Acinetobacter baumannii multi-epitope polypeptide rOmpW and its application

Abstract

The present invention relates to OmpW, and more particularly to rOmpW, a multi-epitope peptide of Acinetobacter baumannii and its use. The amino acid sequence of rOmpW is: EQGVADKVKEDFG X AKYHFKNSTRFTPY X PSEDTTTALGVVKAD, wherein X is 6-aminohexanoic acid. rOmpW can significantly reduce bacterial loads in peripheral blood and lung tissue, inhibit pathological damage to lung tissue, and improve mouse survival rate, exerting an effective immune protective effect, indicating that rOmpW can be used as a candidate vaccine for preventing Ab infection.

Description

Technical Field

[0001] The present invention relates to OmpW, and in particular to a multi-epitope peptide of Acinetobacter baumannii, rOmpW, and its application. Background Art

[0002] Acinetobacter baumannii (Ab) is a strictly aerobic Gram-negative bacillus that is widely present in nature. [1] Ab can cause pneumonia, sepsis, meningitis, post-traumatic infection and urinary tract infection, and is an important conditional pathogen of hospital infection. [2-4] Acinetobacter baumannii is a strictly aerobic Gram-negative bacillus that is highly resistant to heat, humidity, ultraviolet light, and chemical disinfectants. [1] As an ESKAPE pathogen, Ab can cause pneumonia, sepsis, meningitis, post-traumatic infection and urinary tract infection. [2-4] Due to the selective pressure of antibiotics, multi-drug resistant and pan-drug resistant Abs are gradually increasing, which poses a huge threat to human health. [5-7,13] The mechanism of Ab resistance is complex and can produce resistance to commonly used antibiotics in clinical practice. Tigecycline and polymyxin are considered the last line of defense for the treatment of drug-resistant Gram-negative bacteria, but recently strains resistant to tigecycline and polymyxin have appeared and become widespread worldwide. [5,7,14] The development of new antibacterial drugs takes a long time and is prone to induce new drug resistance in Ab. Vaccines are an effective way to prevent and control Ab infection. The development of vaccines targeting Ab has become a hot topic for many inventors. [15,16] .

[0003] The inventors disclosed a recombinant multi-epitope polypeptide of Acinetobacter baumannii Omp22 in CN 110950939B. Although the vaccine has good therapeutic effects, there is still a need to invent more vaccines targeting different epitopes to improve efficacy, reduce drug resistance or side effects, etc.

[0004] OmpW is an important outer membrane protein of Ab, which is related to bacterial adhesion and invasion and plays an important role in regulating bacterial iron homeostasis. [10,11] OmpW is highly conserved and immunogenic. An invention has found that in the Ab infection model, the bacterial load of various organs of mice immunized with OmpW was reduced, the inflammatory factors and chemokines in the serum were reduced, and the survival rate of mice was significantly improved.

[12] However, in vitro experiments showed that OmpW has certain toxicity.

[12] .

[0005] In order to reduce or eliminate the toxicity of OmpW protein and retain its immunogenicity, the structure of OmpW protein with immune protective effect can be screened out. However, the structure of OmpW protein that exerts immune effect is still unclear, so it is difficult to carry out subsequent vaccine invention. Summary of the Invention

[0006] Purpose of the Invention

[0007] The present invention uses bioinformatics methods to predict and screen the T cell epitopes and B cell epitopes of the OmpW protein antigen, conducts immune verification in mice, chemically synthesizes the dominant epitopes, discovers the structure in the OmpW protein that exerts the immune effect, constructs the multi-epitope polypeptide rOmpW, and verifies its safety and effectiveness.

[0008] Technical Solution

[0009] The multi-epitope polypeptide rOmpW of Acinetobacter baumannii is characterized by: an amino acid sequence of EQGVADKVKEDFG X AKYHFKNSTRFTPY X PSEDTTTALGVVKAD; wherein X is 6-aminohexanoic acid.

[0010] The application of the Acinetobacter baumannii multi-epitope peptide rOmpW in the preparation of Acinetobacter baumannii subunit vaccine.

[0011] Beneficial effects

[0012] 1. The present invention constructs a multi-epitope polypeptide rOmpW, which is a completely new polypeptide that has not been reported in the literature and is the first discovery. The details are as follows:

[0013] The present invention successfully constructed, expressed, and purified the full-length OmpW protein. Six B-cell epitopes and four T-cell epitopes of the OmpW protein were predicted by bioinformatics methods, and corresponding peptide segments were constructed by chemical synthesis. Two dominant B-cell epitopes and one dominant T-cell epitope were screened by immunological methods. The two dominant B-cell epitopes and one dominant T-cell epitope were connected in series by 6-aminohexanoic acid, and the multi-epitope polypeptide rOmpW was constructed by chemical synthesis. This polypeptide is a completely new polypeptide that has not been reported in the literature and is the first discovery. rOmpW was verified to be non-toxic in vitro and in vivo and can be used for in vivo experiments. Preliminary verification showed that rOmpW has an immune protective effect on the Ab acute infection mouse model, and the effective dose of rOmpW was preliminarily screened out, which is 50ug, laying the foundation for subsequent experiments.

[0014] The specific results are as follows:

[0015] (1) Enzyme digestion and sequencing confirmed that the recombinant plasmid pET28a-OmpW was successfully constructed and induced to express the full-length OmpW protein. SDS-PAGE results showed that the molecular weight of the OmpW protein was approximately 23 kDa, which was consistent with the theoretical value.

