeruginosa antigens (OprF, OprI and PcrV). Each antigen is expressed from an operon-like construct under the control of the A. baumannii OmpA promoter. PcrV was fused to the N-terminus of OprI as a fusion protein. The expression and location of each antigen in the outer membrane was confirmed by Western blot and ELISA (see Figure 7). Western blot analysis of whole cell lysates and outer membrane extracts with antigen-specific antibodies was also used to confirm the expression and localization of the heterologous antigens in A. baumannii cells.

[0116] Prior to LPS null mutant selection, A. baumannii cells were transformed with a suicide vector (pVXD-50::P-Vax) containing sequences to promote recombination at the cysI target locus into the A. baumannii genome for integration of the expression construct at the cysI locus. This procedure included the design of a suicide plasmid containing the expression construct flanked by homologous regions adjacent to the target gene, as well as a selection marker and a counterselection marker. The ORFs contained in the expression construct include sequences derived from the P. aeruginosa OMPs OprF and OprI, and the P. aeruginosa type 3 secretion apparatus protein PcrV. The oprI and pcrV coding sequences were combined into one coding sequence to generate a novel chimeric gene, oprI::pcrV. Both coding sequences were expressed from an operon-like construct under the control of the A. baumannii OmpA promoter. The expression construct contained the signal sequence of the A. baumannii outer membrane protein OmpA upstream of each coding sequence to facilitate localization of each antigen in the outer membrane of A. baumannii. The expression construct also contained the transcription termination sequence of the A. baumannii protein OmpA.

[0117] The pVXD-50::P-Vax plasmid was constructed by cloning an expression construct flanked by homologous regions adjacent to the Ab283 cysI gene (600 bp upstream and 600 bp downstream of the cysI gene). The suicide plasmid (a non-replicating plasmid in A. baumannii) carries the kanamycin resistance gene, which induces resistance to kanamycin as a selection marker, and the gene sacB as a counter selection marker.

[0118] The pVXD-50::P-Vax suicide plasmid was introduced into Ab283 cells by electroporation, conjugation or chemical transformation followed by two-step allelic exchange. The first recombination event resulted in the insertion of the vector when recombination occurred between the upstream region cloned into the plasmid and the upstream region of the Ab283 genome. Successfully recombined cells with the plasmid integrated into the genome were selected on kanamycin agar plates. Common techniques such as PCR and DNA sequencing were used to confirm whether integration of the plasmid into the target locus occurred as expected.

[0119] A second recombination event was induced by growing the selected kanamycin-resistant Ab283 cells in the presence of sucrose, resulting in recombinant cells in which the vector backbone upstream and downstream of the homologous region was removed and only the expression construct remained in the recombinant Ab283 cells. Such Ab283 cells in which a second recombination event had occurred were obtained by selecting recombinant Ab283 cells that were sucrose-resistant and kanamycin-sensitive. Once selected, common techniques such as PCR and DNA sequencing were used to confirm whether integration of the expression construct into the target locus had occurred as expected.

[0120] Preparation of a fourth drug substance, K1-Vax, by insertion of an expression construct at the cysI locus. The candidates include the OMP selected from K. pneumoniae OmpA under the control of the A. baumannii OmpA promoter. Expression and localization of K. pneumoniae OMP antigens at the outer membrane was confirmed by Western blot and ELISA. Western blot analysis of whole cell lysates and outer membrane extracts using antigen-specific antibodies was also used to confirm the expression and localization of heterologous antigens in A. baumannii cells.

[0121] To construct K1-Vax at the lpxA locus, Acinetobacter baumannii Ab283 cells were transformed with a suicide vector (pVXD-40::K1-Vax) containing sequences to promote recombination at the lpxA target locus into the A. baumannii genome. The LpxA protein is essential for the biosynthesis of LPS in A. baumannii.

[0122] This procedure involved the design of a suicide plasmid containing an expression construct flanked by homology regions adjacent to the target gene, as well as a selection marker and a counter selection marker. The ORF contained in the expression construct contains sequences derived from the K. pneumoniae outer membrane protein OmpA controlled by the A. baumannii OmpA promoter. The signal sequence of the A. baumannii outer membrane protein OmpA was used to promote the location of K. pneumoniae OmpA in the outer membrane of A. baumannii.

[0123] The pVXD-40::K1-Vax plasmid was constructed by cloning an expression construct flanked by homologous regions adjacent to the Ab283 lpxA gene (2000 bp upstream and 2000 bp downstream of the lpxA gene). The suicide plasmid (a non-replicating plasmid in A. baumannii) carries a tetracycline resistance cassette inducing resistance to tetracycline as a selection marker, and the gene sacB as a counter selection marker.

[0124] The pVXD-40::K1-Vax suicide plasmid was introduced into Ab283 cells by electroporation, conjugation or chemical transformation followed by two-step allelic exchange. The first recombination event results in the insertion of the vector when recombination occurs between the upstream region cloned into the plasmid and the upstream region of the Ab283 genome. Successfully recombined cells with the plasmid integrated into the genome are selected on tetracycline agar plates. Common techniques such as PCR and DNA sequencing are used to confirm whether integration of the plasmid into the target locus occurred as expected.

[0125] A second recombination event is induced by growing the selected tetracycline-resistant Ab283 cells in the presence of sucrose, resulting in recombinant cells in which the vector backbone upstream and downstream of the homologous region is removed and only the expression construct remains in the recombinant Aab283 cells. Such Ab283 cells in which a second recombination event has occurred can be obtained by selecting recombinant Ab283 cells that are sucrose-resistant and tetracycline-sensitive. Once selected, common techniques such as PCR and DNA sequencing are used to confirm whether integration of the expression construct into the target locus has occurred as expected.

[0126] The second recombination also resulted in the complete inactivation of lpxA, which is essential for the biosynthesis of LPS in A. baumannii. Thus, the second recombination directly led to recombinant LPS-null Ab283 cells. Therefore, a subsequent step of LPS-null mutant selection on colistin plates was not necessary.

[0127] The expression of the heterologous protein OmpA of K. pneumoniae encoded by the expression construct in recombinant Ab283 cells was analyzed by basic proteomic techniques such as Western blot to confirm its expression. The location of K. pneumoniae OmpA protein in the outer membrane of recombinant A. baumannii cells was analyzed by Western blot when samples were treated with proteinase K.

[0128] Preparation of the fifth drug substance, Eco1-Vax, by insertion of an expression construct at the cysI locus. For construction of Eco1-Vax at the cysI locus, the construct was inserted at the cysI locus in the Ab283 wild-type strain and expression and localization of the heterologous antigen at the outer membrane was demonstrated followed by plating on colistin to select for an LPS-negative mutant carrying a 1 Kb ISAba insertion in the lpxC ORF, followed by selection of an LPS-negative derivative.

[0129] This candidate Eco1-Vax contains two E. coli antigens (OmpA and OmpX). Each antigen is expressed from an operon-like construct under the control of the A. baumannii OmpA promoter. The expression and location of each antigen in the outer membrane was confirmed by basic proteomic techniques including Western blot and ELISA (see FIG. 9). Western blot analysis of whole cell lysates and outer membrane extracts using antigen-specific antibodies was also used to confirm the expression and localization of the heterologous antigens in A. baumannii cells.

[0130] Prior to LPS null mutant selection, A. baumannii cells were transformed with a suicide vector (pVXD-50::Eco1-Vax) containing sequences to facilitate recombination at the cysI target locus into the A. baumannii genome for integration of the expression construct at the cysI locus of A. baumannii Ab283. This procedure included the design of a suicide plasmid containing the expression construct flanked by homologous regions adjacent to the target gene, as well as a selection marker and a counter selection marker. The ORFs contained in the expression construct contain sequences from the E. coli OMPs OmpA and OmpX. Both coding sequences were expressed from an operon-like construct under the control of the A. baumannii OmpA promoter. The expression construct contained the signal sequence of the A. baumannii outer membrane protein OmpA upstream of each coding sequence to facilitate location of each antigen in the outer membrane of A. baumannii. The expression construct also contains the transcription termination sequence of the A. baumannii protein OmpA.

[0131] The pVXD-50::Eco1-Vax plasmid was constructed by cloning an expression construct flanked by homologous regions adjacent to the Ab283 cysI gene (600 bp upstream and 600 bp downstream of the cysI gene). The suicide plasmid (a non-replicating plasmid in A. baumannii) carries the kanamycin resistance gene, which induces resistance to kanamycin as a selection marker, and the gene sacB as a counter selection marker.

