Development of synthetic pseudo-aminic acid-based antibacterial vaccine against acinetobacter baumannii

Abstract

Provided are compositions comprising chemically synthesized pseudamines (Pse) conjugated to carrier proteins using OPA chemistry, methods of using the compositions to stimulate an immune response in a subject and to protect a vaccinated subject from infection by A. baumannii producing Pse.

Description

Background of the Invention

[0002] Acinetobacter baumannii is a Gram-negative bacterium that can cause a range of infections in both hospitals and the community, including bacteremia, pneumonia, meningitis, urinary tract infections, and skin and soft tissue infections. 1 Primarily, it is an opportunistic pathogen that can cause serious hospital-acquired infections, especially in immunocompromised individuals. 2 Acinetobacter baumannii can also colonize the human body without causing infection or symptoms, and it is widely present in the natural environment. 3 Antimicrobial resistance to multiple drugs poses a global threat to public health and severely impacts the effectiveness of public health management. *Acinetobacter baumannii* has been shown to acquire resistance to many classes of antibiotics through various resistance mechanisms. 4 Acinetobacter baumannii is believed to exhibit widespread resistance to most last-line antibiotics developed in recent years, whereas it was sensitive to most antibiotics prior to the 1970s. 5 For many years, carbapenems have been used as a last-resort antibiotic for treating Acinetobacter baumannii infections. However, the rising trend of carbapenem resistance in Acinetobacter baumannii has limited their efficacy and promoted the use of polymyxins and tigecycline as last-line defense drugs. 6 However, the emergence of colistin-resistant and tigecycline-resistant Acinetobacter baumannii has now been reported, exacerbating the clinical problems caused by carbapenem-resistant (CR) Acinetobacter baumannii. 7,8 Treatment of Acinetobacter baumannii infections has become difficult due to the emergence of multidrug-resistant strains, necessitating the development of new strategies for the prevention and treatment of infections caused by this pathogen. In 2017, the World Health Organization (WHO) published a list of 12 "priority pathogens" requiring urgent antimicrobial research and development. 9 Acinetobacter baumannii is at the top of this list as the highest priority for immediate attention. 9 .

[0003] The development of new antibacterial drugs against multidrug-resistant (MDR) Acinetobacter baumannii is ongoing. 10 Besides antibiotics, vaccination or immunotherapy are alternative strategies to protect humans from bacterial infections and combat bacterial multidrug resistance. 11Over the past few decades, a growing number of candidate vaccines against Acinetobacter baumannii have been proposed and studied, including whole bacteria, outer membrane vesicles or complexes, DNA-based vaccines, and purified or recombinant subunits. 12 Bacterial surface sugars have been identified as effective antigens for vaccine development against infectious diseases. 13 , 14 Glycoconjugate vaccines have been successfully developed and are effectively used against Haemophilus influenzae type B. 15 Selected serotypes of Streptococcus pneumoniae 16 Neisseria meningitidis serogroups A, C, W and Y 17 And Salmonella typhi 18 Carbohydrate-based antimicrobial vaccines typically consist of carbohydrate antigens, linkers, and carrier proteins. The carbohydrate antigens can be bacterial surface polysaccharides isolated from cultured bacteria, such as capsular sugars from thirteen serotypes of *Streptococcus pneumoniae* (S. pneumoniae) (1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F) conjugated to the diphtheria CRM197 carrier protein. (Pfizer, New York, NY, approved by the FDA in 2010) 19 However, not all pathogens are easily cultured, and bacterial polysaccharide extraction can be plagued by contamination. Alternatively, synthetic carbohydrate antigens are structurally defined and free from cellular contaminants, such as those containing synthetic phosphoproteosidelantine conjugated with tetanus toxoid carrier proteins. (CIGB, approved in Cuba in 2004) 20 Various synthetic sugar-based vaccine candidates targeting different pathogens are being investigated. 21 .

[0004] Pseudaminic acid (Pse) belongs to the nonulosonic acid family and is widely distributed in various pathogenic bacteria as a component of cell surface-associated glycans, such as lipopolysaccharide (LPS) in Pseudomonas aeruginosa, Shigella boydii, and Vibrio vulnificus; capsular polysaccharide (CPS) in Acinetobacter baumannii; pili in Pseudomonas aeruginosa; and repeating units of flagella in Aeromonascaviae, Helicobacter pylori, and Campylobacter jejuni. 22-27 Although the exact function of Pse on the bacterial cell surface remains unclear, it likely plays an important role in bacterial pathogenicity because it is highly correlated with virulence factors LPS, CPS, and flagella, and it is unique to Gram-negative bacteria and structurally similar to mammalian sialic acid. In summary, CPS from Pse-containing pathogenic bacteria is expected to be an effective target for generating vaccines against pathogens with Pse present on their surfaces. However, the development of Pse-based antimicrobial vaccines is hindered not only by the difficulty of extracting Pse-containing polysaccharide samples with high monodispersity and sufficient quantities, but also by the instability of the pseudaminyl bond. Previously, Wu et al. reported that the phage ΦAB6 tail spike protein could specifically recognize the exopolysaccharide (EPS) of Acinetobacter baumannii strain 54149 and depolymerize it into the oligosaccharide fragment Pse5NAc7NAc-α-(2→6)-Glcp-β-(1→6)-[→3]-Galp-β-(1→3)-GalNAcp-β-(1]2, which is the major product. 28 The resulting oligosaccharides, after conjugation with the carrier protein, were used for vaccination. Booster serum from vaccinated rabbits showed recognition of EPS from *Acinetobacter baumannii* strain 54149, but not from *Acinetobacter baumannii* strain SK44A, which shares most of the sugar components with Ab-54149 except for Pse. 28These studies indicate that Pse is a key epitope for antigenicity. Challenges remain to be considered in the production of *Acinetobacter baumannii*, such as antigenic heterogeneity and batch-to-batch reproducibility encountered when isolating CPS antigens from cultured bacteria, and structural limitations based on phage-based strategies, as naturally occurring Pse forms different structures for pathogenic bacteria. Previous studies have also reported efficient synthesis of Pse and highly stereoselective pseudoaminoylation. 29-31 .

[0005] Therefore, a safe and effective Acinetobacter baumannii vaccine is still needed.

[0006] Invention Summary

[0007] This invention relates to a novel, synthetic pseudoemine (Pse)-based vaccine against Pse-carrying bacterial pathogens. In some embodiments, the Pse conjugated to a carrier protein can stimulate an immune response. In some embodiments, the Pse-carrier protein conjugate can protect vaccinated subjects from infection caused by Pse-carrying Acinetobacter baumannii, particularly Pse-producing Acinetobacter baumannii strain Ab2.

[0008] In some implementations, o-phthalaldehyde (OPA) chemistry can be used to conjugate synthetic sugars onto carrier proteins for glycoconjugate synthesis. Brief description of the attached diagram

[0010] Figure 1A -1E Pse vaccine and antibody titer immunization schedule. Figure 1A Ten C57BL / 6J mice in each group were subcutaneously immunized with three doses of Pse vaccine mixed with aluminum hydroxide: Pse-CRM197 1, Pse-CRM197 2, and Pse-CRM197 3. Control mice received CRM197 mixed with aluminum hydroxide in PBS. The immunizations were completed on day 7. Figure 1B Day 21 Figure 1C ), Day 35 Figure 1D ) and the 65th day ( Figure 1E The collected serum was diluted twice from 100 to determine the endpoint titer of Pse-specific antibodies in the post-immunization serum as analyzed by ELISA.

[0011] Figure 2A-2H The typing of Pse-specific antibodies in serum after immunization was analyzed by ELISA. HRP-conjugated goat anti-mouse IgA (… Figure 2A ), IgM Figure 2B) IgG1 Figure 2C ), IgG2b ( Figure 2D ), IgG2c Figure 2E IgG3 Figure 2F )λ( Figure 2G ) and κ( Figure 2H ) Used for Pse-specific antibodies for typing.

[0012] Figure 3 Flow cytometry analysis of the binding ability of post-immunization serum to Acinetobacter baumannii strain Ab2. Bacteria were incubated with 100-diluted post-immunization serum and Alexa Fluor 647-labeled secondary anti-mouse antibody. Bacteria incubated with the secondary antibody alone served as a negative control.

[0013] Figures 4A-4C Immunization with the Pse vaccine prevents infection with Acinetobacter baumannii. Figure 4A The LD50 of Acinetobacter baumannii strain Ab2 was determined using a mouse sepsis model. The survival rate of mice after intraperitoneal injection of a specified dose of Acinetobacter baumannii strain Ab2 (n = 4-5 mice / group) was determined. Mice were vaccinated with Pse at weeks 0, 2, and 4, followed by a 2.0 × 10⁻⁶ dose two weeks after the last immunization. 7 CFU(2×LD50)( Figure 4B ) and 5.0×10 7 CFU(5×LD50)( Figure 4C The strain Ab2 was used to attack mice.

[0014] Figures 5A-5F Liver samples from mice inoculated 12 hours post-infection and control mice (n = 4 mice / group) Figure 5A ),kidney( Figure 5B ),lung( Figure 5C ),spleen( Figure 5D ),heart( Figure 5E ), and blood ( Figure 5F Bacterial load in ) . Mice were vaccinated with Pse at weeks 0, 2, and 4, and then 2 weeks after the last immunization with 5.0 × 10 ) 7 Mice were attacked with strain Ab2 containing CFU (5×LD50).