[0016] (2) Six B cell epitopes (OmpW B1, B2, B3, B4, B5 and B6) and four T cell epitopes (OmpW T1, T2, T3 and T4) of the OmpW protein were predicted by bioinformatics methods, and the epitope peptides were chemically synthesized. One week after the last immunization, the indirect ELISA method was used to detect the specific IgG antibodies in the mouse serum. The results showed that OmpW B1 and OmpW B6 could react with the serum of mice immunized with the full-length OmpW protein, and their OD450 The nm values ​​were 1.81±0.55 and 1.39±0.38, respectively, which were significantly higher than those in the PBS-immunized group (0.23±0.05 and 0.34±0.15, respectively) (P<0.001), thus confirming that they were effective B cell epitopes of OmpW protein. Subsequently, splenocytes were stimulated with four T cell epitope peptides. Among them, the IFN-γ secreted by splenocytes of the OmpW protein-immunized group stimulated by T4 peptide was 27.06±7.69 pg / ml, which was significantly higher than that of the PBS-immunized group (4.04±1.26) (P<0.001). There was no significant difference in the IFN-γ production of splenocytes between the two groups stimulated by OmpW T1, T2 and T3 peptides, thus confirming that OmpWT4 was the dominant T cell epitope of OmpW protein.

[0017] (3) Two dominant B cell epitope (B1 and B6) and one dominant T cell epitope (T4) peptides of the OmpW protein were concatenated and linked with 6-aminohexanoic acid to construct a multi-epitope peptide rOmpW by chemical synthesis. The peptide has 45 amino acids and a molecular weight of 4974.60 Da. Its purity was 95.50% as determined by HPLC.

[0018] (4) In vitro safety evaluation: rOmpW had no inhibitory effect on A549 cells at different concentrations and time periods. However, at 24 h and 48 h, OmpW at a concentration of 20 μg / ml had a significant inhibitory effect on A549 cell growth.

[0019] (5) In vivo safety assessment: The serum BUN level in OmpW mice was 8.42±0.48 μmol / L, which was significantly increased compared with 4.46±0.087 μmol / L in the control group (P<0.001). However, there were no significant differences in ALT, AST, and Cre levels in the serum of OmpW mice compared with the control group. Different concentrations of rOmpW also had no significant effect on ALT, AST, BUN, and Cre levels in the serum of mice (all P>0.05). HE staining of heart, kidney, liver, spleen, and lung tissues showed that there were no obvious injuries in mice treated with different concentrations of rOmpW and OmpW groups.

[0020] (6) One week after the last immunization, peripheral blood was collected from mice to detect the level of rOmpW-specific IgG in the serum. The OD450 of the OmpW group and rOmpW (25 μg, 50 μg, and 100 μg) groups were 1.28±0.20, 1.19±0.12, 2.85±1.20, and 2.79±1.04, respectively, which were significantly higher than those of the control group (0.19±0.03) (P<0.001). The rOmpW 50 μg and 100 μg groups were significantly higher than those of the OmpW and rOmpW 25 μg groups (P<0.01).

[0021] (7) The colony counts in the peripheral blood of BALB / c mice 24 h after the Ab standard strain (ATCC19606) challenged the OmpW and rOmpW (25 μg, 50 μg, and 100 μg) groups (1 g CFU / ml) were 3.38 ± 0.31, 3.19 ± 0.26, 1.73 ± 0.02, and 1.70 ± 0.01, respectively, which were significantly lower than those in the control group (8.23 ± 0.53) (P < 0.001). The colony counts in the rOmpW 50 μg and rOmpW 100 μg groups (1 g CFU / ml) were significantly lower than those in the OmpW and rOmpW groups. 25μg (P<0.01); the colony counts in the right lung tissue were 7.99±0.04, 7.37±0.51, 7.11±0.13 and 6.90±0.11 in the OmpW group and rOmpW (25μg, 50μg and 100μg) groups (1g CFU / ml), respectively, which were significantly lower than those in the control group (18.33±2.91) (P<0.001). The rOmpW 50μg group (1g CFU / ml) was significantly lower than that in the OmpW group (P<0.01), and the rOmpW 100μg group (1g CFU / ml) was significantly lower than that in the OmpW group (P<0.001). (8) HE staining of the lung tissue of mice showed that the lung tissue structure of the rOmpW 50μg and 100μg groups was relatively intact, the alveolar cavity structure was clear, only a small amount of inflammatory cell infiltration was observed, and no alveolar septum thickening was observed. Scattered inflammatory cell infiltration was observed in the lung tissues of the rOmpW 25μg and OmpW groups, while the lung tissue structure of the control mice was significantly destroyed, with collapsed or disappeared alveolar cavities, obvious exudation, infiltration of numerous inflammatory cells such as lymphocytes and neutrophils, and marked edema and thickening of the alveolar septa. Semi-quantitative analysis using Image pro plus software showed that 24 hours after Ab ATCC19606 challenge, the inflammatory areas in the OmpW and rOmpW 25μg groups were 35.14±20.44% and 37.11±14.67%, respectively, which were significantly lower than those in the control group (76.08±4.68) (P<0.05). The inflammatory areas in the rOmpW 50μg and 100μg groups were 6.25±1.90% and 4.48±4.05%, respectively, which were significantly lower than those in the control group (P<0.001) and the rOmpW 25μg group (P<0.05).