[0132] The pVXD-50::Eco1-Vax suicide plasmid was introduced into Ab283 cells by electroporation, conjugation or chemical transformation followed by two-step allelic exchange. The first recombination event resulted in the insertion of the vector when recombination occurred between the upstream region cloned into the plasmid and the upstream region of the Ab283 genome. Successfully recombined cells with the plasmid integrated into the genome were selected on kanamycin agar plates. Common techniques such as PCR and DNA sequencing were used to confirm whether integration of the plasmid into the target locus occurred as expected.

[0133] A second recombination event was induced by growing the selected kanamycin-resistant Ab283 cells in the presence of sucrose, resulting in recombinant cells in which the vector backbone upstream and downstream of the homologous region was removed and only the expression construct remained in the recombinant Ab283 cells. Such Ab283 cells in which a second recombination event had occurred were obtained by selecting recombinant Ab283 cells that were sucrose-resistant and kanamycin-sensitive. Once selected, common techniques such as PCR and DNA sequencing were used to confirm whether integration of the expression construct into the target locus had occurred as expected (see Figure 5).

[0134] Preparation of the sixth drug substance, Eco2-Vax, by insertion of an expression construct at the cysI locus. For construction of Eco2-Vax at the cysI locus, the construct was inserted at the cysI locus in the Ab283 wild-type strain and expression and localization of the heterologous antigen at the outer membrane was demonstrated followed by plating on colistin to select for an LPS-negative mutant carrying a 1 Kb ISAba insertion in the lpxC ORF, followed by selection of an LPS-negative derivative.

[0135] This candidate Eco2-Vax contains three E. coli antigens (FuyA, HmA, IutA). Each antigen is expressed from an operon-like construct under the control of the A. baumannii OmpA promoter. The expression and location of each antigen in the outer membrane was confirmed by basic proteomic techniques including Western blot and ELISA (see FIG. 9). Western blot analysis of whole cell lysates and outer membrane extracts using antigen-specific antibodies was also used to confirm the expression and localization of the heterologous antigens in A. baumannii cells.

[0136] Prior to LPS null mutant selection, A. baumannii cells were transformed with a suicide vector (pVXD-50::Eco2-Vax) containing sequences to facilitate recombination at the cysI target locus into the A. baumannii genome for integration of the expression construct at the cysI locus of A. baumannii Ab283. This procedure included the design of a suicide plasmid containing the expression construct flanked by homologous regions adjacent to the target gene, as well as a selection marker and a counterselection marker. The ORFs contained in the expression construct include sequences derived from the E. coli OMPs FuyA, HmA, and IutA. These three coding sequences were expressed from an operon-like construct under the control of the A. baumannii OmpA promoter. The expression construct contained the signal sequence of the A. baumannii outer membrane protein OmpA upstream of each coding sequence to facilitate location of each antigen in the outer membrane of A. baumannii. The expression construct also contains the transcription termination sequence of the A. baumannii protein OmpA.

[0137] The pVXD-50::Eco2-Vax plasmid was constructed by cloning an expression construct flanked by homologous regions adjacent to the Ab283 cysI gene (600 bp upstream and 600 bp downstream of the cysI gene). The suicide plasmid (a non-replicating plasmid in A. baumannii) carries the kanamycin resistance gene, which induces resistance to kanamycin as a selection marker, and the gene sacB as a counter selection marker.

[0138] The pVXD-50::Eco2-Vax suicide plasmid was introduced into Ab283 cells by electroporation, conjugation or chemical transformation followed by two-step allelic exchange. The first recombination event resulted in the insertion of the vector when recombination occurred between the upstream region cloned into the plasmid and the upstream region of the Ab283 genome. Successfully recombined cells with the plasmid integrated into the genome were selected on kanamycin agar plates. Common techniques such as PCR and DNA sequencing were used to confirm whether integration of the plasmid into the target locus occurred as expected.

[0139] A second recombination event was induced by growing the selected kanamycin-resistant Ab283 cells in the presence of sucrose, resulting in recombinant cells in which the vector backbone upstream and downstream of the homologous region was removed and only the expression construct remained in the recombinant Ab283 cells. Such Ab283 cells in which a second recombination event had occurred were obtained by selecting recombinant Ab283 cells that were sucrose-resistant and kanamycin-sensitive. Once selected, common techniques such as PCR and DNA sequencing were used to confirm whether integration of the expression construct into the target locus had occurred as expected.

[0140] Preparation of the seventh drug substance Eco3-Vax by insertion of an expression construct at the cysI locus. For construction of Eco3-Vax at the cysI locus, the construct was inserted at the cysI locus in the Ab283 wild-type strain and expression and localization of the heterologous antigen at the outer membrane was demonstrated followed by plating on colistin to select for an LPS-negative mutant carrying a 1 Kb ISAba insertion in the lpxC ORF, followed by selection of an LPS-negative derivative.

[0141] This candidate Eco3-Vax contains three E. coli antigens (FuyA, HmA, OmpA). Each antigen is expressed from an operon-like construct under the control of the A. baumannii OmpA promoter. The expression and location of each antigen in the outer membrane was confirmed by Western blot and ELISA. Western blot analysis of whole cell lysates and outer membrane extracts using antigen-specific antibodies was also used to confirm the expression and localization of the heterologous antigens in A. baumannii cells.

[0142] Prior to LPS null mutant selection, A. baumannii cells were transformed with a suicide vector (pVXD-50::Eco3-Vax) containing sequences to facilitate recombination at the cysI target locus into the A. baumannii genome for integration of the expression construct at the cysI locus of A. baumannii Ab283. This procedure included the design of a suicide plasmid containing the expression construct flanked by homologous regions adjacent to the target gene, as well as a selection marker and a counter selection marker. The ORFs contained in the expression construct include sequences from the E. coli OMPs FuyA, HmA, and OmpA. These three coding sequences were expressed from an operon-like construct under the control of the A. baumannii OmpA promoter. The expression construct contained the signal sequence of the A. baumannii outer membrane protein OmpA upstream of each coding sequence to facilitate location of each antigen in the outer membrane of A. baumannii. The expression construct also contains the transcription termination sequence of the A. baumannii protein OmpA.

[0143] The pVXD-50::Eco3-Vax plasmid was constructed by cloning an expression construct flanked by homologous regions adjacent to the Ab283 cysI gene (600 bp upstream and 600 bp downstream of the cysI gene). The suicide plasmid (a non-replicating plasmid in A. baumannii) carries the kanamycin resistance gene, which induces resistance to kanamycin as a selection marker, and the gene sacB as a counter selection marker.

[0144] The pVXD-50::Eco3-Vax suicide plasmid was introduced into Ab283 cells by electroporation, conjugation or chemical transformation followed by two-step allelic exchange. The first recombination event resulted in the insertion of the vector when recombination occurred between the upstream region cloned into the plasmid and the upstream region of the Ab283 genome. Successfully recombined cells with the plasmid integrated into the genome were selected on kanamycin agar plates. Common techniques such as PCR and DNA sequencing were used to confirm whether integration of the plasmid into the target locus occurred as expected.

[0145] A second recombination event was induced by growing the selected kanamycin-resistant Ab283 cells in the presence of sucrose, resulting in recombinant cells in which the vector backbone upstream and downstream of the homologous region was removed and only the expression construct remained in the recombinant Ab283 cells. Such Ab283 cells in which a second recombination event had occurred were obtained by selecting recombinant Ab283 cells that were sucrose-resistant and kanamycin-sensitive. Once selected, common techniques such as PCR and DNA sequencing were used to confirm whether integration of the expression construct into the target locus had occurred as expected.

[0146] Preparation of the eighth drug substance, Appe1-Vax, by insertion of an expression construct at the cysI locus. For construction of Appe1-Vax at the cysI locus, the construct was inserted at the cysI locus in the Ab283 wild-type strain and expression and localization of the heterologous antigen at the outer membrane was demonstrated followed by plating on colistin to select for an LPS-negative mutant carrying a 1 Kb ISAba insertion in the lpxC ORF, followed by selection of an LPS-negative derivative.

[0147] This candidate Appe1-Vax contains one Actinobacillus pleuropneumoniae antigen (TpbA). OMP antigens are expressed from an operon-like construct under the control of the A. baumannii OmpA promoter. Expression and localization of OMP antigens at the outer membrane was confirmed by Western blot and ELISA. Western blot analysis of whole cell lysates and outer membrane extracts with antigen-specific antibodies was also used to confirm the expression and localization of heterologous antigens in A. baumannii cells.