[0015] Figures 6A-6C IL-1β from mice inoculated 12 hours post-infection and control mice (n = 4 mice / group) Figure 6A ), IL-6 Figure 6B ) and TNF-α Figure 6CSerum levels of pro-inflammatory cytokines were measured. Mice were vaccinated with Pse at weeks 0, 2, and 4, followed by a 5.0 × 10⁻⁶ dose two weeks after the final immunization. 7 Mice were challenged with strain Ab2 containing CFU (5×LD50). IL-1β and TNF-α levels in mice vaccinated with Pse vaccine, Pse-CRM197 1, Pse-CRM197 2, and Pse-CRM197 3 were below the minimum range of measurement (7.8 pg / ml).

[0016] Figure 7 Synthesis of Pse-CRM197 conjugate. Reagents and conditions: (a) NIS, TfOH, DMF, DCM, AW-300 molecular sieve, acceptor 6, -40℃, 6h, 80%. (b) Pd / C, NH4OAc, DCM-MeOH, H2 (1 atm), 30min, then NMM, Ac2O, 1h, 77%. (c)(i) DEA-MeCN, 30min; (ii) acid 7, EDCI, DIPEA, DCM, 87%. (d)(i) LiOH, MeOH-THF-H2O, 24h; (ii) 10% HOAc (aqueous solution), 2h, 75% over step 2. (e) CRM197 carrier protein, PBS buffer (pH 7.4), room temperature, 6h. NIS: N-iodosuccinimide. TfOH: trifluoromethanesulfonic acid. DMF: N,N-dimethylformamide. DCM: Dichloromethane. NMM: N-Methylmorpholine. DEA: Diethylamine. EDCI: 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide. DIPEA: N,N-Diisopropylethylamine. THF: Tetrahydrofuran.

[0017] Figure 8Synthesis of BSA-Pse conjugates. Reagents and conditions: (a) NIS, TfOH, DMF, DCM, AW-300 molecular sieve, acceptor 12, -40℃, 6h, 82%. (b) Pd / C, NH4OAc, DCM-MeOH, H2 (1 atm), 30min, then NMM, Ac2O, 1h, 78%. (c)(i) DEA-MeCN, 30min; (ii) acid 13, EDCI, DIPEA, DCM, 87%. (d) LiOH, MeOH-THF-H2O, 24h, 79%. (e) sulfo-EMCS, PBS (pH 8.0), room temperature, 2h. (f) PBS (pH 7.4), room temperature, 16h. NIS: N-iodosuccinimide. TfOH: trifluoromethanesulfonic acid. DMF: N,N-dimethylformamide. DCM: dichloromethane. NMM: N-methylmorpholine. DEA: Diethylamine. EDCI: 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide. DIPEA: N,N-diisopropylethylamine. THF: Tetrahydrofuran. sulfo-EMCS: N-(ε-maleimide hexanoyloxy)sulfosuccinimide ester.

[0018] Figure 9 Synthesis of N-(2-(2-(2-(2-(4,8-di-O-acetyl-5-azido-7-N-benzyloxycarbonyl-1-isopropyl-α-pseudoaminoyloxy)ethoxy)ethoxy)ethoxy)ethyl)carbamate (9H-fluorene-9-yl)methyl ester (6).

[0019] Figure 10 Synthesis of N-(2-(2-(2-(2-(5,7-diacetamido-4,8-di-O-acetyl-1-isopropyl-α-pseudoamineoxy)ethoxy)ethoxy)ethoxy)ethyl)carbamate (9H-fluorene-9-yl)methyl ester (7).

[0020] Figure 11 Synthesis of 3-(1,3-dimethoxy-1,3-dihydroisobenzofuran-5-yl)-N-(2-(2-(2-(2-(5,7-di-acetamido-4,8-di-O-acetyl-1-isopropyl-β-pseudoamineoxy)ethoxy)ethoxy)ethoxy)ethyl)propionamide (9).

[0021] Figure 12 Synthesis of 3-(3,4-dicarboxyphenyl)-N-(2-(2-(2-(2-(5,7-diacetamido-α-2-pseudoamineoxy)ethoxy)ethoxy)ethyl)propionamide (10).

[0022] Figure 13Synthesis of N-(4,8-di-O-acetyl-5-azido-7-N-benzyloxycarbonyl-1-isopropyl-α-pseudoaminooxy)pentylcarbamate (9H-fluorene-9-yl) methyl ester (12).

[0023] Figure 14 Synthesis of N-(5,7-diacetamido-4,8-di-O-acetyl-1-isopropyl-α-pseudoaminooxy)pentylcarbamate (9H-fluorene-9-yl) methyl ester (13).

[0024] Figure 15 Synthesis of S-(2-((5-(5,7-di-acetamido-4,8-di-O-acetyl-1-isopropyl-α-pseudoaminooxy)pentyl)amino)-2-oxoethyl)thioacetate (15).

[0025] Figure 16 Synthesis of 2-mercapto-N-(5-(5,7-diacetamido-α-pseudoamineoxy)pentyl)acetamide (16).

[0026] Figure 17A-17J SDS-PAGE of CRM197 before reaction, conjugated with 20, 30, and 50 equivalents of Pse, respectively. Figure 17A SDS-PAGE and Western blot of BSA before reaction, after activation, and after conjugation (using anti-α-Pse antibody) Figure 17B-17C ). ( Figure 17D-17J MALDI-TOF analysis of Pse-protein conjugates was performed to measure the average molecular size. Recombinant CRM197 and BSA were determined as standards. Invention Details

[0028] Select definition

[0029] As used herein, the singular forms “a,” “an,” and “the” are intended to also include the plural forms, unless the context clearly indicates otherwise. Furthermore, for the purposes of the terms “including,” “includes,” “having,” “has,” “with,” or variations thereof used in the detailed description and / or claims, these terms are intended to be included in a manner similar to the term “comprising.” The transitional terms / phrases (and any grammatical variations thereof) “comprising,” “comprises,” “comprise,” “consisting essentially of,” “consists essentially of,” “consisting,” and “consists” are used interchangeably.

[0030] The phrase "consisting essentially of" or "consists essentially of" indicates that the claims cover embodiments that include the specified materials or steps and do not substantially affect the basic and novel features (multiple features) of the claims.

[0031] The term "about" refers to an acceptable range of error for a particular value as determined by a person skilled in the art, which depends in part on how the value is measured, i.e., the limitations of the measurement system. In the context of compositions containing an amount of an ingredient, the term "about" is used when the composition contains that amount of the ingredient, and its variation (error range) around the value (X ± 10%) is 0-10%. In other contexts, the term "about" provides for a variation (error range) of 0-10% around a given value (X ± 10%). It is evident that this variation represents a range of up to 10% above or below the given value, such as X ± 1%, X ± 2%, X ± 3%, X ± 4%, X ± 5%, X ± 6%, X ± 7%, X ± 8%, X ± 9%, or X ± 10%.

[0032] In this disclosure, ranges are stated in abbreviated form to avoid the need to state and describe every single value within the range in detail. Where appropriate, any suitable value within the range may be chosen as the upper limit, lower limit, or end of the range. For example, a range of 0.1–1.0 represents the end values ​​of 0.1 and 1.0, and the intermediate values ​​of 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, as well as all intermediate ranges contained within 0.1–1.0 such as 0.2–0.5, 0.2–0.8, 0.7–1.0, etc. Values ​​having at least two significant figures within a range are envisioned; for example, a range of 5–10 represents all values ​​from 5.0 to 10.0 and from 5.00 to 10.00, including the end values. When ranges are used herein, combinations and sub-combinations of ranges (e.g., sub-ranges within the scope of the disclosure) are explicitly included. Detailed Implementation Plan

[0033] As used herein, the term "subject" refers to an animal that needs or expects to receive the benefits provided by the vaccine. Animals can be, for example, humans, pigs, horses, goats, cats, mice, rats, dogs, apes, fish, chimpanzees, orangutans, guinea pigs, hamsters, cattle, sheep, birds, chickens, and any other vertebrates or invertebrates. These benefits may include, but are not limited to, treating a health condition, disease, or ailment; preventing a health condition, disease, or ailment; improving immune health; or enhancing the function of organs, tissues, or systems in the body. In the context of this invention, a human subject is preferred. A subject can be of any age or developmental stage, including infants, toddlers, adolescents, teenagers, adults, or the elderly.

[0034] As used herein, the terms “therapeutic effective amount,” “therapeutic effective dose,” “effective amount,” and “effective dose” refer to the amount or dose of a compound or composition that, when administered to a subject, is capable of treating, preventing, or improving a subject’s condition, disease, or ailment, or of providing health or functional enhancement to the immune system or organs, tissues, or body systems. In other words, the amount is “therapeutic effective” when administered to a subject. The actual amount will vary depending on a number of factors, including but not limited to the specific condition, disease, or ailment being treated, prevented, or improved; the severity of the condition; the specific organ, tissue, or body system for which health or function is to be enhanced; the patient’s weight, height, age, and health status; and the route of administration.

[0035] As used herein, the term "treatment" means to eradicate, reduce, improve, or reverse the signs or symptoms of a health condition, disease, or symptom to any extent, and includes, but does not require, a complete cure of the condition, disease, or symptom. Treatment can cure, improve, or partially reduce a symptom. "Treatment" can also include improving or enhancing a symptom or characteristic, for example, bringing a particular system in the body to a healthy or homeostatic state.

[0036] As used herein, “prevention” of a health condition, disease, or symptom means avoiding, delaying, preventing, or minimizing the onset of a specific sign or symptom of the condition, disease, or symptom. Prevention can, but is not required to, be absolute or complete; meaning that the sign or symptom may still develop at a later time. Prevention may include reducing the severity of the onset of such a condition, disease, or symptom, and / or inhibiting its progression to a more severe condition, disease, or symptom.