[0022] (9) After ATCC challenge, mice were observed for one week and their survival status was recorded daily. The survival rates of the rOmpW 25 μg group, rOmpW 50 μg group, rOmpW 100 μg group, and OmpW group were 66.67% (4 / 6), 66.67% (4 / 6), 83.33% (5 / 6), and 50% (3 / 6), respectively, which were significantly higher than those of the control group (the survival rate on day 3 was 0).

[0023] 2. OmpW is an important outer membrane protein of Ab. Hypoxia can lead to downregulation of OmpW expression, which reduces the adhesion and invasion of Ab to human lung epithelial cells and also reduces the formation of Ab biofilm.

[10] OmpW plays an important role in regulating bacterial iron homeostasis

[11] . The OmpW protein is highly conserved in the Baumanii strain. An inventor reported that the homology of OmpW in 804 Ab strains is greater than 91%. OmpW has a slight inhibitory effect on the growth of 293FT and A549 cells, indicating that OmpW may have certain toxicity. In the Ab infection model, the bacterial load of various organs of mice immunized with OmpW was reduced, the inflammatory factors and chemokines in the serum were reduced, and the survival rate of mice was significantly improved. After Ab attack, the survival rate of actively immunized mice was 100% after seven days, and the survival rate of passively immunized mice was 83.3%.

[12] . In summary, OmpW is highly conserved and immunogenic, making it one of the ideal candidate subunit vaccine proteins for Ab. However, using the full-length OmpW protein as a vaccine antigen has certain toxicity. Predicting and screening the antigenic epitopes of the OmpW protein not only retains the immunogenicity of the OmpW protein, but also reduces the molecular weight of the vaccine protein, and may also reduce or eliminate the toxic structure of the OmpW protein. Therefore, screening the antigenic epitopes of Ab candidate vaccine proteins has become a hot topic in the invention of subunit vaccines.

[0024] In the present invention, the B cell epitope of OmpW is obtained from Optimum Antigen TM Design Tool software predicts. Optimum Antigen TM Design Tool software is a B-cell epitope prediction software developed by GenScript Biotech. It uses bioinformatics methods to analyze the secondary structure, surface accessibility, hydrophilicity, flexibility, antigenic index, and other characteristics of B-cell epitopes, and then combines them with the physicochemical properties of the protein to finally screen suitable candidate B-cell epitopes. The T-cell epitopes of OmpW were predicted using the IEDB system, a free resource funded by the National Institute of Allergy and Infectious Diseases. [17,18]. The OmpW gene was amplified by PCR, and the recombinant plasmid was constructed and expressed and purified to obtain a high-purity OmpW protein. The serum of OmpW-immunized mice was detected by indirect ELISA, and two B cell epitope peptides, OmpW B1 and OmpW B6, which could react specifically with the serum of immune mice were screened out. The spleen cells of OmpW-immunized mice were cultured in vitro and stimulated with T cell epitope peptides. The concentration of IFN-γ in the supernatant of spleen cells was detected by double antibody sandwich ELISA. The concentration of IFN-γ in the OmpW T4 stimulation group was significantly increased compared with that in the control group, thus screening OmpW T4 as an effective T cell epitope of the OmpW protein; the two dominant B cell epitopes and one dominant T cell epitope screened out were connected in series by 6-aminohexanoic acid, and the multi-epitope peptide rOmpW was successfully constructed by chemical synthesis. This epitope polypeptide retains the antigenicity of the OmpW protein while avoiding the toxic effects of its structural protein. It has no effect on the proliferation of A549 cells and is non-cytotoxic. In vivo verification shows that rOmpW has no damage to the liver and kidney functions and major organs of mice, and can be used for in vivo experiments.

[0025] In order to detect the immune effect of rOmpW, we measured the content of rOmpW-specific antibody IgG in serum and the expression levels of Th1 (IFN-γ) and Th2 (IL-4) cytokines in spleen cell supernatant. BALB / c mice were immunized by subcutaneous injection once every 2 weeks for a total of 3 times. One week after the last immunization, the expression level of specific antibody IgG in mouse serum was detected. High titers of antigen-specific IgG antibodies were detected in the serum of mice in the rOmpW immunization group with the same dose, indicating that the nanovaccine can induce a stronger humoral immune response. Compared with the OmpW group and the control group; rOmpW vaccination induced a significant increase in the secretion of IFN-γ and IL-4 in the spleen cell supernatant (P<0.01). The results of the invention show that rOmpW can induce the body to produce a stronger humoral and cellular immune response and establish a comprehensive protective effect.

[0026] The successful establishment of a mouse infection model is the basis for evaluating the immune protection effect of vaccines. The most common infection caused by Ab in clinical practice is acute severe pneumonia caused by respiratory tract infection.

[19] To evaluate the protective effect of this nanovaccine against infection with different clinical Ab strains, we established an acute Ab pneumonia model in mice using a standard Ab strain (ATCC19606). The results showed that rOmpW significantly reduced bacterial loads in peripheral blood and lung tissue. The lung tissue structure of mice in the rOmpW group was relatively intact, with clear alveolar cavities, only a small amount of inflammatory cell infiltration, and no thickening of the alveolar septa. In contrast, mice in the control group showed significant damage to the lung tissue structure, with collapsed or absent alveolar cavities, prominent exudates, numerous inflammatory cell infiltrations, including lymphocytes and neutrophils, and marked edema and thickening of the alveolar septa. The 7-day survival rate and symptom scores of mice in the rOmpW group were significantly higher than those in the control group. This suggests that rOmpW can exert effective immune protection and could serve as a candidate vaccine for preventing Ab infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 , Sequencing map of recombinant plasmid pET-28a-OmpW;