[0148] Prior to LPS null mutant selection, A. baumannii cells were transformed with a suicide vector (pVXD-50::Appel-Vax) containing sequences to facilitate recombination at the cysI target locus into the A. baumannii genome for integration of the expression construct at the cysI locus of A. baumannii Ab283. This procedure included the design of a suicide plasmid containing the expression construct flanked by homologous regions adjacent to the target gene, as well as a selection marker and a counter selection marker. The ORF contained in the expression construct contains sequences derived from the A. pleuropneumoniae OMP, TpbA. The coding sequences were expressed from an operon-like construct under the control of the A. baumannii OmpA promoter. The expression construct contained the signal sequence of the A. baumannii outer membrane protein OmpA upstream of each coding sequence to facilitate location of each antigen in the outer membrane of A. baumannii. The expression construct also contains a transcription termination sequence for the A. baumannii protein OmpA.

[0149] The pVXD-50::Appe1-Vax plasmid was constructed by cloning an expression construct flanked by homologous regions adjacent to the Ab283 cysI gene (600 bp upstream and 600 bp downstream of the cysI gene). The suicide plasmid (a non-replicating plasmid in A. baumannii) carries the kanamycin resistance gene, which induces resistance to kanamycin as a selection marker, and the gene sacB as a counter selection marker.

[0150] The pVXD-50::Appe1-Vax suicide plasmid was introduced into Ab283 cells by electroporation, conjugation or chemical transformation followed by two-step allelic exchange. The first recombination event resulted in the insertion of the vector when recombination occurred between the upstream region cloned into the plasmid and the upstream region of the Ab283 genome. Successfully recombined cells with the plasmid integrated into the genome were selected on kanamycin agar plates. Common techniques such as PCR and DNA sequencing were used to confirm whether integration of the plasmid into the target locus occurred as expected.

[0151] A second recombination event was induced by growing the selected kanamycin-resistant Ab283 cells in the presence of sucrose, resulting in recombinant cells in which the vector backbone upstream and downstream of the homologous region was removed and only the expression construct remained in the recombinant Ab283 cells. Such Ab283 cells in which a second recombination event had occurred were obtained by selecting recombinant Ab283 cells that were sucrose-resistant and kanamycin-sensitive. Once selected, common techniques such as PCR and DNA sequencing were used to confirm whether integration of the expression construct into the target locus had occurred as expected.

[0152] Preparation of the ninth drug substance, Appe2-Vax, by insertion of an expression construct at the cysI locus. For construction of Appe2-Vax at the cysI locus, the construct was inserted at the cysI locus in the Ab283 wild-type strain and expression and localization of the heterologous antigen at the outer membrane was demonstrated followed by plating on colistin to select for an LPS-negative mutant carrying a 1 Kb ISAba insertion in the lpxC ORF, followed by selection of an LPS-negative derivative.

[0153] This candidate Appe2-Vax contains one Actinobacillus pleuropneumoniae antigen (ApfA). The OMP antigens are expressed from an operon-like construct under the control of the A. baumannii OmpA promoter. The expression and localization of each antigen in the outer membrane was confirmed by Western blot and ELISA. Western blot analysis of whole cell lysates and outer membrane extracts with antigen-specific antibodies was also used to confirm the expression and localization of the heterologous antigens in A. baumannii cells.

[0154] Prior to LPS null mutant selection, A. baumannii cells were transformed with a suicide vector (pVXD-50::Appe2-Vax) containing sequences to facilitate recombination at the cysI target locus into the A. baumannii genome for integration of the expression construct at the cysI locus of A. baumannii Ab283. This procedure included the design of a suicide plasmid containing the expression construct flanked by homologous regions adjacent to the target gene, as well as a selection marker and a counter selection marker. The ORF contained in the expression construct contains sequences derived from ApfA, an OMP of A. pleuropneumoniae. The coding sequences were expressed from an operon-like construct under the control of the A. baumannii OmpA promoter. The expression construct contained the signal sequence of the A. baumannii outer membrane protein OmpA upstream of each coding sequence to facilitate location of each antigen in the outer membrane of A. baumannii. The expression construct also contains a transcription termination sequence of the A. baumannii protein OmpA.

[0155] The pVXD-50::Appe2-Vax plasmid was constructed by cloning an expression construct flanked by homologous regions adjacent to the Ab283 cysI gene (600 bp upstream and 600 bp downstream of the cysI gene). The suicide plasmid (a non-replicating plasmid in A. baumannii) carries the kanamycin resistance gene, which induces resistance to kanamycin as a selection marker, and the gene sacB as a counter selection marker.

[0156] The pVXD-50::Appe2-Vax suicide plasmid was introduced into Ab283 cells by electroporation, conjugation or chemical transformation followed by two-step allelic exchange. The first recombination event resulted in the insertion of the vector when recombination occurred between the upstream region cloned into the plasmid and the upstream region of the Ab283 genome. Successfully recombined cells with the plasmid integrated into the genome were selected on kanamycin agar plates. Common techniques such as PCR and DNA sequencing were used to confirm whether integration of the plasmid into the target locus occurred as expected.

[0157] A second recombination event was induced by growing the selected kanamycin-resistant Ab283 cells in the presence of sucrose, resulting in recombinant cells in which the vector backbone upstream and downstream of the homologous region was removed and only the expression construct remained in the recombinant Ab283 cells. Such Ab283 cells in which a second recombination event had occurred were obtained by selecting recombinant Ab283 cells that were sucrose-resistant and kanamycin-sensitive. Once selected, common techniques such as PCR and DNA sequencing were used to confirm whether integration of the expression construct into the target locus had occurred as expected.

[0158] Preparation of the tenth drug substance, Appe3-Vax, by insertion of an expression construct at the cysI locus. For construction of Appe3-Vax at the cysI locus, the construct was inserted at the cysI locus in the Ab283 wild-type strain and expression and localization of the heterologous antigen at the outer membrane was demonstrated followed by plating on colistin to select for an LPS-negative mutant carrying a 1 Kb ISAba insertion in the lpxC ORF, followed by selection of an LPS-negative derivative.

[0159] This candidate Appe3-Vax contains one Actinobacillus pleuropneumoniae antigen (OmpA). The OMP antigens are expressed from an operon-like construct under the control of the A. baumannii OmpA promoter. The expression and location of each antigen in the outer membrane was confirmed by basic proteomic techniques including Western blot and ELISA (see Figure 9). Western blot analysis of whole cell lysates and outer membrane extracts using antigen-specific antibodies was also used to confirm the expression and localization of the heterologous antigens in A. baumannii cells.

[0160] Prior to LPS null mutant selection, A. baumannii cells were transformed with a suicide vector (pVXD-50::Appe3-Vax) containing sequences to facilitate recombination at the cysI target locus into the A. baumannii genome for integration of the expression construct at the cysI locus of A. baumannii Ab283. This procedure included the design of a suicide plasmid containing the expression construct flanked by homologous regions adjacent to the target gene, as well as a selection marker and a counter selection marker. The ORFs contained in the expression construct contain sequences derived from OmpA, the OMP of A. pleuropneumoniae. The coding sequences were expressed from an operon-like construct under the control of the A. baumannii OmpA promoter. The expression construct contained the signal sequence of the A. baumannii outer membrane protein OmpA upstream of each coding sequence to facilitate location of each antigen in the outer membrane of A. baumannii. The expression construct also contains a transcription termination sequence of the A. baumannii protein OmpA.

[0161] The pVXD-50::Appe3-Vax plasmid was constructed by cloning an expression construct flanked by homologous regions adjacent to the Ab283 cysI gene (600 bp upstream and 600 bp downstream of the cysI gene). The suicide plasmid (a non-replicating plasmid in A. baumannii) carries the kanamycin resistance gene, which induces resistance to kanamycin as a selection marker, and the gene sacB as a counter selection marker.

[0162] The pVXD-50::Appe3-Vax suicide plasmid was introduced into Ab283 cells by electroporation, conjugation or chemical transformation followed by two-step allelic exchange. The first recombination event resulted in the insertion of the vector when recombination occurred between the upstream region cloned into the plasmid and the upstream region of the Ab283 genome. Successfully recombined cells with the plasmid integrated into the genome were selected on kanamycin agar plates. Common techniques such as PCR and DNA sequencing were used to confirm whether integration of the plasmid into the target locus occurred as expected.

[0163] A second recombination event was induced by growing the selected kanamycin-resistant Ab283 cells in the presence of sucrose, resulting in recombinant cells in which the vector backbone upstream and downstream of the homologous region was removed and only the expression construct remained in the recombinant Ab283 cells. Such Ab283 cells in which a second recombination event had occurred were obtained by selecting recombinant Ab283 cells that were sucrose-resistant and kanamycin-sensitive. Once selected, common techniques such as PCR and DNA sequencing were used to confirm whether integration of the expression construct into the target locus had occurred as expected.