[0037] In some embodiments of the invention, the method includes administering multiple doses of the compound of the invention. The method may include administering 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, or more therapeutically effective doses of a composition comprising the compound of the invention as described herein. In some embodiments, the doses are administered over a period of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 14 days, 21 days, 30 days, or longer than 30 days. The frequency and duration of administering multiple doses of the composition are, for example, to enhance immune system function and / or prevent or treat bacterial infections. Furthermore, treating a subject with a therapeutically effective amount of the compound of the invention may include a single treatment or may include a series of treatments. It will also be understood that the effective dose of the compound used for treatment may be increased or decreased during a specific treatment. Changes in dose may result in and become apparent based on the results of diagnostic assays or imaging techniques known in the art for detecting tumor size. In some embodiments of the invention, the method includes administering the compound several times a day, including but not limited to administering the compound twice a day, three times a day, and four times a day.

[0038] As used herein, an “isolated” or “purified” compound is substantially free of other compounds. In some embodiments, the purified compound is at least 60% by weight (dry weight) of the compound of interest. Preferably, the formulation is at least 75% by weight of the compound of interest, more preferably at least 90% by weight, and most preferably at least 99% by weight. For example, the purified compound is at least 90%, 91%, 92%, 93%, 94%, 95%, 98%, 99%, or 100% by weight of the desired compound. Purity is measured by any suitable standard method, such as column chromatography, thin-layer chromatography, or high-performance liquid chromatography (HPLC).

[0039] "Reduction" refers to a negative change of at least 1%, 5%, 10%, 25%, 50%, 75%, or 100%.

[0040] "Increase" refers to a positive change of at least 1%, 5%, 10%, 25%, 50%, 75%, or 100%.

[0041] As used herein, "medicine" refers to a compound manufactured for use as a pharmaceutical and / or therapeutic agent. Pseudoenic acid (Pse) conjugate composition and method of using the composition.

[0042] This invention relates to a method for generating an immune response against Pse-producing bacteria such as Acinetobacter baumannii. In some embodiments, the method includes administering a subject a composition comprising Pse conjugated to an immunogenic carrier protein.

[0043] The composition can be given as a prophylactic measure to subjects who do not have a bacterial infection at the time of administration to prevent or delay the onset of a bacterial infection. The composition can also be given as a therapeutic measure to subjects who do have a bacterial infection at the time of administration to reduce or eliminate one or more symptoms of the infection.

[0044] In some implementations, the glycoconjugate construct may be administered to a subject to generate an immune response in the subject, the method comprising conjugating pseudoamine (Pse) to a carrier protein.

[0045] In some embodiments, the composition, which can be administered to a subject to elicit an immune response in the subject, comprises Pse conjugated to a carrier protein. In some embodiments, a drug carrier, excipient, and / or adjuvant may be used in the composition.

[0046] In some embodiments, the target Acinetobacter spp., particularly Acinetobacter baumannii and its strains, includes, for example, Acinetobacter baumannii strain Ab2 or other Acinetobacter baumannii strains containing Pse, such as, for example, serotypes K2, K6, K16, K23, K31, K33, K42, K46, K58, K77, K81, K90, K93 and K120.

[0047] In some embodiments, o-phthalaldehyde (OPA)-pseudoamine linkers can be synthesized. In some embodiments, OPA can be used to react with primary amines to conjugate pseudoamines to carrier proteins. In some embodiments, Pse donors such as, for example, Pse donor 4 can be stereoselectively glycosylated with Fmoc-protected PEG linkers 5 in 80% yield. Figure 7 In some embodiments, the resulting N5-azide and N7-carbamate 6 can be converted to acetamide 7 via hydrogenolysis and acetylation. The Fmoc group can then be removed to produce a free amine (using diethylamine or other secondary amine reagents, such as, for example, piperidine and 4-methylpiperidine), which can be coupled with an acid. Figure 8The Pse-OPA moiety, such as, for example, is coupled with an acid 8 containing a methyl acetal protected with phthalaldehyde. Finally, deprotection is performed by treatment 9 with lithium hydroxide (or, for example, potassium hydroxide, sodium hydroxide, tetrabutylammonium hydroxide, and other hydroxide sources), followed by treatment with an aqueous solution of acetic acid at a concentration ranging from 10% to about 5% to about 75%, to produce a Pse-OPA moiety, such as, for example, a Pse-OPA moiety 10. The Pse-OPA moiety is then reacted with a carrier protein, such as, for example, CRM197, in phosphate-buffered saline (PBS, pH 7.4) to produce a CRM197-Pse conjugate. In some embodiments, the carrier protein may be diphtheria toxoid (DT), tetanus toxoid (TT), meningococcal outer membrane protein complex (OMPC), Haemophilus influenzae (H. influenzae) protein D (HiD), keyhole hemocyanin (KLH), bovine serum albumin (BSA), or human serum albumin (HSA).

[0048] In some embodiments, Pse conjugates can be synthesized using about 1 to about 50, about 2 to about 50, about 3 to about 50, about 4 to about 50, about 5 to about 50, about 10 to about 50, about 20 to about 50, about 30 to about 50, about 20, about 30, or about 50 equivalents of OPA-Pse to produce Pse-carrier protein compounds. In some embodiments, the Pse-carrier protein compound may be Pse-CRM197 1 (sugar / protein ratio: 4.76), Pse-CRM197 2 (sugar / protein ratio: 8.27), or Pse-CRM197 3 (sugar / protein ratio: 14.34) as measured by MOLDI-TOF mass spectrometry.

[0049] In some embodiments, the compounds of the present invention are provided by formula (I).

[0050] Formula (I):

[0051]

[0052] The compounds of the present invention may comprise at least one pseudoamine (Pse) moiety conjugated to a carrier protein, wherein the pseudoamine moiety moiety is conjugated to the carrier protein via a linker and / or linker. In some embodiments, R at the N5 position 1 The group can be acetyl, formyl, or (R)-3-hydroxybutyryl. In some embodiments, the R at the N7 position... 2 The group can be acetyl, formyl, or (R)-3-hydroxybutyryl. In some embodiments, the connection between the pseudoamine moiety and the linker is a glycosidic bond, including α- or β-linking.

[0053] In some embodiments, the compounds of the present invention according to formula (II) may have PEG-based connectors with a variable number of (CH2CH2O) units, wherein m is from about 1 to about 5.

[0054] Equation (II):

[0055]

[0056] In some embodiments, according to formula (III), the compounds of the present invention may have a connector, which may be a saturated hydrocarbon chain of variable length having a length of about 2 to about 10. The value of m may range from about 0 to about 8.

[0057] Equation (III):

[0058]

[0059] In some embodiments, the compounds of the present invention may have a linker with a cyclic lactam structure, which may optionally be generated by a linkage reaction between the orthophthalaldehyde (OPA) moiety and the lysine side chain. The m value may range from about 0 to about 5.

[0060] Formula (IV):

[0061]

[0062] In some embodiments, the compounds of the present invention according to formula (V) may have a linker, which may be a maleimide thiol adduct, optionally generated via Michael addition of a thiol to a maleimide-modified lysine side chain. In some embodiments, the lysine side chain maleimide modification may be implemented using SMCC or other reagents containing maleimide and an amine-reactive NHS ester. The m value may range from about 1 to about 5.

[0063] Equation (V):

[0064]

[0065] In some embodiments, the compounds of the present invention may have a linker that is based on a triazole structure, optionally generated from a sugar-derived alkyne and an azide-modified protein side chain. The azide modification can be attached to the lysine side chain of the carrier protein using an azide containing an NHS ester or other active ester. The m value can range from about 0 to about 5, while the p value can range from about 1 to about 5.

[0066] Formula (VI):

[0067]

[0068] In some embodiments, the compounds of the present invention according to formula (VII) may have a linker that can be based on a triazole structure, optionally generated from a sugar-derived azide substance and an alkyne-modified protein side chain. The alkyne modification can be attached to the lysine side chain of the carrier protein using an alkyne containing an NHS ester or other active ester. The m value can range from about 1 to about 5, while the p value can range from about 0 to about 5.

[0069] Equation (VII):

[0070]

[0071] In some embodiments, the compounds of the present invention according to formula (VIII) may have a linker that can optionally be a thiol-yne adduct generated by free radical addition. The yne modification can be attached to the lysine side chain of the carrier protein using an yne containing an NHS ester or other active ester. The m value can range from about 1 to about 5, while the p value can range from about 0 to about 5.

[0072] Formula (VIII):

[0073]

[0074] In some embodiments, the compounds of the present invention are Pse-CRM197 1 (Formula (IX)), Pse-CRM197 2 (Formula (X)), Pse-CRM197 3 (Formula (XI)), Pse-BSA 17 (Formula (XII)) or other compounds having variations at the above sites:

[0075] Formula (IX):

[0076]

[0077] Equation (X):

[0078]

[0079] Formula (XI):

[0080]

[0081] Formula (XII):

[0082]

[0083] In some embodiments, the composition further comprises a suitable carrier, diluent, or buffer. Compositions contemplated within the scope of this invention may contain one or more other compounds for generating an immune response and / or for treating or preventing bacterial infections. For example, the Pse-carrier protein conjugate of this invention may be provided in a composition having one or more adjuvants and / or antibiotics. In one embodiment, the composition comprises a Pre-carrier protein conjugate in a pharmaceutically or physiologically acceptable carrier, buffer, or diluent.