[0028] Figure 2 Protein quantitative SDS-PAGE image; Lane 1: OmpW 1 μl; Lane 2: OmpW 2 μl; Lane 3: Bovine Serum Albumin (BSA) 1 μg; Lane 4: BSA 2 μg; Lane 5: BSA 4 μg; The concentration of OmpW is approximately 1.8 mg / ml;

[0029] Figure 3 , indirect ELISA results of B cell epitope peptides and mouse serum (***P<0.001, n=6);

[0030] Figure 4 , IFN-γ content in the supernatant of spleen cells of mice in each group (***P<0.001, n=6);

[0031] Figure 5 , mass spectrometry analysis of recombinant multi-epitope peptide rOmpW;

[0032] Figure 6 , liquid chromatogram of recombinant multi-epitope polypeptide rOmpW;

[0033] Figure 7 , OmpW (A) and rOmpW (B) on the proliferation level of A549 cells;

[0034] Figure 8 , in vivo safety evaluation of rOmpW; including detection of changes in ALT (A), AST (B), BUN (C) and Cre (D) in mouse serum, and HE staining results (E);

[0035] Figure 9, detection of rOmpW-specific IgG in the serum of mice in each immunization group (n=3, **P<0.01, ***P<0.001);

[0036] Figure 10 , peripheral blood colony counts of mice 24 hours after Ab tracheal challenge (**P<0.01, ***P<0.001);

[0037] Figure 11 , colony counts in mouse lung tissues 24 h after Ab tracheal challenge (n=3, **P<0.01, ***P<0.001);

[0038] Figure 12 , survival of mice within 1 week after Ab tracheal challenge;

[0039] Figure 13 , changes in mouse body weight within 1 week of Ab challenge;

[0040] Figure 14 , changes in symptom scores of mice within 1 week after Ab 19606 challenge;

[0041] Figure 15 2. Lung tissue pathology (HE staining, ×200) (A) and statistical results (B) 24 hours after Ab tracheal challenge; DETAILED DESCRIPTION

[0042] Example 1

[0043] 1. Materials and Methods

[0044] 1.1 Experimental Materials

[0045] The Ab standard strain ATCC19606 was a gift from the Department of Laboratory, The Second Affiliated Hospital of Nanjing Medical University; restriction enzymes, Taq enzymes, dNTPs, and T4 ligase were purchased from Biolab (USA); Ni-NTA Agarose was a product of Novagen; HRP-labeled IgG was purchased from Sigma; mouse interferon-γ (IFN-γ, IL-4) pre-coated ELISA kits were purchased from Shenzhen Xinbosheng; and CCK-8 ELISA kits were purchased from Nanjing Keygen Biotechnology Co., Ltd. SPF-grade BALB / c female mice, 6–8 weeks old, were purchased from Shanghai Bikeway Biotechnology Co., Ltd. Their use was approved by the relevant ethics committee (Ethics Number: IACUC-2204012).

[0046] 1.2 Experimental methods

[0047] 1.2.1 Prediction of OmpW antigenic epitopes

[0048] Use Optimum Antigen developed by GenScript Biotech TM The B cell epitopes of OmpW were predicted by the Design Tool software. The amino acid sequence of OmpW was input into the Optimum Antigen TM Design Tool software web page ( https: / / www.genscript.com / antigen-design.html Based on the physicochemical properties of proteins, the software comprehensively analyzes the secondary structure, surface accessibility, hydrophilicity, flexibility, and antigenic index of B cell epitopes, screening for candidate B cell epitopes with a length of 14 amino acids and no overlap. The IEDB (Immune Epitope Database Analysis Resource) database (http: / / tools.iedb.org / mhcii / ) was used to predict the T cell epitopes of OmpW. The amino acid sequence of the OmpW protein was input, the species was selected as mouse, and the genotypes were selected as H2-IAb, H2-IAd, and H2-IEd. The MHC-II class II binding peptides were predicted. Sequences with high scores and no overlap were selected as candidate T cell epitopes.

[0049] 1.2.2 Expression, purification and quantification of OmpW protein

[0050] Specific PCR primers were designed based on the gene sequence encoding the OmpW protein: upstream primer: 5'-AAGGATCCGGTAATTGGCAAGTAAAATTTG-3'; downstream primer: 5'-TACTCGAGTTAGAATTTATAGCTATAACC-3'. PCR reaction conditions were: 98°C denaturation for 1 minute, followed by 30 cycles of 98°C denaturation for 15 seconds, 58°C annealing for 30 seconds, and 72°C extension for 30 seconds, followed by 72°C extension for 1 minute, and storage at 10°C. The target gene and empty plasmid were double-digested with restriction endonucleases BamHI and XhoI, respectively. After ligation, the target gene and empty plasmid were transformed into competent E. coli BL21(DE3) cells. Colonies were picked, plasmids were extracted, and enzyme digestion was performed for identification. Bacterial expression of the recombinant protein was induced with IPTG, and protein expression was analyzed by 15% SDS-PAGE. The target recombinant protein was purified by Ni-NTA affinity chromatography. After removing endotoxin from the recombinant protein solution, the concentration of OmpW protein was determined using a bicinchoninic acid (BCA) protein quantification kit.