[0164] Preparation of the eleventh drug substance, Appe4-Vax, by insertion of an expression construct at the cysI locus. For construction of Appe4-Vax at the cysI locus, the construct was inserted at the cysI locus in the Ab283 wild-type strain and expression and localization of the heterologous antigen at the outer membrane was demonstrated followed by plating on colistin to select for an LPS-negative mutant carrying a 1 Kb ISAba insertion in the lpxC ORF, followed by selection of an LPS-negative derivative.

[0165] This candidate Appe4-Vax contains one Actinobacillus pleuropneumoniae antigen (OmpW). The OMP antigens are expressed from an operon-like construct under the control of the A. baumannii OmpA promoter. The expression and localization of each antigen in the outer membrane was confirmed by Western blot and ELISA. Western blot analysis of whole cell lysates and outer membrane extracts with antigen-specific antibodies was also used to confirm the expression and localization of the heterologous antigens in A. baumannii cells.

[0166] Prior to LPS null mutant selection, A. baumannii cells were transformed with a suicide vector (pVXD-50::Appe4-Vax) containing sequences to facilitate recombination at the cysI target locus into the A. baumannii genome for integration of the expression construct at the cysI locus of A. baumannii Ab283. This procedure included the design of a suicide plasmid containing the expression construct flanked by homologous regions adjacent to the target gene, as well as a selection marker and a counter selection marker. The ORFs contained in the expression construct contain sequences derived from OmpW, an OMP of A. pleuropneumoniae. The coding sequences were expressed from an operon-like construct under the control of the A. baumannii OmpA promoter. The expression construct contained the signal sequence of the A. baumannii outer membrane protein OmpA upstream of each coding sequence to facilitate location of each antigen in the outer membrane of A. baumannii. The expression construct also contains a transcription termination sequence of the A. baumannii protein OmpA.

[0167] The pVXD-50::Appe4-Vax plasmid was constructed by cloning an expression construct flanked by homologous regions adjacent to the Ab283 cysI gene (600 bp upstream and 600 bp downstream of the cysI gene). The suicide plasmid (a non-replicating plasmid in A. baumannii) carries the kanamycin resistance gene, which induces resistance to kanamycin as a selection marker, and the gene sacB as a counter selection marker.

[0168] The pVXD-50::Appe4-Vax suicide plasmid was introduced into Ab283 cells by electroporation, conjugation or chemical transformation followed by two-step allelic exchange. The first recombination event resulted in the insertion of the vector when recombination occurred between the upstream region cloned into the plasmid and the upstream region of the Ab283 genome. Successfully recombined cells with the plasmid integrated into the genome were selected on kanamycin agar plates. Common techniques such as PCR and DNA sequencing were used to confirm whether integration of the plasmid into the target locus occurred as expected.

[0169] A second recombination event was induced by growing the selected kanamycin-resistant Ab283 cells in the presence of sucrose, resulting in recombinant cells in which the vector backbone upstream and downstream of the homologous region was removed and only the expression construct remained in the recombinant Ab283 cells. Such Ab283 cells in which a second recombination event had occurred were obtained by selecting recombinant Ab283 cells that were sucrose-resistant and kanamycin-sensitive. Once selected, common techniques such as PCR and DNA sequencing were used to confirm whether integration of the expression construct into the target locus had occurred as expected.

[0170] Example 2. Protocol for OMP sensitivity to proteinase K Objective: To examine the location of heterologous OMP antigens of other bacterial pathogens in the outer membrane of LPS-deficient Acinetobacter baumannii.

[0171] Brief description: A. baumannii LPS-whole cells expressing OMP antigens from other bacterial pathogens or parental LPS-forms of A. baumannii strains not expressing OMP antigens are treated with proteinase K. Membrane preparations of treated or untreated samples (control) are run on a 12% acrylamide gel. Subsequently, Western blots are performed using polyclonal or monoclonal antibodies raised against specific heterologous OMP proteins. Proteins located at the membrane surface are degraded by proteinase K treatment. This part of the protocol is also performed on whole cell lysates and the results show that polyclonal antibodies recognize cytoplasmic proteins with cross-reactivity in both proteinase K-treated and untreated samples. On the other hand, OMPs expected to be located in the outer membrane are only detected in those untreated samples.

[0172] Methodology: Proteinase K treatment: 1. Grow a pre-inoculum from a single colony overnight at 30° C. with shaking. 2. The culture is diluted to an OD of 0.05 and then grown at 30° C. with shaking to an OD of 2. Concentrate 3.25 ml of culture by centrifugation and discard the supernatant. 4. Resuspend the pellet in 10 ml of PBS containing 250 μg / ml proteinase K in the treated sample. 5. Incubate at 37°C with shaking for 2 hours. 6. Concentrate the pellet by centrifugation and then resuspend in 10 ml of PBS or PBS + 3 mM PMFS (protease inhibitor) to stop the reaction in samples treated with Proteinase K. 7. Incubate at room temperature for 10 minutes. membrane preparation 8. Sonicate as follows: 10 seconds at 20% amplitude, 30 seconds pause. Repeat this cycle 7 times. 9. Ultracentrifuge at 4000 rpm and 8°C for 1 hour. 10. Discard the supernatant and resuspend in 250 μl PBS Triton 1%. 11. Incubate overnight at 4°C for membrane solubilization. SDS-PAGE and WB 12. Sample preparation: 25 μl membrane prep + 25 μl loading buffer 4x + 50 μl milliQ water. Boil at 13.98°C for 20 minutes. 14. Load 10 μl onto a 12% acrylamide gel. 15. Operate at 170V for 1 hour. Wet transfer to nitrocellulose membrane for 1:30 h at 16.360 mA. 17. Ponceau staining for loading control. 18. Block with PBS tween 1% milk 5% for 1 hour. 19. Incubate overnight at 4°C with primary antibody at 1:5000 in PBS tween 1% milk 5%. 20. Wash the membrane with PBS tween 1%. Repeat washing two more times. 21. Incubate with secondary antibody at 1:5000 in PBS tween 1% milk 5% for 1 hour at room temperature. 22. Wash the membrane with PBS tween 1%. Repeat washing five more times. 23. Treat and visualize with chemiluminescence reagent for 5 minutes.

[0173] Examples of the final results with some of the drug substances delivered using the technology of the present invention are shown in Figures 6, 7, and 8. Using this technology, the outer membrane location of all of the heterologous antigens expressed for the drug substance construction described in Example 1 was confirmed.

[0174] Example 3. Efficacy of drug substances made in A. baumannii for protection against infections caused by other bacterial pathogens. Vaccine candidates were prepared from various drug substances by laboratory-scale fermentation of drug substance cells and subsequent heat inactivation.

[0175] In an in vivo proof-of-concept study, we employed a previously developed murine sepsis model, as described in Garcia-Quintanilla et al. 2014, to test the in vivo efficacy of our monovalent vaccine, AcinetoVax, against A. baumannii. Briefly, 6- to 8-week-old C57BL / 6 animals (8-12 animals / group) were immunized by IM injection of two doses of inactivated drug substance cells formulated with 0.83 mg / dose of aluminum hydroxide as an adjuvant. The doses were administered 2 weeks apart. Seven days after the second immunization, animals were challenged by intraperitoneal injection of an inoculum of a reference strain of the bacterial pathogen to be tested. The inoculum was equivalent to the previously characterized minimum lethal dose (MLD) for each strain in this model. We tested the in vivo efficacy in independent challenge experiments against each of the target pathogens. Animals were monitored for 7-12 days to determine survival. At the end of the termination or follow-up period, bacterial burdens were determined in the lungs, spleen and kidneys as a quantitative measure of bacterial spread, as well as in the blood to assess bacteremia associated with experimental sepsis. Separate groups of animals (8-10 animals / group) were sacrificed 12 (or 18) hours post-infection to assess bacterial burden in various organs, as well as serum levels of TNF-α, IL-1β and IL-6 to characterize the inflammatory response that occurred during infection in vaccinated versus non-vaccinated control animals.

[0176] The primary variable for the evaluation of the efficacy of each candidate was the survival rate in the post-challenge period in vaccinated mice compared to control mice vaccinated with the vaccine vehicle (adjuvant only). As a secondary variable, a comparison of protection between the drug substance "LPS-null Acinetobacter baumannii Ab283 with expression of heterologous OMP antigens on the outer membrane" and the drug substance "LPS-null Acinetobacter baumannii Ab283 carrier cells without heterologous OMP antigens on the outer membrane" was performed to evaluate the contribution of the heterologous OMP antigens to protection against the target pathogen.

[0177] An example of KapaVax protection against lethal sepsis caused by Klebsiella pneumoniae KapaVax2 (KapaVax) is made from LPS-null A. baumannii Ab283 cells expressing the OMP antigens OmpA and OmpK36 from K. pneumoniae, and the OMP antigen OprF from P. aeruginosa and the fusion protein OprI::PcrV.