[0084] In one embodiment, the compositions of the present invention are formulated as oral consumables, such as, for example, food products, capsules, pills, or drinkable liquids. Orally deliverable drugs are any physiologically active substances delivered through initial absorption in the gastrointestinal tract or through initial absorption into the mucous membranes of the oral cavity. Topical compositions can also be formulated as solutions that can be administered, for example, by injection, including intravenous injection, intraperitoneal injection, intramuscular injection, intrathecal injection, or subcutaneous injection. In other embodiments, the compositions of the present invention are formulated for administration via skin through patches or directly to the skin for local or systemic action. The compositions can be administered sublingually, orally, rectally, or vaginally. Furthermore, the compositions can be sprayed into the nose for absorption through the nasal membrane, atomized, inhaled through the mouth or nose, or administered through the eyes or ears.

[0085] An oral consumable according to the invention is any formulation or composition suitable for consumption, nutrition, oral hygiene, or pleasure, and is intended to be introduced into the oral cavity of a human or animal to remain there for a period of time and then either swallowed (e.g., food or pills prepared for consumption) or removed from the oral cavity again (e.g., chewing gum or oral hygiene products or medical mouthwash). Although orally deliverable drugs can be formulated into oral consumables, and oral consumables can contain orally deliverable drugs, these two terms are not intended to be used interchangeably herein.

[0086] Oral consumables include all substances or products intended for ingestion by humans or animals in a processed, semi-processed, or unprocessed state. This also includes substances added to oral consumables (particularly food and pharmaceuticals) during their production, processing, or manufacturing and intended to be introduced into the oral cavity of humans or animals.

[0087] Oral consumables may also include substances intended to be swallowed by humans or animals and then digested in an unmodified, prepared or processed state; therefore, oral consumables according to the invention also include casings, coatings or other encapsulations intended to be swallowed with the product or intended for use in swallowing.

[0088] In one embodiment, the oral consumable is a capsule, pill, syrup, emulsion, or liquid suspension containing a desired orally deliverable substance. In another embodiment, the oral consumable may comprise an orally deliverable substance in powder form, which may be mixed with water or another liquid to produce a drinkable oral consumable.

[0089] In some embodiments, the oral consumables according to the invention may contain one or more formulations intended for nutrition or pleasure. These specifically include baked goods (e.g., bread, biscuits, cakes, and other pastries), confectionery (e.g., chocolate, chocolate bar products, other bar products, pectin, coated sheets, hard caramel, toffee, and caramel, as well as chewing gum), and alcoholic or non-alcoholic beverages (e.g., cocoa, coffee, green tea, black tea, black tea or green tea beverages rich in green tea or black tea extracts, Rooibos tea, other herbal teas, fruit-containing lemonade, isotonic beverages, soft drinks, nectar, fruit and vegetable juices, and fruit or vegetable juice products). Instant beverages (such as instant cocoa drinks, instant tea drinks, and instant coffee drinks), meat products (such as ham, fresh or raw sausage products, and seasoned or cured fresh or cured meat products), eggs or egg products (such as dried whole eggs, egg whites, and egg yolks), cereal products (such as breakfast cereals, cereal bars, and pre-cooked instant rice products), dairy products (such as full-fat, reduced-fat, or non-fat dairy beverages, rice pudding, yogurt, kefir, cream cheese, soft cheese, hard cheese, dry milk powder, whey, buttermilk, and some other dairy products). Products containing milk protein (or fully hydrolyzed milk protein), products derived from soy protein or other soy fractions (e.g., soy milk and products made from it, beverages containing isolated or enzymatically treated soy protein, beverages containing soy flour, products containing soy lecithin, fermented products such as tofu or fermented soy products made from it, and mixtures with fruit products and optional flavorings), fruit products (e.g., jams, fruit ice cream, fruit sauces and fruit fillings), vegetable products (e.g., tomato sauces, sauces, dried vegetables, frozen vegetables, pre-prepared vegetables and boiled vegetables), confectionery products (e.g., baked or fried potato chips (slices) or potato dough products and extrusions based on corn or peanuts), products based on fats and oils or their emulsions (e.g., mayonnaise, remoulade and seasonings), other ready-to-eat meals and soups (e.g., dry soups, instant soups and pre-prepared soups), seasonings (e.g., spray seasonings), sweetener compositions (e.g., tablets, sachets and other products for sweetening or whitening beverages or other foods). The compositions of the present invention can also be used as semi-finished products for the production of other compositions intended for nutritional or pleasurable purposes.

[0090] The compositions of the present invention may further comprise one or more pharmaceutically acceptable carriers and / or excipients, and may be formulated into formulations such as solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants and aerosols.

[0091] As used herein, the term "pharmaceutically acceptable" means compatible with other components of a pharmaceutical composition and harmless to its recipient.

[0092] The carriers and / or excipients according to the invention may include any and all solvents, diluents, buffers (such as, for example, neutral buffered saline, phosphate buffered saline, or optionally Tris-HCl, acetate, or phosphate buffers), oil-in-water or water-in-oil emulsions, aqueous compositions containing or not containing organic cosolvents suitable for, for example, IV use, solubilizers (e.g., polysorbate 65, polysorbate 80), colloids, dispersion media, mediators, fillers, chelating agents (e.g., EDTA or glutathione), amino acids (e.g., glycine), proteins, disintegrants, binders, lubricants, wetting agents, emulsifiers, sweeteners, colorants, flavoring agents, aroma agents, thickeners (e.g., carbomer, gelatin, or sodium alginate), coatings, preservatives (e.g., thimerosal, benzyl alcohol, polyquaternium), antioxidants (e.g., ascorbic acid, sodium metabisulfite), tension control agents, absorption delay agents, adjuvants, fillers (e.g., lactose, mannitol), etc. The use of carriers and / or excipients in the pharmaceutical and supplement fields is well known. The use of carriers or excipients in the compositions of this invention may be considered, in addition to any conventional media or reagents incompatible with the target health-promoting substance or the composition.

[0093] In one embodiment, the compositions of the present invention can be formulated as aerosol formulations such that they can be nebulized or inhaled, for example. Suitable pharmaceutical formulations for administration in aerosol or spray form are, for example, powders, granules, solutions, suspensions, or emulsions. Formulations for oral or nasal aerosol or inhalation administration can also be formulated with a carrier comprising, for example, saline, polyethylene glycol or ethylene glycol, DPPC, methylcellulose, or formulated as a mixture with a powdered dispersant or fluorocarbon. Aerosol formulations can be contained in pressurized propellants such as dichlorodifluoromethane, propane, nitrogen, fluorocarbons, and / or other solubilizers or dispersants known in the art. Exemplarily, delivery can be achieved using a single-use delivery device, a nebulizer, a respiratory-activated powder inhaler, a metered-dose aerosol inhaler (MDI), or any other of the many nebulizer delivery devices available in the art. Alternatively, a mist tent or direct administration via an endotracheal tube can also be used.

[0094] In one embodiment, the compositions of the present invention can be formulated for administration by injection, for example, as a solution or suspension. The solution or suspension may contain a suitable non-toxic, parenteral acceptable diluent or solvent such as mannitol, 1,3-butanediol, water, Ringer's solution or isotonic sodium chloride solution, or a suitable dispersant or wetting agent and suspending agent such as a sterile, non-irritating fixative oil, including synthetic monoglycerides or diglycerides, and fatty acids including oleic acid. An illustrative example of a carrier for intravenous use includes a mixture of 10% USP ethanol, 40% USP propylene glycol or polyethylene glycol 600, and the balance USP water for injection (WFI). Other exemplary carriers for intravenous use include 10% USP ethanol and USP WFI; 0.01-0.1% triethanolamine in USP WFI; or 0.01-0.2% dipalmitoylphosphatidylcholine in USP WFI; and 1-10% squalene or a parenteral water-in-oil emulsion. Aqueous or saline solutions and aqueous solutions of dextran and glycerol are preferred as carriers, especially for injectable solutions. Illustrative examples of carriers for subcutaneous or intramuscular use include phosphate-buffered saline (PBS) solutions, 5% dextran in WFI and 0.01-0.1% triethanolamine in 5% dextran or 0.9% sodium chloride in USP WFI or a 1:2 or 1:4 mixture of 10% USP ethanol, 40% propylene glycol and the balance being an acceptable isotonic solution such as 5% dextran or 0.9% sodium chloride; or 0.01-0.2% dipalmitoyldiphosphatidylcholine in USP WFI and 1 to 10% squalene or parenteral water-in-oil emulsions.

[0095] In one embodiment, the compositions of the present invention can be formulated for administration by topical application to the skin, for example, as topical compositions, comprising rinses, sprays or drops, lotions, gels, ointments, creams, foams, powders, solids, sponges, strips, vapors, pastes, tinctures, or transdermal patches. Suitable formulations for topical application, in addition to any pharmaceutically active carrier, may contain, for example, emollients such as carnauba wax, cetyl alcohol, cetyl ester wax, emulsified wax, aqueous lanolin, lanolin, lanolin alcohol, microcrystalline wax, paraffin, petrolatum, polyethylene glycol, stearic acid, stearyl alcohol, white beeswax, or yellow beeswax. Additionally, the compositions may contain wetting agents such as glycerin, propylene glycol, polyethylene glycol, sorbitol solution, and 1,2,6-hexanetriol, or penetration enhancers such as ethanol, isopropanol, or oleic acid.

[0096] The Pse-carrier protein conjugates of the present invention can be formulated into pharmaceutically acceptable salt forms. Pharmaceutically acceptable salts of the Pse-carrier protein conjugates of the present invention can be prepared using conventional techniques. "Pharmaceutically acceptable salt" includes both acid addition salts and base addition salts. The pharmaceutically acceptable salt of any Pse-carrier protein conjugate described herein is intended to include any and all pharmaceutically suitable salt forms. Preferred pharmaceutically acceptable salts described herein are pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts. In some embodiments, the pharmaceutically acceptable salts include acetate, chloride, or trifluoroacetic acid (TFA) salts.