[0051] 1.2.3 Synthesis of OmpW epitope peptide

[0052] According to Optimum Antigen TMBased on the OmpW epitopes predicted by the Design Tool software and the IEDB system, high-scoring, non-overlapping B-cell and T-cell epitopes were selected for synthesis. These peptides were synthesized manually by Suzhou Qiangyao Biotechnology Co., Ltd., achieving a purity exceeding 90%. To ensure binding to the ELISA plate, bovine serum albumin (BSA) was coupled to the C-terminus of each epitope peptide.

[0053] 1.2.4 Immunization of mice with purified OmpW protein

[0054] 6-8 week old SPF-grade BALB / c mice were randomly divided into experimental and control groups, with 6 mice in each group. For the first immunization, the experimental group received a solution of OmpW protein (100 μg / mouse) mixed with an equal volume of Freund's complete adjuvant, followed by a thorough emulsification. The control group received a PBS solution. The quadriceps femoris and back skin of the mice were disinfected, and the mice were immunized subcutaneously at multiple sites (100 μl / mouse). Immunizations were repeated every two weeks for a total of three times. For the second and third immunizations, the experimental group received a solution of OmpW protein (100 μg / mouse) mixed with an equal volume of Freund's incomplete adjuvant, followed by a PBS solution.

[0055] 1.2.5 Screening of B cell epitopes by indirect ELISA

[0056] One week after the third immunization, the eyeballs of the mice were removed to collect blood. After centrifugation, the upper serum was aspirated and stored in a -80°C refrigerator. ELISA plates were coated with B cell epitope peptides (OmpW B1, OmpW B3, OmpW B5) and OmpW full-length protein at a concentration of 10 μg / ml, and 100 μl was added to each well for coating. An equal volume of PBS solution was used for each well of the negative control group. Three replicates were set up for each group and coated overnight in a 4°C refrigerator. Then washed, blocked, and washed again before adding mouse serum. The Omp W full-length protein group, B cell epitope group, and negative control group were all added with 1:200 diluted experimental group mouse serum and control group mouse serum. Incubate at 37°C for 2 hours. After washing, add 1:10,000 diluted HRP-labeled goat anti-mouse IgG and incubate for 1 hour. After 30 minutes of color development, the stop solution was added and the plate was analyzed using an enzyme reader at OD 450 Read the number.

[0057] 1.2.6 Isolation of mouse spleen, culture of spleen cells and screening of T cell epitopes of OmpW protein by double antibody sandwich ELISA:

[0058] One week after the third immunization, mice were sacrificed and their spleens were isolated aseptically. Cells were collected after grinding and then lysed with red blood cell lysis buffer for 2 minutes before collection of spleen cells. The concentration of spleen cells was adjusted to 1×10 6Splenocytes were cultured in 96-well plates, with 200 μl added to each well, and triplicate wells were used for each group. Splenocytes from each mouse were stimulated with either the full-length OmpW protein or a T cell epitope peptide at a concentration of 20 μg / ml. A negative control group was also established, with an equal volume of PBS added to each well. Splenocytes were cultured in a 37°C, 5% CO2 incubator, and splenocyte supernatant was collected after 3 days.

[0059] 1.2.7 Chemical synthesis of rOmpW

[0060] Two dominant B cell epitopes (B1 and B6) and one dominant T cell epitope (T4) peptide of the OmpW protein were concatenated and separated by 6-aminohexanoic acid. The multi-epitope peptide rOmpW was constructed by chemical synthesis and commissioned to Shanghai Jier Biochemical Co., Ltd. for artificial synthesis.

[0061] 1.2.8 Safety and effectiveness of rOmpW

[0062] 1.2.8.1 In vitro safety assessment of rOmpW

[0063] Different concentrations of OmpW (10, 20, 40 and 80 μg / ml) and rOmpW (10, 20, 40 and 80 μg / ml) were co-cultured with A549 cells for 6, 24 or 48 h, respectively. The cytotoxicity of OmpW and rOmpW peptides on A549 cells was detected by CCK-8 assay.

[0064] 1.2.8.2 In vivo evaluation of the safety and efficacy of rOmpW

[0065] 6-8 week old SPF BALB / c mice were randomly divided into a control group, an OmpW group (50 μg), and different concentrations of rOmpW (25, 50, and 100 μg), with 12 mice per group. The control group received 100 μl of Freund's adjuvant, while the OmpW group received OmpW (50 μg / 100 μl) and rOmpW (25, 50, and 100 μg / 100 μl). Immunizations were repeated every two weeks for three times. One week after the third immunization, mouse serum and major organs were collected, and spleen cells were isolated and cultured. The changes of ALT, AST, BUN and Cre in the serum of mice were detected. The pathological changes of the main organs (heart, liver, spleen, lung and kidney) of mice were evaluated by HE staining. The levels of specific IgG antibodies in the serum of mice were detected by indirect ELISA. The concentrations of IFN-γ and IL-4 secreted by splenocytes after OmpW and rOmpW stimulation were detected by double antibody sandwich ELISA. Two weeks after the last immunization, lethal doses of Ab ATCC19606 (5×10 8Mice were challenged with the strain (CFU / mouse) to establish an acute Ab infection pneumonia model. Twenty-four hours after challenge, peripheral blood and right lung tissue were collected and homogenized for colony culture and count. Left lung tissue was stained with HE staining to observe pathological changes. The remaining mice were observed for one week, and their survival, weight changes, and clinical symptom scores were recorded daily.

[0066] 1.3 Statistical analysis: GraphPad Prism 9 software was used for statistical analysis, and two-way analysis of variance was used for comparison. P < 0.05 indicated statistically significant differences.