[0178] As shown in Figure 10, mice vaccinated with KapaVax2 (KapaVax) were significantly protected against lethal sepsis caused by the highly virulent hyperencapsulated K. pneumoniae clinical isolate ATCC43816 compared to mice vaccinated with adjuvant alone (vehicle). Furthermore, mice vaccinated with carrier cells alone (LPS-null A. baumannii Ab283) showed partial cross-protection. Protection with KapaVax was significantly higher than that obtained with Ab283 LPS-, thus the presence of heterologous OMP antigens was necessary to increase protection against the target pathogen K. pneumoniae.

[0179] An example of KapaVax protection against lethal sepsis caused by Pseudomonas aeruginosa KapaVax2 (KapaVax) is made from LPS-null A. baumannii Ab283 cells expressing the OMP antigens OmpA and OmpK36 from K. pneumoniae, and the OMP antigen OprF from P. aeruginosa and the fusion protein OprI::PcrV.

[0180] As shown in FIG. 11, mice vaccinated with KapaVax2 (KapaVax) were significantly protected against lethal sepsis caused by P. aeruginosa clinical isolate PA14 compared to mice vaccinated with adjuvant alone (vehicle).

[0181] An example of K-Vax protection against lethal sepsis caused by Klebsiella pneumoniae K-Vax is made from LPS-null A. baumannii Ab283 cells that express the K. pneumoniae OMP antigens OmpA and OmpK36.

[0182] As shown in Figure 12, mice vaccinated with K-Vax (DS3) were significantly protected against lethal sepsis caused by the highly virulent, highly encapsulated K. pneumoniae clinical isolate ATCC43816 compared to mice vaccinated with adjuvant alone (vehicle). Furthermore, mice vaccinated with carrier cells alone (LPS-null A. baumannii Ab283) showed partial cross-protection. Protection with K-Vax was significantly higher than that obtained with Ab283 LPS-, thus the presence of heterologous OMP antigens was necessary to increase protection against the target pathogen K. pneumoniae.

[0183] An example of P-Vax protection against lethal sepsis caused by Pseudomonas aeruginosa P-Vax is produced from LPS-null A. baumannii Ab283 cells expressing the P. aeruginosa OMP antigen OprF and the fusion protein OprI::PcrV.

[0184] As shown in Figure 13, mice vaccinated with P-Vax (DS4) were significantly protected against lethal sepsis caused by the virulent P. aeruginosa clinical isolate PA14 compared to mice vaccinated with adjuvant alone (vehicle). Furthermore, mice vaccinated with carrier cells alone (LPS-null A. baumannii Ab283) showed partial cross-protection. Protection with P-Vax was significantly higher than that obtained with Ab283 LPS-, thus the presence of heterologous OMP antigens was necessary to increase protection against the target pathogen P. aeruginosa.

[0185] Example 4. Drug Substances Constructed in LPS-Null A. baumannii Expressing OMP Antigens from Other Bacterial Pathogens Preserved the Ability of LPS-Null A. baumannii Cells to Protect Against Infection Caused by A. baumannii

[0186] AcinetoVax Protection Against Fatal Sepsis Caused by Diverse A. baumannii Clinical Isolates Vaccination with VXD001 (AcinetoVax, a vaccine based on inactivated whole cells of an LPS-deficient A. baumannii strain featuring complete inactivation of lpxC as taught in EP 2942389 plus the adjuvant aluminum hydroxide) provided protection against lethal sepsis caused by diverse A. baumannii clinical isolates. Protection was supported by a rapid humoral response established after the first dose and by detected T cell-mediated responses (Th1, Th2, Th17) established after the first dose and significantly boosted after the second dose (see FIG. 2).

[0187] As shown in Figure 1, VXD001 (AcinetoVax) protects against infection with A. baumannii clinical isolates. The same sepsis model described in Example 3 was used. Mice were infected with the indicated strains of A. baumannii (ATCC19606, Ab-154, and Ab-113-16) 7 days after the second immunization with AcinetoVax (day 21) and survival was monitored for 7 days. ATCC19606 is a urinary infection clinical isolate used as a reference strain in sepsis and pneumonia models of infection in mice. Ab-154 is a carbapenem-susceptible clinical isolate from the 2002 outbreak at Virgen del Rocio Hospital in Seville. Ab-113-16 is a pan-resistant clinical isolate from a patient who died as a result of the 2002 outbreak.

[0188] KapaVax protection against lethal sepsis caused by A. baumannii AcinetoVax is made from LPS-null A. baumannii AB-001 cells. As mentioned above, the immunity raised by AcinetoVax can prevent infection with A. baumannii. With LPS removed from the outer membrane, the authors found that the immunodominant OMP proteins responsible for the protective immunity raised by AcinetoVax are A. baumannii proteins OmpA and Omp22. The authors also found by mass spectrometry of the outer membrane of AcinetoVax and other A. baumannii LPS-strains made in various genetic backgrounds that OmpA and Omp22 are the most abundant OMP proteins in the outer membrane. Once a drug substance based on LPS-A. baumannii cells expressing heterologous OMP antigens from other bacterial pathogens in the outer membrane was constructed, the question was whether the presence of heterologous OMP antigens would affect the immunity raised against A. baumannii.

[0189] As shown in Figure 14, the presence of OMP antigens OmpA and OmpK36 from K. pneumoniae and OMP antigens OprF and fusion OprI::PcrV from P. aeruginosa in drug substance KapaVax did not affect the ability of the vaccine to protect mice against lethal sepsis caused by A. baumannii compared to that of AcinetoVax as described above. This result was consistent with the observation that although constructed on a different A. baumannii strain (Ab283) than AcinetoVax (AB001), mass spectrometry of the outer membrane of KapaVax showed that A. baumannii OmpA and Omp22 were still the most abundant proteins in the outer membrane. The same results were found for all drug substances constructed as shown in Example 1.

[0190] Example 5. Immunogenicity analysis showed that vaccination with drug substances constructed in LPS-null A. baumannii expressing OMP antigens from other bacterial pathogens enhances specific immunity against other bacterial pathogens and also against A. baumannii

[0191] Examples of KapaVax that enhance specific immunity to K. pneumoniae challenge strains and K. pneumoniae antigens contained in KapaVax KapaVax2 (KapaVax) is made from LPS-null A. baumannii Ab283 cells expressing the OMP antigens OmpA and OmpK36 from K. pneumoniae, and the OMP antigen OprF from P. aeruginosa and the fusion protein OprI::PcrV.

[0192] Immunogenicity was determined by immunizing C57BL / 6 mice (8-10 mice / group) by IM injection with two doses on days 0 and 14. KapaVax candidates were adjuvanted with aluminum hydroxide.

[0193] The primary variables for the assessment of immunogenicity were vaccine cells, K. pneumoniae and P. aeruginosa (target pathogen) whole cells, A. baumannii whole cells and total IgG against each of the antigens measured by indirect ELISA. All assays included control mice immunized with vehicle only at the same time points.

[0194] As shown in Figures 15 and 16, KapaVax enhanced specific immunity against the challenge strain of K. pneumoniae ATCC 43816 as well as the K. pneumoniae antigens included in KapaVax, i.e., K. pneumoniae OMPs OmpA and OmpK36. The immunogenicity data supported the contribution and requirement of K. pneumoniae antigens for complete protection against K. pneumoniae ATCC 43186 as described in Example 3.

[0195] Examples of KapaVax that enhance specific immunity to P. aeruginosa challenge strains and K. pneumoniae antigens contained in KapaVax KapaVax2 (KapaVax) is made from LPS-null A. baumannii Ab283 cells expressing the OMP antigens OmpA and OmpK36 from K. pneumoniae, and the OMP antigen OprF from P. aeruginosa and the fusion protein OprI::PcrV.

[0196] Figure 17 shows IgG levels against P. aeruginosa cells. ELISA recognition of P. aeruginosa PA14 cells by antisera from animals immunized with KapaVax (N=5) or vehicle only (N=5) sampled on day 21 (day 7 after the second immunization). Statistical significance: (ns p>0.05, * p<0.05, ** p<0.005, *** p<0.001). The red dashed line indicates the limit of detection.

[0197] Figure 18 shows IgG levels against P. aeruginosa antigens OprF, OprI, and PcrV. ELISA recognition of recombinant proteins by antisera from animals immunized with two doses of KapaVax (N=4) or vehicle only (N=4), sampled on day 21 (day 7 after the second immunization). Statistical significance: (ns p>0.05, * p<0.05, ** p<0.005, *** p<0.001). The red dashed line indicates the limit of detection.