[0097] "Pharmaceutically acceptable acid addition salts" refer to those salts that retain the biological effectiveness and properties of the free base, are not biologically or otherwise undesirable, and form with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, hydroiodic acid, hydrofluoric acid, phosphorous acid, etc. This also includes salts formed with organic acids such as aliphatic monocarboxylic acids and dicarboxylic acids, phenyl-substituted alkanes, hydroxyalkanes, alkanedioic acids, aromatic acids, aliphatic and aromatic sulfonic acids, etc., including, for example, acetic acid, trifluoroacetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, etc. Therefore, exemplary salts include sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, nitrates, phosphates, monohydrogen phosphates, dihydrogen phosphates, metaphosphates, pyrophosphates, chlorides, bromides, iodides, acetates, trifluoroacetates, propionates, caprylates, isobutyrates, oxalates, malonates, succinates, caprylates, sebacic acid salts, fumarates, maleates, mandelates, benzoates, chlorobenzoates, methylbenzoates, dinitrobenzoates, phthalates, benzenesulfonates, toluenesulfonates, phenylacetates, citrates, lactates, malates, tartrates, methanesulfonates, etc. Salts of amino acids such as arginine salts, gluconates, and galacturons are also considered (see, for example, Berge SM et al., “Pharmaceutical Salts,” Journal of Pharmaceutical Science, 66:1–19 (1997), which is incorporated herein by reference in its entirety). Acid addition salts of basic compounds can be prepared by contacting a free base with a sufficient amount of the desired acid to produce a salt, according to methods and techniques familiar to those skilled in the art.

[0098] "Pharmaceutically acceptable base addition salts" are those salts that retain the bioavailability and properties of the free acid, and are not biologically or otherwise undesirable. These salts are prepared by adding an inorganic or organic base to the free acid. Pharmaceutically acceptable base addition salts can be formed from metals or amines such as alkali metals and alkaline earth metals or organic amines. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, and aluminum salts. Salts derived from organic bases include, but are not limited to, salts of the following substances: primary, secondary, and tertiary amines; substituted amines, including naturally occurring substituted amines; cyclic amines; and basic ion exchange resins such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, diethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, N,N-dibenzylethylenediamine, chloroprocaine, heparin, choline, betaine, ethylenediamine, ethylenediphenylamine, N-methylglucosamine, glucosamine, methylglucosamine, theobromine, purine, piperazine, piperidine, N-ethylpiperidine, polyamine resins, etc. See Berge et al., ibid.

[0099] The therapeutic and preventative application of the Pse-carrier protein conjugates and compositions thereof of the present invention can be achieved by any suitable treatment methods and techniques currently known or anticipated by those skilled in the art. The Pse-carrier protein conjugates can be administered via any suitable route known in the art, including, for example, local, oral, mucosal (e.g., nasal), rectal, parenteral, subcutaneous, or intravascular (e.g., intravenous) administration. Therefore, administration can be local or systemic at the desired anatomical site of the subject (e.g., the site of current or potential infection). As readily determined by those skilled in the art, the administration of the Pse-carrier protein conjugates of the present invention can be continuous or at varying intervals.

[0100] The Pse-carrier protein conjugates and compositions of the present invention can be administered to a subject together with one or more adjuvants. The adjuvant can be administered simultaneously or sequentially with the Pse-carrier protein conjugates and compositions thereof. The adjuvant can be administered in the same composition as the Pse-carrier protein conjugate or as a separate composition. In some embodiments, the adjuvant is an alum salt or other mineral adjuvant, a bacterial product or bacterial-derived adjuvant, a tonicotinic agent (e.g., saponin), an oil / water (o / w) or water / oil (w / o) emulsion, a liposome adjuvant, a cytokine (e.g., IL-2, GM-CSF, IL-12, and IFN-γ), an α-galactosylceramide analog, or a Toll-like receptor (TLR) ligand. In another embodiment, the adjuvant is QS21, Freund's complete or incomplete adjuvant, aluminum phosphate, aluminum hydroxide, BCG, or alum. Other specific examples of adjuvants are provided in Pasquale AD et al., “Vaccine Adjuvants: from 1920 to 2015 and Beyond”, Vaccines, 2015, 3:320-343; Petrovsky N. et al., “Vaccine Adjuvants: Current State and Future Trends”, Immunology and Cell Biology, 2004, 82:488-496; and Vogel FR, “Improving Vaccine Performance with Adjuvants”, Clin Infect Dis., 2000, 30(Supplement 3):S266-S270, which are incorporated herein by reference in their entirety.

[0101] The Pse-carrier protein conjugates and compositions of the present invention can also be administered using liposome technology, sustained-release capsules, implantable pumps, and biodegradable containers. These delivery methods can advantageously provide a uniform dose over an extended period of time.

[0102] Pse-carrier protein conjugates can be covalently bound or otherwise linked to molecules that increase the half-life, solubility, bioavailability, or immunogenicity of antigens (e.g., adjuvants). Molecules that can covalently bind to antigens include sugars, biotin, polyethylene glycol (PEG), polysialic acid, N-propionyl polysialic acid, nucleic acids, polysaccharides, and PLGA. Many different types of PEG exist, with molecular weights ranging from below 300 g / mol to above 10,000,000 g / mol. PEG chains can be linear, branched, or have comb-like or star-shaped geometries. In some embodiments, the naturally occurring form of the protein is covalently bound to a portion that stimulates the immune system.

[0103] The present invention also relates to packaged dosage forms containing at least one pharmaceutically acceptable dose of the Pse-carrier protein conjugate and / or composition of the present invention in one or more containers. The package may contain discrete amounts of the dosage form, such as tablets, capsules, lozenges, and powders. The amount of the Pse-carrier protein conjugate in the dosage form that can be administered to a patient may vary from about 1 mg to about 5000 mg, or from about 1 mg to about 2000 mg, or more generally from about 1 mg to about 500 mg, or from about 5 mg to about 250 mg, or from about 10 mg to about 100 mg.

[0104] The present invention also relates to kits comprising one or more Pse-carrier protein conjugates, compositions, compounds, or molecules of the present invention in one or more containers. In one embodiment, the kit contains the Pse-carrier protein conjugates and / or compositions of the present invention.

[0105] In addition to the Pse-carrier protein conjugates and / or compositions of the present invention, the kits of the present invention may also contain one or more compounds, biomolecules or drugs for treating pathogenic infections such as Acinetobacter baumannii infection.

[0106] In one embodiment, the kit of the present invention includes instructions or packaging materials describing how to administer the Pse-carrier protein conjugate, composition, compound, or molecule to the kit. The container of the kit can be any suitable material, such as glass, plastic, metal, etc., and can be any suitable size, shape, or construction. In one embodiment, the Pse-carrier protein conjugate, composition, compound, or molecule of the present invention is provided in the kit as a solid such as a tablet, pill, or powder. In another embodiment, the Pse-carrier protein conjugate, composition, compound, or molecule of the present invention is provided in the kit as a liquid or solution. In one embodiment, the kit comprises an ampoule or syringe containing the Pse-carrier protein conjugate, composition, compound, or molecule of the present invention in liquid or solution form. In one embodiment, the kit further comprises one or more adjuvants, such as those disclosed herein.

[0107] Any method of the present invention may optionally include the step of identifying a person or animal who needs or may need treatment or prevention of a disease, symptom, or condition (e.g., Acinetobacter baumannii infection).

[0108] Biological samples refer to fluid or tissue compositions obtained from humans or animals. Biological samples within the scope of this invention include, but are not limited to, cells, whole blood, peripheral blood, plasma, bone marrow, spleen, serum, urine, tears, saliva, sputum, exhaled breath, nasal secretions, pharyngeal secretions, bronchoalveolar lavage, tracheal inspiration, interstitial fluid, lymph, meningeal fluid, amniotic fluid, glandular fluid, feces, sweat, mucus, vaginal or urethral secretions, cerebrospinal fluid, and transdermal secretions. Biological samples also include experimentally separated fractions of all the aforementioned solutions or mixtures containing homogenized solid materials such as feces, cells, tissues, and biopsy samples.

[0109] In some embodiments, the Pse-carrier protein conjugate can elicit a humoral response. In some embodiments, the Pse-carrier protein conjugate can induce the production of IgG1, IgG2b, IgG3, and IgG2c.

[0110] The description of a list of chemical groups in any definition of a variable herein includes the definition of that variable as any single group or combination of the listed groups. The description of embodiments of a variable or aspect herein includes embodiments as any single embodiment or in combination with any other embodiment or part thereof.

[0111] Any composition or method provided herein may be combined with one or more of any other compositions and methods provided herein.

[0112] Other features and advantages of the invention will be apparent from the following description of its preferred embodiments and from the claims.

[0113] Materials and methods

[0114] The bacterial strains and mice used in this study

[0115] Previously reported strain Ab2 of Acinetobacter baumannii, which produces pseudoamine. 29 The bacteria were cultured at 37°C in Luria-Bertani (LB) broth or brain and heart infusion (BHI) agar.

[0116] Male C57BL / 6J inbred mice (6-8 weeks old, approximately 20g) were obtained from the Laboratory Animal Research Unit (LARU) of City University of Hong Kong. Animal rest and handling were strictly in accordance with the Hong Kong Animal (Experimental Control) Ordinance (Cap. 340). All animal experiments were approved by the Animal Research Ethics Subcommittee (ARESC) of City University of Hong Kong. Animals were housed under specific pathogen-free conditions during experiments. Every effort was made to minimize animal suffering.