[0067] 2. Results

[0068] 2.1 Sequencing and identification of recombinant plasmid pET-28a-OmpW

[0069] The recombinant plasmid pET28a-OmpW was amplified by PCR and digested with enzymes, and the results were positive. Figure 1 It showed that the coding gene OmpW of OmpW protein was completely inserted into the pET28a plasmid;

[0070] 2.2 Expression and identification of target protein OmpW

[0071] The constructed recombinant plasmid pET-28a-Omp22 was transformed into Escherichia coli BL21. After IPTG induction, the OmpW protein was expressed in the form of inclusion bodies. The final concentration of the OmpW protein was 1.8 mg / mL by protein quantitative electrophoresis. The expression of the purified OmpW protein was detected by SDS-PAGE. A specific protein band was observed at 23 kDa, which was consistent with the expected size. Figure 2 shown.

[0072] 2.3 Prediction results of antigenic epitopes of Acinetobacter baumannii OmpW protein

[0073] Using OptimumAntigen TM Design Tool software was used to predict candidate B-cell epitopes of the OmpW protein. Bioinformatics methods were used to analyze the secondary structure, surface accessibility, hydrophilicity, flexibility, and antigenic index of the epitopes. Combined with the properties of the amino acids in the Omp22 protein antigen, six B-cell epitopes, OmpW B1, OmpW B2, OmpWB3, OmpWB4, OmpWB5, and OmpWB6, were screened as candidate peptides. The IEDB system was used to predict T-cell epitopes. Based on the prediction software scores, peptides with a score > 10 were selected as candidate epitopes. The four T-cell epitopes screened were OmpW T1, OmpW T2, OmpW T3, and OmpW T4. The locations and amino acid sequences of the six B-cell epitopes and four T-cell epitopes are shown in Table 1.

[0074] Table 1 Candidate antigen epitope sites and sequences of OmpW protein

[0075]

[0076] Note:location indicates the position of amino sequence in OmpWprotein.

[0077] 2.4 Screening of B cell epitopes of OmpW protein by ELISA

[0078] ELISA plates were coated with 6 candidate B cell epitope peptides of OmpW, OmpW full-length protein and PBS, and the sera of mice immunized with OmpW full-length protein or PBS (negative control) were added. The results showed that OmpW B1 and OmpW B6 epitope peptides and OmpW protein could react with the sera of mice immunized with OmpW protein, and their OD450 values ​​were 1.81±0.55 and 1.39±0.38, respectively, which were significantly higher than those of mice immunized with PBS (0.23±0.05 and 0.34±0.15, respectively) (P<0.001). However, after the OmpW B2, B3, B4, and B4 epitope peptides and PBS were coated with the ELISA plates, there was no significant difference in the OD450 nm values ​​of the serum reaction wells between the OmpW immunized group and the corresponding PBS group (see Figure 3 ), therefore, we believe that the dominant B cell antigen epitopes of OmpW protein are OmpW B1 (EQGVADKVKEDFG) and OmpW B6 (AKYHFKNSTRFTPY).

[0079] 2.5 Screening of T cell epitopes of OmpW protein by ELISA

[0080] The PBS immunization group was used as a control. One week after the mice were last immunized with the full-length OmpW protein, the spleens of the two groups of mice were collected and prepared into splenocyte suspensions. The splenocytes of the two groups were stimulated with the four candidate T cell epitope peptides of the Omp22 protein, the full-length OmpW protein, and PBS, respectively. After 72 hours of stimulation, the ELISA method was used to detect the secretion level of IFN-γ in the supernatant of the splenocytes. The results showed that the IFN-γ secreted by the splenocytes of the mice in the OmpW protein immunization group stimulated by the T4 peptide was 27.06±7.69pg / ml, which was significantly higher than that in the PBS immunization group (4.04±1.26) (P<0.001). However, there was no significant difference in the IFN-γ produced by the splenocytes of the two groups stimulated by the OmpW T1, T2, and T3 peptides (see the results). Figure 4 OmpWT4 was identified as the dominant T cell epitope of OmpW protein, and its amino acid sequence was PSEDTTTALGVVKAD.

[0081] 2.6 Construction and identification of multi-epitope peptide rOmp22

[0082] The two dominant B cell epitopes (OmpW B1 and B6) and one T cell epitope (OmpW T4) peptides screened were concatenated through 6-aminohexanoic acid and commissioned to Jier Biochemical (Shanghai) Co., Ltd. to chemically synthesize the multi-epitope peptide rOmpW, which has 45 amino acids. The molecular weight of the multi-epitope peptide rOmpW was identified by mass spectrometry to be 4974.60Da. Figure 1-10 The purity of the synthesized peptide was 95.50% as determined by HPLC. Figure 5-6 .

[0083] 2.7 Effect of rOmpW polypeptide on the proliferation level of A549 cells

[0084] After stimulating A549 cells with PBS (control group) and different concentrations of OmpW and rOmpW for 24 and 48 h, the proliferation level of A549 cells was detected by CCK-8 method. The results are shown in Figure 7 Different concentrations of rOmpW had no inhibitory effect on A549 cells, while OmpW had a significant inhibitory effect on the growth of A549 cells at a concentration of 20 μg / ml.