[0198] An example of KapaVax boosting specific immunity against A. baumannii KapaVax2 (KapaVax) is made from LPS-null A. baumannii Ab283 cells expressing the OMP antigens OmpA and OmpK36 from K. pneumoniae, and the OMP antigen OprF from P. aeruginosa and the fusion protein OprI::PcrV.

[0199] Figure 19 shows IgG levels against A. baumannii cells. ELISA recognition of A. baumannii ATCC19606 cells by antisera from animals immunized with two doses of KapaVax (N=9) or vehicle only (N=9), sampled on day 21 (day 7 after the second immunization). Boxes indicate IQR, horizontal lines and crosses indicate median and mean, respectively. Error bars extend to CI95%. Statistical analysis by unpaired two-tailed Mann-Whitney test (ns p>0.05, * p<0.05, ** p<0.005, *** p<0.001). The red dashed line indicates the limit of detection.

[0200] Example 6. Detection of a panel of global isolates by ELISA confirms global strain coverage One of the many sought features of the present invention was to overcome one of the major caveats of bacterial vaccines based on specific saccharide antigens, typically from LPS or capsular polysaccharides. These vaccines are the predominant type of vaccine on the market or in clinical development, and while they raise specific immunity against a limited number of bacterial strains (serotypes), others escape the vaccine-raised immunity. The technology of the present invention can raise immunity against a variety of bacterial OMP antigens, as shown extensively in the previous examples. OMPs are highly conserved proteins among all bacterial strains of a particular bacterial species, and even across bacterial species. The selection of vaccine alleles (see also SEQ ID NOs: 2-16, which are sequences of vaccine alleles, excluding the N-terminal signal peptide) for all drug substances shown in Example 1 was done based on the most prevalent and conserved OMP alleles among all circulating strains of each pathogen. However, the hypothesis is that the immunity raised by the vaccines made in the present invention will have high coverage among all the diversity of circulating strains for each pathogen, i.e., it will be universal. The following example, done with antisera produced by KapaVax, is presented to illustrate that the authors have obtained results that support this hypothesis: KapaVax2 (KapaVax) is made from LPS-null A. baumannii Ab283 cells expressing the OMP antigens OmpA and OmpK36 from K. pneumoniae, and the OMP antigen OprF and the fusion protein OprI::PcrV from P. aeruginosa.

[0201] Figure 20 shows that KapaVax2 antisera show specific recognition of 93% of a panel of 15 diverse global clinical isolates of K. pneumoniae (73% higher than or equal to the ELISA titers against challenge strain K. pneumoniae ATCC 43816). Additionally, KapaVax2 antisera show specific recognition of 100% of a panel of 15 diverse global clinical isolates of P. aeruginosa (93% higher than or equal to the ELISA titers against challenge strain PA14).

[0202] To confirm the universality of the vaccine antisera, information on the diversity of the strain panel used in Figure 20 is shown in Tables 2 and 3. The strain panel covers all major types of K. pneumoniae and P. aeruginosa clinical isolates currently circulating worldwide.

[0203] Figure 21 also shows that the universality shown by KapaVax antisera against K. pneumoniae and P. aeruginosa also applies to the third pathogen targeted by the multi-pathogen vaccine generated by applying the present invention, namely A. baumannii. Information on the diversity of the strain panel used in Figure 21 to confirm the universality of the vaccine antisera is provided in Table 4.

[0204] [Table 2]

[0205] Table 2. Collection of K. pneumoniae clinical isolates. The diagram shows the country of origin of the clinical isolates, sequence type, K locus (capsule) type and O type (O polysaccharide serotype) of each bacterial strain.

[0206] [Table 3]

[0207] Table 3. Collection of P. aeruginosa clinical isolates. The chart shows the country of origin of the clinical isolates, sequence type, and O type (O polysaccharide serotype) of each bacterial strain.

[0208] [Table 4]

[0209] Table 4. Collection of A. baumannii clinical isolates. The diagram shows the country of origin of the clinical isolates, sequence type, K locus (capsule) type and OC type of each bacterial strain.

[0210] Example 7. Exposure of A. baumannii immunodominant antigens OmpA and Omp22 on the outer membrane of KapaVax2 KapaVax2 (KapaVax) is made from LPS-null A. baumannii Ab283 cells expressing the K. pneumoniae OMP antigens OmpA and OmpK36, and the P. aeruginosa OMP antigen OprF and the fusion protein OprI::PcrV. The immunodominant A. baumannii OMPs OmpA and Omp22 are detected by Western blot from crude extracts made from vaccine batches of KapaVax.

[0211] FIG. 22 shows expression of A. baumannii antigens in KapaVax2 and AcinetoVax vaccine batches: total lysate preparations of the vaccine batches (2×10 10 Cells / ml). Samples were run on gels with a 4% to 16% acrylamide gradient. Panels show signals obtained with monoclonal antibodies raised against OmpA (left) and Omp22 (right).

[0212] Figure 23 shows the surface exposure of A. baumannii Omp22 on the outer membrane of KapaVax and carrier cells Ab283 LPS-. Cultures were washed after growth, resuspended in PBS and treated with 0.5 mg / ml proteinase K for 1 h at 37°C. Western blot with a monoclonal antibody raised against Ab-Omp22. Treatment with proteinase K is indicated by + (treated) or - (untreated) in each sample. Mb: outer membrane extract, S: extract supernatant. Protocol described in Example 2.

[0213] Example 8. Purification of outer membrane vesicles is also possible from LPS-null cells of A. baumannii and contains OMP heterologous antigens from other bacterial pathogens. As taught by the authors in EP 2942389 and shown herein, outer membrane vesicles (OMVs) can be purified from cultures of A. baumannii LPS null derivatives and can be used alternatively as drug substance instead of whole cells. The authors found that when heterologous OMP antigens from other bacterial pathogens are expressed on the outer membrane of LPS null A. baumannii cells, the ability to release OMVs is not impaired. The authors purified OMVs from the drug substance cells described in Example 1.

[0214] The protocol used for OMV purification is as follows: OMVs are obtained from 200 ml overnight cultures grown in a Mueller Hilton II at 30 °C with shaking at 150 rpm. After growth, the cultures are centrifuged at 10,000 × g for 15 min at 4 °C, the supernatant is saved and the pellet is discarded. This step is repeated three times. Concentration of OMVs is first performed by filtration using a 0.22 micron filter. The filtered OMVs are resuspended in 10-15 ml of solvent-resistant buffer. The OMVs are then ultracentrifuged at 100,000 × g for 6 h at 4 °C. The ultracentrifugation supernatant is carefully discarded and the pellet is resuspended in 2 ml of sterile PBS. Purification of OMVs is performed by size exclusion chromatography (SEC), fractions are collected and the presence of OMVs in each fraction is assessed by Western blot.

[0215] As an example, the results of OMV purification from DS3 (K-Vax) and carrier cells LPS-null A. baumannii Ab283 are shown in Figure 24. K-Vax are LPS-null A. baumannii Ab283 cells that express the K. pneumoniae OMPs OmpA and OmpK36 in their outer membrane.