[0117] mouse immunity

[0118] Male 6–8 week old inbred C57BL / 6J mice were subcutaneously (sc) immunized with Pse vaccine (2.4 μg glucose per dose) mixed with 1:1 (v / v) aluminum hydroxide (Thermo Fisher Scientific, Waltham, MA, USA). Control mice received CRM197 mixed with aluminum hydroxide in PBS. Mice received booster injections using the same formulation on days 14 and 28. Blood (50 μl) was aspirated from the tail vein on days 0, 21, 35, and 65 and centrifuged (5000 x g, 10 min, room temperature) to recover serum. Antibody responses in serum were measured using ELISA.

[0119] ELISA assay for detecting the immunogenicity of vaccines

[0120] Highly bound 96-well polystyrene microtiter plates were coated overnight at 4°C with BSA-Pse (2 μg / ml, 100 μl per well) in sodium carbonate-bicarbonate buffer (pH 9.6). The next day, the plates were washed three times with PBS (PBST) containing 0.1% Tween-20 and blocked at 37°C for 2 hours with 2% BSA-PBS (200 μl per well). After washing three times with PBST, the plates were incubated at 37°C for 1 hour with post-immunization serum (100 μl per well) at a 2-fold dilution starting from 100. Again, the plates were washed three times with PBST and further incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse antibody (Abcam, Cambridge, UK) at a 1:10000 dilution in PBS (100 μl per well), followed by incubation at 37°C for 1 hour. The plates were washed three times with PBST and developed with tetramethylbenzidine (TMB). The reaction was terminated by adding 2% sulfuric acid, and the absorbance was recorded at 450 nm. Pse-specific mouse antibody isotypes were performed using the SBA clone typing system for C57BL / 6 mice (SouthernBiotech, Birmingham, AL, USA). BSA-Pse was used to coat 96-well polystyrene microtiter plates and capture Pse-specific antibodies from serum. Pse-specific antibodies were typed using HRP-conjugated goat anti-mouse IgA, IgG1, IgG2b, IgG2c, IgG3, IgM, κ, and λ.

[0121] Flow cytometry

[0122] Flow cytometry was performed to determine the binding capacity of post-immunization serum to Pse-producing Acinetobacter baumannii strains. Briefly, overnight cultures of Acinetobacter baumannii strain Ab2 were collected, washed with PBS, and adjusted to OD ≈ 0.2 with PBS. 500 μl of bacterial suspension was incubated for 1 h with 100-fold diluted post-immunization serum collected on day 35. After washing with PBS, the bacteria were incubated for 1 h with Alexa Fluor 647-labeled secondary goat anti-mouse antibody (Abcam). After further washing, the bacteria were resuspended in 2 ml of PBS and analyzed by BDFACSVia flow cytometry (BD Biosciences, Franklin Lakes, NJ). Bacteria incubated with only the secondary antibody served as a negative control.

[0123] The efficacy of Pse-based vaccines

[0124] The efficacy of the Pse vaccine was characterized using a mouse sepsis model. 32 First, the 50% lethal dose (LD50) of Acinetobacter baumannii strain Ab2 was determined by infecting mice with serially diluted bacteria. 50 Acinetobacter baumannii strain Ab2 was cultured in LB medium at 37°C to the logarithmic developmental phase (OD ~0.6), and then adjusted to an appropriate concentration in PBS. The bacterial concentration of the inoculum was determined by plating on BHI agar plates. Male 6-8 week old inbred C57BL / 6J mice were intraperitoneally infected with 0.2 ml of bacterial suspension. Mice survival was observed and recorded at 12-hour intervals for 7 days post-infection. Mice survival was determined by infecting mice with Acinetobacter baumannii strain Ab2. Mice were immunized on days 0, 14, and 28 as previously described. Two weeks after the last immunization, four groups of inoculated mice and control mice were inoculated with Acinetobacter baumannii strain Ab2 at high concentrations of 2-fold (2×LD50) and 5-fold (5×LD50), respectively. Mice survival was observed and recorded 7 days post-infection. Appearance and behavior were also evaluated.

[0125] Bacterial load in infected and control mice was determined at a high bacterial load of 5×LD50 for Acinetobacter baumannii strain Ab2. Mice were anesthetized 12 hours after bacterial inoculation. Blood and tissues, including spleen, kidney, lung, liver, and heart, were aseptically collected. The tissues were weighed and homogenized in sterile PBS. Serial dilutions of tissues and blood were plated on BHI agar and incubated at 37°C for bacterial quantification. Serum levels of interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and IL-6 were measured in mice 12 hours after infection with Acinetobacter baumannii strain Ab2 at 5×LD50 using a mouse ELISA kit (Thermo Fisher Scientific).

[0126] All patents, patent applications, provisional applications and publications mentioned or cited herein are incorporated herein by reference in their entirety, including all figures and tables, to the extent that they do not contradict the express teachings of this specification.

[0127] The following are examples illustrating the procedures for implementing the present invention. These examples should not be construed as limiting. Unless otherwise specified, all percentages are by weight and all solvent mixture proportions are by volume.

[0128] Example 1 – Synthesis of Pse-based antibacterial vaccine

[0129] Since sugars are T-cell-independent antigens that cannot generate strong, durable, and memorized IgG antibodies, they must be conjugated to immunogenic carrier proteins capable of activating helper T cells to enhance anti-glycan antibody titers. The nontoxic mutant of diphtheria toxin, CRM197, is currently widely used as a carrier protein for glycans to impart immunogenicity. We designed an o-phthalaldehyde (OPA)-pseudoamine liner, in which OPA is reported to react with primary amines to conjugate pseudoamine to the carrier protein. For this purpose, Pse donor 4 was stereoselectively glycosylated with a Fmoc-protected PEG linker 5 to produce α-glycoside 6 in 80% yield. Figure 7 The glycosylation conditions used here are NIS / TfOH with DMF as an additive in DCM. Other activation conditions, such as NIS / TMSOTf, NIS / AgOTf, TolSCl / AgOTf, and PhSCl / AgOTf, can also be used alternatively, yielding lower variable yields and selectivity. The additive DMF can be changed to other amides such as N-formylpiperidine and N,N-dimethylacetamide. After converting N5-azide and N7-carbamate to acetamide 7 via hydrogenolysis and acetylation, the Fmoc group is then removed to produce a free amine, which is coupled to acid 8 containing methyl acetal phthalaldehyde. Figure 8The coupling conditions (EDCI / DIPEA) used here can be changed to other amide coupling conditions such as DCC, DIC, HATU, HBTU, PyBOP, PyBroP, DEPBT, EEDQ, and COMU. Finally, deprotection is performed by treatment with lithium hydroxide followed by a 10% aqueous acetic acid solution (or other concentrations from 5% to 75%) to obtain the Pse-OPA moiety. The Pse-OPA moiety is then reacted with the CRM197 carrier protein in phosphate-buffered saline (PBS, pH 7.4) to produce the CRM197-Pse conjugate. To investigate the differences in immune responses induced by different antigen loads, we used 20, 30, and 50 equivalents of OPA-Pse partially synthetic conjugates to generate Pse-CRM197 1 (glucose / protein ratio: 4.76), Pse-CRM197 2 (glucose / protein ratio: 8.27), and Pse-CRM197 3 (glucose / protein ratio: 14.34), respectively.

[0130] In addition, we synthesized bovine serum albumin (BSA)-Pse conjugate 17 as a substitute for natural glycans to validate anti-Pse antibodies generated by the vaccine. Different alkyl linkers and thiol-maleimide strategies were used to reduce unintended recognition of carrier proteins and linkers by the booster serum. Using the same strategy as above, compound 13 was obtained using Pse donor 4 and FmocNH(CH2)6OH11, coupled with 2-(acetylthio)acetic acid 14 after Fmoc removal to give 15 (… Figure 8 Finally, Pse-thiol linker 16 was obtained after saponification. Commercially available BSA was treated with N-(ε-maleimide hexoxy)sulfosuccinimide ester (sulfon-EMCS) in PBS (pH 7.4) to attach maleimide to the protein, which was further reacted with Pse-thiol linker 16 in PBS (pH 7.4) to give BSA-Pse conjugate 17.

[0131] Synthesis of N-(2-(2-(2-(2-(4,8-di-O-acetyl-5-azido-7-N-benzyloxycarbonyl-1-isopropyl-α-pseudoaminooxy)ethoxy)ethoxy)ethoxy)ethyl)carbamate (9H-fluorene-9-yl)methyl ester (6) Figure 9 ).

[0132] Under argon atmosphere, flame-dried AW-300 molecular sieve (100 mg) was added to a flame-dried Schlenk tube, followed by Pse donor 4 (41.7 mg, 0.0500 mmol, 1.0 equivalent), acceptor 5 (46.0 mg, 0.100 mmol, 2.0 equivalent), anhydrous DCM (freshly distilled on CaH2, 1.0 mL), and anhydrous DMF (17.0 μL, 0.250 mmol, 5.0 equivalent). After stirring at room temperature for 1 hour, the mixture was cooled to -78 °C, and N-iodosuccinimide (27.0 mg, 0.120 mmol, 2.4 equivalent) was added. Finally, trifluoromethanesulfonic acid (0.450 μL, 0.1 equivalent) was added dropwise to initiate the reaction, and the mixture was stirred at -40 °C for 8 hours. Complete conversion was achieved by quenching the reaction with Et3N, as shown by TLC. The mixture was diluted with ethyl acetate and filtered through diatomaceous earth. The organic phase was then washed with saturated NaHCO3 (aqueous solution), dried over anhydrous Na2SO4, and concentrated under vacuum. Product 6 was purified by silica gel column chromatography using a 1:1 volume / volume ratio of n-hexane:ethyl acetate as the eluent. f (n-Hexane:ethyl acetate 1:1) = 0.15. Only the α-terminal isomer was obtained (J Cl-H3a =0Hz, on an Advance DRX Bruker 500MHz NMR spectrometer via non-decoupling 13 The sample was measured by a C-spectrum and was a white solid (37.3 mg, 80%).