[0085] 2.8 In vivo safety assessment of rOmpW

[0086] One week after the last immunization, the serum and major organs of the mice were collected to detect the changes in ALT, AST, BUN and Cre in the serum of the mice. HE staining was used to evaluate the pathological changes in the major organs of the mice (heart, liver, spleen, lung and kidney). The BUN level in the serum of OmpW mice was 8.42±0.48μmol / L, which was significantly increased compared with 4.46±0.087μmol / L in the control group (P<0.001). Figure 8 A, while there was no significant difference in ALT, AST and Cre levels in the serum of OmpW group mice compared with the control group. Figure 1-13 There was no significant difference in the levels of ALT, AST, BUN and Cre in the serum of mice between different concentrations of rOmpW (all P>0.05). Figure 8 AD. HE staining of heart, kidney, liver, spleen and lung tissues showed that no obvious damage was observed in mice in the rOmpW and OmpW groups. Figure 8 E. The above results indicate that the full-length protein OmpW can damage renal function, while rOmpW at different concentrations has a high safety in vivo.

[0087] 2.9 Detection of rOmp22-specific IgG in mice in each immunization group

[0088] One week after the last immunization, peripheral blood was collected from mice to detect the level of rOmpW-specific IgG in serum. The OD450 of the OmpW group and rOmpW (25 μg, 50 μg and 100 μg) groups were 1.28±0.20, 1.19±0.12, 2.85±1.20 and 2.79±1.04, respectively, which were significantly higher than those of the control group (0.19±0.03) (P<0.001). The rOmpW 50 μg and 100 μg groups were significantly higher than those of the OmpW and rOmpW 25 μg groups (P<0.01). Figure 9 .

[0089] 2.11 Peripheral blood colony count

[0090] 24 hours after the mice were challenged with the Ab standard strain (ATCC19606), peripheral blood was collected for colony count. The colony counts (1g CFU / ml) of rOmpW 50μg and rOmpW 100μg were significantly lower than those of the OmpW group and rOmpW 25μg group (**P<0.01). Figure 10 .

[0091] 2.12 Colony counts in lung tissue homogenate

[0092] 24 hours after the BALB / c mice were challenged with the Ab standard strain (ATCC19606), the right lung tissues of the mice were prepared into homogenates for colony counting. The colony counts (1g CFU / ml) of the OmpW group and the rOmpW (25μg, 50μg, and 100μg) groups were 7.99±0.04, 7.37±0.51, 7.11±0.13, and 6.90±0.11, respectively, which were significantly higher than those of the control group (18.33±2.91) (P<0.001). The colony counts (1g CFU / ml) of the rOmpW 50μg group were significantly lower than those of the OmpW group (P<0.01), and the colony counts (1g CFU / ml) of the rOmpW 100μg group were significantly lower than those of the OmpW group (P<0.001). Figure 11 .

[0093] 2.13 Survival of mice within one week of ATCC19606 tracheal challenge

[0094] After ATCC19606 challenge, mice were observed for one week and their survival status was recorded daily. The survival rates of the rOmpW 25 μg group, rOmpW 50 μg group, rOmpW 100 μg group, and OmpW group were 66.67% (4 / 6), 66.67% (4 / 6), 83.33% (5 / 6), and 50% (3 / 6), respectively, which were significantly higher than those of the control group (survival rate on day 3 was 0). See the survival curve. Figure 12 .

[0095] 2.14Ab tracheal challenge and changes in mouse body weight within 1 week

[0096] After ATCC19606 challenge, the weight of mice was recorded daily. The weight of mice dropped to the lowest on the second day, and then the weight of surviving mice in the OmpW and rOmpW immunization groups gradually increased. The weight of mice could basically return to the pre-challenge level on the 6th to 7th day. Figure 13 .

[0097] 2.15 Changes in symptom scores of mice within 1 week after ATCC19606 challenge

[0098] Symptoms were scored on a scale of 0 to -5 based on the mice's activity, bending, closing eyes, hair condition, and movements. 0 was for healthy mice and -5 for dead mice. The score was 0 before ATCC19606 attack and was lowest on the second day after the attack. All mice in the NS+adjuvant control group died within 3 days. Afterwards, the symptoms of the surviving mice gradually eased and they resumed their diet. By the fifth day of observation, the clinical symptom scores of the surviving mice had basically returned to 0. Figure 14 .

[0099] 2.16Ab tracheal challenge 24 hours after lung pathology severity

[0100] HE staining of mouse lung tissue showed that the lung tissue structure of rOmpW 50μg and 100μg groups was relatively intact, the alveolar cavity structure was clear, only a small amount of inflammatory cell infiltration was observed, and no alveolar septum thickening was observed. In the rOmpW 25μg and OmpW groups, scattered inflammatory cell infiltration was observed in the lung tissue, while in the control group, the lung tissue structure was obviously destroyed, the alveolar cavity collapsed or disappeared, obvious exudation was observed in the alveolar cavity, a large number of inflammatory cells such as lymphocytes and neutrophils infiltrated, and obvious edema and thickening of the alveolar septum were observed. Semi-quantitative analysis by Image pro plus software was shown in the figure. 24 hours after Ab ATCC19606 challenge, the inflammatory areas in the OmpW and rOmpW 25μg groups were 35.14±20.44% and 37.11±14.67%, respectively, which were significantly lower than those in the control group (76.08±4.68) (P<0.05). The inflammatory areas in the 50μg and 100μg groups were 6.25±1.90% and 4.48±4.05%, respectively, which were significantly lower than those in the control group (P<0.001) and the rOmpW 25μg group (P<0.05). Figure 15 .

[0101] References

[0102] [1]ANTUNES L C,VISCA P,TOWNER K J.Acinetobacter baumannii:evolutionof aglobal pathogen[J].Pathog Dis,2014,71(3):292-301.DOI:10.1111 / 2049-632X.12125.