[0216] References 1. Aguilera-Herce J, Garcia-Quintanilla M, Romero-Flores R, McConnell MJ, Ramos-Morales F. A live Salmonella vaccine delivering PcrV through the type III secretion system protects against Pseudomonas aeruginosa. mSphere 2019; 4:e00116-19. 2. Ali, S, Yu XQ, Robbie GJ, Shoemaker K. et al. Phase 1 study of MEDI3902, an investigational anti-Pseudomonas aeruginosa PcrV and Psl bispecific human monoclonal antibody, in healthy adults. Clin. Microbiol. Infect. 2019, 25(5):629.e1-629.e6 3. Antenucci F, Ovsepian A, Wrobel A, Winther-Larsen H, and Bojesen, A. Design and Characterization of a Novel Tool for the Antigenic Enrichment of Actinobacillus pleuropneumoniae Outer Membrane. Pathogens. 2020, 9: 1014. 4. Babu L, Uppalapati SR, Sripathy MH, Reddy PN. Evaluation of Recombinant Multi-Epitope Outer Membrane Protein-Based Klebsiella pneumoniae Subunit Vaccine in Mouse Model. Front. Microbiol. 2017; 8:1-12. 5. Carretero-Ledesma M, Garcia-Quintanilla M, Martin-Pena R, Pulido MR, Pachon J, McConnell MJ. Phenotypic changes associated with Colistin resistance due to Lipopolysaccharide loss in Acinetobacter baumannii. Virulence. 2018, 9:930-42. 6. Chen X, Shao Z, Wu L, He B, Yang H, Chen J, Jin E, Huang Q, Lei L, Xu J, Li, H, Zhang H, Wan, Y, Liu W, and Zhou R. Involvement of the Actinobacillus pleuropneumoniae ompW Gene in Confrontation of Environmental Pressure. Front. Vet Sci. 2022, 9: 846322. 7. Cote-Sierra J, Bredan A, Toldos CM, et al. Bacterial lipoprotein-based vaccines induce tumor necrosis factor-dependent type 1 protective immunity against Leishmania major. Infection and Immunity 2002;70(1):240-8. 8. Finke M, Duchene M, Eckhardt A, Domdey H. Protection against Experimental Pseudomonas aeruginosa Infection by Recombinant P. aeruginosa Lipoprotein I Expressed in Escherichia coli. Infection and Immunity. 1990;58(7):2241-4. 9. Finke M, Muth G, Reichhelm T, Thoma M, Duchene M et al. Protection of Immunosuppressed Mice against Infection with Pseudomonas aeruginosa by Recombinant P. aeruginosa Lipoprotein I and Lipoprotein I-Specific Monoclonal Antibodies. Infection and Immunity. 1991; 59:1251-1254. 10. Forsyth VS, Himpsl SD, Smith SN, Sarkissian CA, Mike LA, Stocki JA, Sintsova A, Alteri CJ, Mobley HLT. Optimization of an Experimental Vaccine To Prevent Escherichia coli Urinary Tract Infection. mBIO 2020 11(2):e00555-20. 11. Francois, B, Luyt CE, Dugard A, Wolff M. et al. Safety and pharmacokinetics of an anti-PcrV PEGylated monoclonal antibody fragment in mechanically ventilated patients colonized with Pseudomonas aeruginosa: a randomized, double-blind, placebo-controlled trial. Critical Care Medicine. 2012; 40, 2320-2326. 12. Garcia-Quintanilla M, Pulido MR, Pachon J, McConnell MJ. Immunization with lipopolysaccharide-deficient whole cells provides protective immunity in an experimental mouse model of Acinetobacter baumannii infection. PLoS One 2014 9(12):e114410. 13. Gartner T, Baeten M, Otieno S, Revets H, Baetselier P et al. Mucosal prime-boost vaccination for tuberculosis based on TLR triggering OprI lipoprotein from Pseudomonas aeruginosa fused to mycolyl-transferase Ag85A. Immunology letters. 2007; 111:26-35. 14. Gu H, Liao Y, Zhang J, Wang Y, Liu Z, Cheng P, Wang X, Zou Q, and Gu J. Rational Design and Evaluation of an Artificial Escherichia coli K1 Protein Vaccine Candidate Based on the Structure of OmpA. Front. Cell. Infect. Microbiol. 2018, 8:172. 15. Guilleland Jr, Parker MG, Matthews JM et al. Use of a Purified Outer Membrane Protein F (Porin) Preparation of Pseudomonas aeruginosa as a Protective Vaccine in Mice. Infection and Immunity. 1984;44:49-54. 16. Habibi, M, Reza M, Karam A, Bouzari S. Evaluation of prevalence, immunogenicity and efficacy of FyuA iron receptor in uropathogenic Escherichia coli isolates as a vaccine target against urinary tract infection. Microb. Pathog. 2017, 110:477-83. 17. Hagan EC and Mobley HLT. Heme acquisition is facilitated by a novel receptor Hma and required by uropathogenic Escherichia coli for kidney infection. Mol. Microbiol. 2009, 71: 79-91. 18. Hamaoka S, Naito Y, Katoh H, Shimizu M, Kinoshita M et al. Efficacy comparison of adjuvants in PcrV vaccine against Pseudomonas aeruginosa pneumonia. Microbiology and Immunology. 2017; 61:64-67. 19. Hancock V, Ferrieres L, Klemm P. The ferric yersiniabactin uptake receptor FyuA is required for efficient biofilm formation by urinary tract infectious Escherichia coli in human urine. Microbiology (Reading) 2008, 154:167-175. 20. Hassan R, El-naggar W, El-aziz AM, Saaban M, Kenawy HI, Ali YM. Immunization with outer membrane proteins (OprF and OprI) and flagellin B protects mice from pulmonary infection with mucoid and nonmucoid Pseudomonas aeruginosa. J. Microbiol. Immunol. Infect. 2018; 51(3):312-320. 21. Hussein KE, Bahey-el-din M, Sheweita SA. Immunization with the outer membrane proteins OmpK17 and OmpK36 elicits protection against Klebsiella pneumoniae in the murine infection model. Microbial Pathogenesis 2018; 119:12-8. 22. Juncker AS, Willenbrock H, von Heijne G., Brunak S, Nielsen H, and Krogh A. Prediction of lipoprotein signal peptides in Gram-negative bacteria. Protein Sci. 2003 12: 1652-62. 23. Koebnik R, Locher KP, and Van Gelder, P. Structure and function of bacterial outer membrane proteins: barrels in a nutshell. Mol. Microbiol. 2000 37:239-53. 24. Kurupati P, Ramachandran NP, Poh CL. Protective Efficacy of DNA Vaccines Encoding Outer Membrane Protein A and OmpK36 of Klebsiella pneumoniae in Mice. Clin. Vaccine Immunol. 2011;18(1):82-8. 25. Kurupati P, Teh BK, Kumarasinghe G, Poh CL. Identification of vaccine candidate antigens of an ESBL producing Klebsiella pneumoniae clinical strain by immunoproteome analysis. Proteomics. 2006; 836-44. 26. Maisnier-Patin K, Malissard M, Jeannin P, Haeuw JF, Corbiere JC, Hoeffel G, Gauchat JF, Nguyen T, Saez JM, and Delneste Y. The outer membrane protein X from Escherichia coli exhibits immune properties. Vaccine 2003, 21:3765-74. 27. Mellata, M, Mitchell NM, Schoedel F, Curtiss R, and Pierd GB. Novel vaccine antigen combinations elicit protective immune responses against Escherichia coli sepsis. Vaccine. 2016 34: 656-662. 28. Meynet E, Laurin D, Luc J, Camara B, Toussaint B, Le A. Killed but metabolically active Pseudomonas aeruginosa-based vaccine induces protective humoral- and cell-mediated immunity against Pseudomonas aeruginosa pulmonary infections. Vaccine. 2018; 36(14):1893-1900. 29. Milla CE, Chmiel JF, Accurso FJ, VanDevanter DR, Konstan MW, Yarranton G, et al. Anti-PcrV antibody in cystic fibrosis: a novel approach targeting Pseudomonas aeruginosa airway infection. Pediatric Pulmonology 2014; 49:650-8. 30. Moffatt JH, Harper M, Harrison P, Hale JDF, Vinogradov E, Seemann T, Henry R, Crane B, St Michael F, Cox AD, Adler B, Nation RL, Li J, and Boyce JD. Colistin resistance in Acinetobacter baumannii is mediated by complete loss of lipopolysaccharide production. Antimicrob Agents Chemother. 2010, 54(12):4971-7. 31. Moriyama K, Wiener-Kronish JP, Sawa T. Protective effects of affinity-purified antibody and truncated vaccines against Pseudomonas aeruginosa V-antigen in neutropenic mice. Microbiol. Immunol. 2009; 53: 587-594. 32. Pulido MR, Garcia-Quintanilla M, Pachon J, McConnell MJ. Immunization with lipopolysaccharide-free outer membrane complexes protects against Acinetobacter baumannii infection. Vaccine 2018, 36:4153-56. 33. Rau H, Revets H, Cornelis P, Titzmann A, Ruggli N et al. Efficacy and functionality of lipoprotein OprI from Pseudomonas aeruginosa as adjuvant for a subunit vaccine against classical swine fever. Vaccine. 2006;24:4757-4768. 34. Sawa T, Ito E, Nguyen VH, Haight M. Anti-PcrV antibody strategies against virulent Pseudomonas aeruginosa. Hum. Vaccin. Immunother. 2014; 10:2843-52. 35. Sawa T, Yahr TL, Ohara M, Kurahashi K, Gropper MA, Wiener-Kronish JP, Frank DW. Active and passive immunization with the Pseudomonas V antigen protects against type III intoxication and lung injury. Nat. Med. 1999; 5:392-398. 36. Smithers L, Olatunji S, and Caffrey M. Bacterial Lipoprotein Posttranslational Modifications. New Insights and Opportunities for Antibiotic and Vaccine Development. Front. Microbiol 2021 12:788445. 37. Vogt J, and Schulz GE. The structure of the outer membrane protein OmpX from Escherichia coli reveals possible mechanisms of virulence. Structure 1999, 7:1301-9. 38. von Specht BU, Licking HC, Schmitt A, Hungerert KD, Domdey H. Safety and immunogenicity of a Pseudomonas aeruginosa outer membrane protein I vaccine in human volunteers. Vaccine. 1996;14(12):1111-7. 39. Wang Y. The function of OmpA in Escherichia coli. Biochem. Biophys. Res. Commun. 2002, 292:396-401. 40. Zhang Y, Tennent JM, Ingham A, Beddome G, Prideaux C, and Michalski WP. Identification of type 4 fimbriae in Actinobacillus pleuropneumoniae. FEMS Microbiol. Lett. 2000, 189: 15-18.