[0133] 1 H NMR (500MHz, CDCl3): δ=7.75(d,J=7.5Hz,2H,ArH),7.52-7.68(m,2H,ArH),7.39(t,J=7.5Hz,2H,ArH),7.26-7.36(m,7H,A rH),5.59(d,J=10.0Hz,1H,NH),5.30-5.41(m,2H,H-4,NH),5.00-5.15(m,3H,PhCH2O,CH(CH3)2),4.33-4.45(m,3H,H-7,C 12 H8CHCH2),4.20(t,J=6.5Hz,1H,C 12H8CHCH2),4.11(d,J=9.5Hz,1H,H-6),3.90(s,1H,H-5),3.76-3.84(m,1H,OCH2CH2O) ,3.53-3.70(m,10H,OCH2CH2O),3.36-3.52(m,4H,OCH2CH2O),3.26-3.31(m,1H,NHCH 2),2.13-2.17(m,2H,H-3a,H-3e),2.10(s,3H,CH3CO),2.02(s,3H,CH3CO),1.35(d,J =6.5Hz,3H,H-9),1.27(d,J=6.0Hz,3H,CH(CH3)2),1.27(d,J=6.0Hz,3H,CH(CH3)2).

[0134] 13 C NMR (125MHz, CDCl3): δ=170.8,170.3,166.4,16.4,156.7,156.2,144.2,141.4,136.5,128.6,128.4,128.3,127.8,127.2,125.2,120. 1,98.2,71.6,70.8,70.6,70.4,70.3,70.1,69.9,69.8,69.3,67.2,66.7,63.1,59.4,53.9,47.4,40.9,32.1,21.80,21.77,21.3,20.9.

[0135] HR-ESI-MS(m / z): For C 47 H 59 N5O 15 Na + (M+Na) Calculated value: 956.3900, measured value: 958.3858.

[0136] Synthesis of N-(2-(2-(2-(2-(5,7-diacetamido-4,8-di-O-acetyl-1-isopropyl-α-pseudoamineoxy)ethoxy)ethoxy)ethoxy)ethyl)carbamate (9H-fluorene-9-yl)methyl ester (7) Figure 10 ).

[0137] DCM (2.0 mL) and MeOH (2.0 mL) were added to a 25 mL round-bottom flask containing 6 (103 mg, 0.108 mmol, 1.0 equivalent), Pd / C (Pd on 10% activated carbon, 50 mg), and NH4OAc (30 mg, 0.38 mmol, 4.0 equivalent). The mixture was stirred for 1 hour under a H2 atmosphere at 1 atm, and then filtered through diatomaceous earth to remove the catalyst. NMM (2.0 mL, excess) and Ac2O (1.0 mL, excess) were added to the filtrate. After stirring at room temperature for 2 hours, the mixture was concentrated under vacuum. The residue was dissolved in ethyl acetate (50 mL), and the solution was washed successively with 1 M HCl (aqueous solution) and saturated NaHCO3 (aqueous solution). The organic phase was dried over anhydrous Na2SO4, and the solvent was removed under vacuum. The residue was purified by silica gel column chromatography using EtOAc:methanol 20:1 as the eluent. f (EtOAc:methanol 20:1) = 0.21. Product 7 was obtained, which was a colorless syrup (68.4 mg, 77%).

[0138] 1 H NMR (500MHz, CDCl3): δ = 7.76 (d, J = 7.5Hz, 2H, ArH), 7.61 (d, J = 7.0Hz, 2H, ArH), 7.40 (t, J = 7.5Hz, 2H, ArH), 7. 31(t,J=7.5Hz,2H,ArH),6.31(d,J=10.5Hz,1H,NH),6.25(d,J=10.0Hz,1H,NH),5.70(t,J=5.0Hz,1H,NH),1. 28(d,J=6.5Hz,3H,H-9),1.29(d,J=6.0Hz,3H,(CH3)2CH),5.24(dt,J1=12.0Hz,J2=4.5Hz,1H,H-4),5.19(qd ,J1=6.5Hz,J2=3.5Hz,1H,H-8),5.06-5.13(m,1H,(CH3)2CH),4.48-4.60(m,2H,H-5,H-7),4.36-4.43(m,2H,C 12 H8CHCH2),4.31(d,J=10.5Hz,1H,H-6),4.22(t,J=6.5Hz,1H,C 12H8CHCH2),3.55-3.76(m,13H,OCH2CH2O),3.51(dt,J1=10.5Hz,J2=2.5Hz,1H,OCH2CH2O),3.36-3.43(m,2H,OCH2CH2O),2.12(dd,J1=13.0Hz,J2=5.0Hz,1 H,H-3e),2.03(s,3H,CH3CO),2.00(s,3H,CH3CO),1.96(s,3H,CH3CO),1.91( s, 3H, CH3CO), 1.83 (t, J = 13.0Hz, 1H, H-3a), 1.30 (d, J = 6.0Hz, 3H, (CH3) 2CH).

[0139] 13 C NMR (125MHz, CDCl3): δ=171.3,170.9,170.6,170.3,167.2,156.8,144.1,141.4,127.8,127.1,125.2,120.0,98.6,71.0,7 0.6,70.25,70.17,70.10,70.07,69.8,67.3,66.7,63.4,50.2,47.4,45.7,41.0,32.5,23.3,23.1,21.8,21.4,21.1,15.4.

[0140] HR-ESI-MS(m / z): For C 43 H 59 N3O 15 Na + (M+Na) Calculated value: 880.3838, measured value: 880.3850.

[0141] Synthesis of 3-(1,3-dimethoxy-1,3-dihydroisobenzofuran-5-yl)-N-(2-(2-(2-(2-(5,7-di-acetamido-4,8-di-O-acetyl-1-isopropyl-β-pseudoaminooxy)ethoxy)ethoxy)ethoxy)ethyl)propionamide (9) Figure 11 ).

[0142] Add 3 mL of 20% diethylamine in MeCN solution (v / v) to a 10 mL round-bottom flask containing 7 (21.8 mg, 0.254 mmol, 1.0 equivalence) and stir the solution at room temperature for 6 hours. Concentrate the mixture under vacuum to remove the diethylamine and redissolve the residue in DCM (4 mL). Add acid 8 (32 mg, 5.0 equivalence) to the above solution, followed by EDCI (25 mg, 5.0 equivalence) and DIPEA (45 μL, 10.0 equivalence), and monitor the reaction by TLC. When complete, dilute the mixture with ethyl acetate (20 mL), followed by washing with NaHCO3 (aqueous solution) and brine. Dry the organic phase with anhydrous Na2SO4 and remove the solvent under vacuum. Purify the residue by silica gel column chromatography using EtOAc:methanol 10:1 as the eluent. Product 9 is given as a white solid (15.5 mg, 71%). f (EtOAc:methanol 10:1) = 0.25. Due to extensive overlap of chemical shifts, the proportion of each isomer cannot be determined using NMR.

[0143] 1 H NMR (400MHz, CDCl3): δ = 7.15-7.54 (m, 5.8H), 6.99-7.13 (m, 1H), 6.59-6.78 (m, 1H), 6.26 (d, J = 3.5Hz, 0.7H), 5.98-6.03 (m, 1H), 5.83-5. 92(m,1H),5.59-5.65(m,0.7H),5.23(qd,J1=6.5Hz,J2=3.0Hz,1H),5.03-5.13(m,1H),4.73(d,J=16.5Hz,1H),4.46-4.56(m,2H),3.92- 4.06(m,2.4H),3.75(t,J=8.5Hz,1.2H),3.48-3.68(m,14H),3.34-3.48(m,11H),3.25-3.34(m,6H),2.84-3.06(m,6H),2.70(s,2.6H),2 .61(t,J=9.5Hz,0.7H),2.38(dt,J1=16.5Hz,J2=5.0Hz,1H),2.25-2.33(m,0.7H),1.93-2.05(m,11H),1.90(s,4H),1.20-1.34(m,19H).

[0144] 13C NMR (100MHz, CDCl3): δ=172.3,172.1,171.5,170.58,170.50,170.1,167.3,143.3,143.1,141.3,139.07,138.99,1 38.90,138.81,138.79,136.71,136.59,136.45,136.34,136.31,136.15,135.92,135.88,133.7,130.40,130.28,13 0.20,130.06,128.2,127.24,127.00,126.99,126.76,126.68,126.59,123.13,123.10,122.95,122.90,122.87,122.7,110.0,106.52,106.50,105.48,105.44,105.43,105.41,101.2,99.2,72.5,70.35,70.32,70.29,70.27,70.06, 69.96,69.94,69.83,69.81,67.8,63.7,54.64,54.54,54.37,54.33,54.28,54.27,54.19,54.12,54.09,53.7,53.6,53.5,50.6,45.9,45.3,43.3,41.5,39.2,38.05,38.04,37.76,37.67,35.7,35.6,35.46,35.44,34.0,32.9,31.90, 31.88,31.51,31.45,30.9,30.7,30.56,30.53,29.76,29.67,29.63,29.60,29.50,29.47,29.33,29.29,29.25,29.17,29.10,27.20,27.15,25.3,24.93,24.85,23.2,22.94,22.92,22.7,21.7,21.2,20.9,14.7,14.09,14.03,14.02.