[0103] [2]AYOUB MOUBARECK C,HAMMOUDI HALAT D.Insights intoAcinetobacterbaumannii:A Review of Microbiological,Virulence,and ResistanceTraits in aThreatening Nosocomial Pathogen[J].Antibiotics(Basel),2020,9(3).DOI:10.3390 / antibiotics9030119.

[0104] [3]HOWARD A,O'DONOGHUE M,FEENEY A,et al.Acinetobacter baumannii:anemerging opportunistic pathogen[J].Virulence,2012,3(3):243-50.DOI:10.4161 / viru.19700.

[0105] [4]NIEDERMAN M S,BARON R M,BOUADMA L,et al.Initialantimicrobialmanagement of sepsis[J].Crit Care,2021,25(1):307.DOI:10.1186 / s13054-021-03736-w.

[0106] [5]VAZQUEZ-LOPEZ R,SOLANO-GALVEZ S G,JUAREZ VIGNON-WHALEY J J,etal.Acinetobacter baumannii Resistance:A Real Challenge for Clinicians[J].Antibiotics(Basel),2020,9(4).DOI:10.3390 / antibiotics9040205.

[0107] [6]NASR P.Genetics,epidemiology,and clinical manifestations ofmultidrug-resistantAcinetobacter baumannii[J].J Hosp Infect,2020,104(1):4-11.DOI:10.1016 / j.jhin.2019.09.021.

[0108] [7]NANG S C,AZAD M A K,VELKOV T,et al.Rescuing the Last-LinePolymyxins:Achievements and Challenges[J].Pharmacol Rev,2021,73(2):679-728.DOI:10.1124 / pharmrev.120.000020.

[0109] [8]BASSETTI M,KANJ S S,KIRATISIN P,et al.Early appropriatediagnostics andtreatment of MDR Gram-negative infections[J].JAC AntimicrobResist,2022,4(5):dlac089.DOI:10.1093 / jacamr / dlac089.

[0110] [9]GARCIA-QUINTANILLA M P M,MCCONNELL MJ.First Steps Towards aVaccineagainst Acinetobacter baumannii[J].Curr Pharm Biotechnol,2013,14(10):897-902.DOI:10.2174 / 1389201014666131226123511.

[0111]

[10] GIL-MARQUéS ML,PACHóN J,Y S.iTRAQ-Based Quantitative ProteomicAnalysis ofAcinetobacter baumannii under Hypoxia and Normoxia Reveals theRole of OmpW as aVirulence Factor[J].Microbiol Spectr,2022,10(2):e0232821.DOI:10.1128 / spectrum.02328-21.

[0112]

[11] CATEL-FERREIRA M,MARTI S,GUILLON L,et al.The outer membraneporinOmpW of Acinetobacter baumannii is involved in iron uptake and colistinbinding[J].FEBS Lett,2016,590(2):224-31.DOI:10.1002 / 1873-3468.12050.

[0113]

[12] HUANG W,WANG S,YAO Y,et al.OmpW is a potential target foreliciting protectiveimmunity against Acinetobacter baumannii infections[J].Vaccine,2015,33(36):4479-85.DOI:10.1016 / j.vaccine.2015.07.031.

[0114]

[13] CHOKSHI A,SIFRI Z,CENNIMO D,et al.Global Contributors toAntibiotic Resistance[J].J Glob Infect Dis,2019,11(1):36-42.DOI:10.4103 / jgid.jgid_110_18.

[0115]

[14] JO J,KWON K T,KO K S.Multiple heteroresistance to tigecycline andcolistin inAcinetobacter baumannii isolates and its implications for combinedantibiotic treatment[J].J Biomed Sci,2023,30(1):37.DOI:10.1186 / s12929-023-00914-6.

[0116]

[15] KENNEDY D A,READ A F.Why does drug resistance readily evolve butvaccineresistance does not?[J].Proc Biol Sci,2017,284(1851).DOI:10.1098 / rspb.2016.2562.

[0117]

[16] AHMAD T A,TAWFIK D M,SHEWEITA S A,et al.Development ofimmunizationtrials against Acinetobacter baumannii[J].Trials in Vaccinology,2016,5:53-60.DOI:10.1016 / j.trivac.2016.03.001.

[0118]

[17] VITA R,OVERTON J A,GREENBAUM J A,et al.The immune epitopedatabase(IEDB)3.0[J].Nucleic Acids Res,2015,43(Database issue):D405-12.DOI:10.1093 / nar / gku938.

[0119]

[18] VITA R,MAHAJAN S,OVERTON J A,et al.The Immune Epitope Database(IEDB):2018 update[J].Nucleic Acids Res,2019,47(D1):D339-D43.DOI:10.1093 / nar / gky1006.

[0120]

[19] Chuang YC,Cheng A,et al.Microbiological and clinicalcharacteristics of Acinetobacterbaumannii bacteremia:Implications of sequencetype for prognosis[J].J Infect,2019,78:106-112。

Claims

1. A multi-epitope peptide rOmpW of Acinetobacter baumannii, characterized by: Its amino acid sequence is: EQGVADKVKEDFG X AKYHFKNSTRFTPY X PSEDTTTALGVVKAD; wherein X is 6-aminohexanoic acid.

2. Use of the multi-epitope peptide rOmpW of Acinetobacter baumannii according to claim 1 in the preparation of Acinetobacter baumannii subunit vaccine.

Images