Claims

1. a lipopolysaccharide (LPS)-deficient A. baumannii strain characterized by partial or complete inactivation of one or various cellular nucleic acid molecules encoding endogenous LPS biosynthetic genes, wherein said lipopolysaccharide (LPS)-deficient A. baumannii strain is characterized by partial or complete inactivation of a gene selected from the group consisting of lpxA, lpxB, lpxC, lpxD, lpxK, lpxL and / or lpxM, and wherein said lipopolysaccharide (LPS)-deficient A. baumannii strain expresses one or more heterologous antigens or proteins having targeting sites in the outer membrane; and / or b. Outer membrane vesicles (OMVs) derived from the lipopolysaccharide (LPS)-deficient A. baumannii strain described in item a) above; A composition comprising: the one or more heterologous antigens having target sites in the outer membrane I. at the N-terminus of the one or more heterologous antigens, a signal sequence derived from an outer membrane protein (OMP) that is processed by A. baumannii to direct the location of the expressed protein to the outer membrane of A. baumannii; II. A. baumannii, an OMP transmembrane domain unique to an endogenous OMP protein or a bacterial lipoprotein, which allows insertion of the expressed protein at the outer membrane of B. baumannii, III. An immunogenic domain; The method according to claim 1, the strain has been modified to express the one or more heterologous antigens having target sites in the outer membrane by insertion of an expression construct comprising at least one or more transcription promoter sequences, one or more ORFs (open reading frames) encoding the one or more heterologous antigens, and one or more transcription termination sequences at a locus in the A. baumannii chromosome capable of integrating inserts by recombination selected from the group consisting of cysI, trpE, lpxA, lpxC, lpxD, lpxB, lpxK, lpxL, lpxM, and / or Tn5 / Tn7 sites; the one or more heterologous antigens having a target site in the outer membrane are outer membrane proteins (OMPs) from Gram-negative bacteria; The composition, wherein the signal sequence is the outer membrane protein OmpA of A. baumannii (SEQ ID NO: 1) and the promoter is the A. baumannii OmpA promoter.

2. 2. The composition of claim 1, wherein the locus in the A. baumannii chromosome into which an insert can be integrated by recombination is cysI.

3. 3. The composition of claim 1 or 2, wherein the heterologous antigen or protein expressed in the outer membrane of the lipopolysaccharide (LPS)-deficient A. baumannii strain is derived from at least K. pneumoniae, P. aeruginosa, E. coli, and / or A. pleuropneumoniae.

4. 2. The composition of claim 1, wherein the heterologous antigen or protein expressed in the outer membrane of the lipopolysaccharide (LPS)-deficient A. baumannii strain is at least derived from K. pneumoniae and is selected from the group consisting of Kp-OmpA (SEQ ID NO: 31 or 17) and Kp-Ompk36 (SEQ ID NO: 32 or 18), or any sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the full / length sequence of any of SEQ ID NOs: 31, 32, 17, or 18, and when expressed or exposed in one or more copies in the outer membrane of A. baumannii by using a signal sequence that can be processed by A. baumannii cells to facilitate the location of the expressed protein in the outer membrane of A. baumannii, when inoculated into a subject in need thereof, results in immunization against not only A. baumannii infections but also infections caused by K. pneumoniae.

5. the heterologous antigen expressed in the outer membrane of the lipopolysaccharide (LPS)-deficient Acinetobacter baumannii strain is at least derived from P. aeruginosa and is selected from the group consisting of Pa-OprF (SEQ ID NO: 33 or 19), Pa-OprI (SEQ ID NO: 35 or 21), Pa-PcrV (SEQ ID NO: 45), or Pa-OprI:PcrV (SEQ ID NO: 34 or 20), or any sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the full / length sequence of any of SEQ ID NOs: 33-35, 19-21, or 45, and is expressed by using a signal sequence capable of being processed by A. baumannii cells to promote the location of the expressed protein in the outer membrane of A. baumannii.

10. The composition of claim 1, which, when expressed or exposed in one or more copies in the outer membrane of P. baumannii, when inoculated into a subject in need thereof, immunizes against not only A. baumannii infections but also infections caused by P. aeruginosa.

6. the heterologous antigen expressed in the outer membrane of the lipopolysaccharide (LPS)-deficient Acinetobacter baumannii strain is at least derived from E. coli and is selected from the group consisting of Ec-OmpA (SEQ ID NO: 36 or 22), Ec-OmpX (SEQ ID NO: 37 or 23), Ec-FuyA (SEQ ID NO: 38 or 24), Ec-Hma (SEQ ID NO: 39 or 25), or Ec-IutA (SEQ ID NO: 40 or 26), comprising any sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the full / length sequence of any of SEQ ID NOs: 22-26 or 36-40, and by using a signal sequence capable of being processed by A. baumannii cells to promote the location of the expressed protein in the outer membrane of A. baumannii; 10. The composition of claim 1, which, when expressed or exposed in one or more copies in the outer membrane of E. baumannii, when inoculated into a subject in need thereof, immunizes against not only A. baumannii infections but also infections caused by E. coli.

7. the heterologous antigen expressed in the outer membrane of the lipopolysaccharide (LPS)-deficient Acinetobacter baumannii strain is at least derived from A. pleuropneumoniae and is selected from the group consisting of Ap-OmpA (SEQ ID NO: 27 or 41), Ap-OmpW (SEQ ID NO: 28 or 42), Ap-TbpA (SEQ ID NO: 29 or 43), or Ap-ApfA (SEQ ID NO: 30 or 44), comprising any sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the full / length sequence of any of SEQ ID NOs: 27-30 or 41-44, and by using a signal sequence capable of being processed by A. baumannii cells to promote the location of the expressed protein in the outer membrane of A. baumannii; 10. The composition of claim 1, which, when expressed or exposed in one or more copies in the outer membrane of A. baumannii, when inoculated into a subject in need thereof, immunizes against not only A. baumannii infections but also infections caused by A. pleuropneumoniae.

8. The heterologous antigen expressed in the outer membrane of the lipopolysaccharide (LPS)-deficient A. baumannii strain is at least derived from K. pneumoniae and selected from the group consisting of Kp-OmpA and / or Kp-Ompk36, and / or any of the identical sequences thereof according to claim 4, and / or derived from P. aeruginosa and selected from the group consisting of Pa-OprF and / or Pa-OprI, and / or the fusion protein Pa-OprI::PcrV, and / or any of the identical sequences thereof according to claim 5, and / or derived from E. coli and selected from the group consisting of Ec-OmpA and / or Ec-OmpX and / or Ec-FuyA and / or Ec-Hma and / or Ec-IutA, and / or any of the identical sequences thereof according to claim 6, and / or derived from A.

3. The composition of claim 1 or 2, which is derived from Saccharomyces pleuropneumoniae and is selected from the list of Ap-OmpA and / or Ap-OmpW and / or Ap-TbpA and / or Ap-ApfA, and / or any identical sequences thereof as set forth in claim 7.

9. 8. The composition of any one of claims 1, 2, and 4-7, wherein the lipopolysaccharide (LPS)-deficient A. baumannii strain further comprises expression of the A. baumannii antigens Ab-OmpA and Ab-Omp22.

10. 8. The composition of any one of claims 1, 2, and 4-7, wherein the lipopolysaccharide (LPS)-deficient A. baumannii strain is characterized by partial or complete inactivation of the gene selected from the group consisting of lpxA, lpxB, lpxC.

11. A composition according to any one of claims 1, 2 and 4 to 7 for use in therapy.

12. The composition of any one of claims 1, 2 and 4 to 7, for use in delivering a bacterial outer membrane antigen in a subject in need thereof.

13. 8. The composition of any one of claims 1, 2, and 4-7, for use in delivering bacterial outer membrane antigens to induce an immune protective response against at least K. pneumoniae, P. aeruginosa, E. coli, and / or A. pleuropneumoniae, and optionally A. baumannii, in a subject in need thereof.

14. A vaccine composition comprising the lipopolysaccharide (LPS)-deficient A. baumannii strain of any one of claims 1, 2 and 4 to 7.

15. The vaccine is ６ pieces ~ 10 １２ 15. The vaccine of claim 14, comprising an A. baumannii strain that is deficient in a lipopolysaccharide (LPS).

16. 15. The vaccine of claim 14, wherein the vaccine comprises an adjuvant.

Images