[0145] HR-ESI-MS(m / z): For C 41 H 63 N3O 17 Na + (M+Na + Calculated value: 892.4050, measured value: 892.4061.

[0146] Synthesis of 3-(3,4-dicarboxyphenyl)-N-(2-(2-(2-(2-(5,7-diacetamido-α-2-pseudoamineoxy)ethoxy)ethoxy)ethyl)propionamide (10) Figure 12 ).

[0147] Lithium hydroxide monohydrate (9.4 mg, 0.224 mmol, 10.0 equivalent) was dissolved in a mixture of THF, MeOH, and H₂O (3 mL + 0.75 mL + 0.75 mL) to obtain a 0.5 mM LiOH solution. This solution was added to a 10 mL round-bottom flask containing compound 9 (19.5 mg, 0.0224 mmol, 1.0 equivalent), and the mixture was stirred at room temperature for 48 hours. The mixture was then passed through a Dowex 50H filter. + The resin was neutralized and filtered. The filtrate was evaporated under vacuum, and 3 mL of 10% HOAc aqueous solution (v / v) was added to the residue. The mixture was stirred at room temperature for 6 hours. The mixture was then evaporated under vacuum and purified by RP HPLC. R = 21.2 minutes (Column: Vydac 218TP C18) Column (Grace Davison Discovery Sciences); eluent A: MECN and B: H2O; gradient: sample run at 10 mL / min, gradient 5–20% A, duration 35 min; detection: UV 200 nm. Compound 10 was obtained as a mixture of dialdehyde and hemiacetal as a white solid (13.2 mg, 77%).

[0148] 1 H NMR (500MHz, D2O): δ=7.22-7.98(m,3H),6.42(s,1H),4.05-4.18(m,5H),3.75(d,J=10.0Hz,1H),3.31-3.69(m,15H),3.2(s,2H),2.93( t,J=7.0Hz,1H),2.51(t,J=7.0Hz,1H),2.01(d,J=11.0Hz,1H),1.90(s,3H),1.88(s,3H),1.48(t,J=12.5Hz,1H),1.04(d,J=6.0Hz,3H).

[0149] 13C NMR (125MHz, D2O): δ=175.5,174.8,174.6,173.7,142.8,139.2,130.4,122.8,117.4,115.2,100.4,7 0.4,69.7,69.5,69.3,68.8,66.9,64.9,62.5,53.6,48.6,38.8,37.3,35.1,31.2,22.04,21.92,15.6.

[0150] HR-ESI-MS(m / z): For C 32 H 47 N3O 14 Na + (M+Na + Calculated value: 720.2950, ​​measured value: 720.2959.

[0151] Synthesis of N-(4,8-di-O-acetyl-5-azido-7-N-benzyloxycarbonyl-1-isopropyl-α-pseudoaminooxy)pentylcarbamate (9H-fluorene-9-yl)methyl ester (12) Figure 13 ).

[0152] Under argon atmosphere, flame-dried AW-300 molecular sieve (100 mg) was added to a flame-dried Schlenk tube, followed by Pse donor 4 (41.7 mg, 0.0500 mmol, 1.0 equivalence), acceptor 11 (46.0 mg, 0.100 mmol, 2.0 equivalence), anhydrous DCM (freshly distilled on CaH2, 1.0 mL), and anhydrous DMF (17.0 μL, 0.250 mmol, 5.0 equivalence). After stirring at room temperature for 1 hour, the mixture was cooled to -78 °C, and N-iodosuccinimide (27.0 mg, 0.120 mmol, 2.4 equivalence) was added. Finally, trifluoromethanesulfonic acid (0.450 μL, 0.1 equivalence) was added dropwise to initiate the reaction, and the mixture was stirred at -40 °C for 8 hours. As shown by TLC, complete conversion was achieved by quenching the reaction with the addition of Et3N. The mixture was diluted with ethyl acetate and filtered through diatomaceous earth. The organic phase was then washed with saturated NaHCO3 (aqueous solution), dried over anhydrous Na2SO4, and concentrated under vacuum. Product 12 was purified by silica gel column chromatography using a 2:1 volume / volume ratio of n-hexane:ethyl acetate as the eluent. f (n-Hexane:ethyl acetate 1:1) = 0.34. Only the α-terminal isomer was obtained (J Cl-H3a =0Hz, on an Advance DRX Bruker 500MHz NMR spectrometer via non-decoupling 13 (Measured by C-spectroscopy), it is a white solid (35.5 mg, 83%).

[0153] 1 ¹H NMR (500 MHz, CDCl₃): δ = 7.74 (d, J = 10.5 Hz, 2H, ArH), 7.54 (d, J = 6.7 Hz, 2H, ArH), 7.35-7.41 (m, 3H, ArH), 7.19-7.33 (m, 6H, ArH), 5.77 (d, J = 9.6 Hz, 1H, NH), 5.34 (dt, J₁ = 11.0 Hz, J₂ = 4.1 Hz, 1H, H-4), 5.21-5.30 (m, 1H, H-8), 4.99-5.12 (m, 3H, PhCH₂O, CH(CH₃)₂), 4.94 (t, J = 5.7 Hz, 1H, NH), 4.30-4.42 (m, 3H, H-7, C 12 H₈CHCH₂), 4.09-4.13 (m, 1H, C 12 H₈CHCH₂), 3.98 (s, 1H, H-5), 3.88 (d, J = 8.7 Hz, 1H, H-6), 3.48-3.58 (m, 1H), 3.27-3.38 (m, 1H), 3.06-3.22 (m, 2H), 2.21 (dd, J₁ = 12.6 Hz, J₂ = 4.7 Hz, 1H, H-3e), 2.11 (t, J = 12.6 Hz, 1H, H-3a), 2.09 (s, 3H, CH₃CO), 2.00 (s, 3H, CH₃CO), 1.46-1.61 (m, 4H), 1.36 (d, J = 6.2 Hz, 3H, H-9), 1.23-1.27 (m, 8H).

[0154] 13 ¹³C NMR (125 MHz, CDCl 3) ): δ = 170.3, 170.0, 166.4, 156.7, 143.95, 143.91, 141.29, 141.27, 136.3, 128.5, 128.44, 128.08, 127.6, 127.0, 124.93, 124.85, 119.9, 71.78, 71.69, 69.85, 69.70, 69.62, 69.39, 69.30, 66.96, 66.57, 63.18, 59.23, 59.13, 59.9, 47.17, 47.09, 41.1, 32.1, 29.7, 28.8, 23.4, 21.67, 21.63, 21.58, 20.7.

[0155] HR-ESI-MS (m / z): for C 44 H 53 N₅O 12 Na+ (M+Na + Calculated value: 866.3583, measured value: 866.3588.

[0156] Synthesis of N-(5,7-diacetamido-4,8-di-O-acetyl-1-isopropyl-α-pseudoamineoxy)pentylcarbamate (9H-fluorene-9-yl) methyl ester (13) Figure 14 ).

[0157] To a 25 mL round-bottom flask containing 12 (79.2 mg, 0.094 mmol, 1.0 equivalent), Pd / C (Pd on 10% activated carbon, 50 mg), and NH4OAc (29.2 mg, 0.38 mmol, 4.0 equivalent), DCM (2.0 mL) and MeOH (2.0 mL) were added. The mixture was stirred for 1 hour under a H2 atmosphere (1 atm), and then filtered through diatomaceous earth to remove the catalyst. NMM (2.0 mL, excess) and Ac2O (1.0 mL, excess) were added to the filtrate. After stirring at room temperature for 2 hours, the mixture was concentrated under vacuum. The residue was dissolved in ethyl acetate (50 mL), and the solution was washed successively with 1 M HCl (aqueous solution) and saturated NaHCO3 (aqueous solution). The organic phase was dried over anhydrous Na2SO4, and the solvent was removed under vacuum. The residue was purified by silica gel column chromatography using EtOAc:methanol 20:1 as the eluent. f (EtOAc:methanol 20:1) = 0.33. Product 13 was obtained as a white solid (55.4 mg, 77%).

[0158] 1 H NMR (500MHz, CDCl3) δ7.76(d,J=7.5Hz,2H,ArH),7.67(d,J=7.5Hz,1H,ArH),7.63(d,J=7.5Hz,1H,A rH),7.40(t,J=7.5Hz,2H,ArH),7.36-7.28(m,2H,ArH),6.22(d,J=9.0Hz,1H,NH),6.20(d,J=8.6Hz ,1H,NH),5.23(dt,J1=12.2Hz,J2=4.2Hz,1H,H-4),5.16(qd,J1=6.5Hz,J2=2.8Hz,1H,H-8),5.13-5 .07(m,1H,CH(CH3)2),5.03(t,J=5.8Hz,1H,NH),4.57(d,J=9.1Hz,1H,H-5),4.55-4.47(m,2H,H-7,C 12 H8CHCH2), 4.31-4.22(m,2H,C 12H8CHCH2),4.04(d,J=10.4Hz,1H,H-6),3.54-3.46(m,1H,OCH2),3.41-3.34(m,1H, OCH2),3.29-3.10(m,2H,NHCH2),2.16(dd,J1=13.0Hz,J2=4.7Hz,1H,H-3e),2.04(s ,3H,CH3CO),2.00(s,3H,CH3CO),1.97(s,3H,CH3CO),1.85(s,3H,CH3CO),1.80(t, J=12.8Hz,1H,H-3a),1.68-1.38(m,6H,(CH2)3),1.33-1.25(m,9H,H-9,CH(CH3)2).

[0159] 13 C NMR (125MHz, CDCl3) δ171.03,170.61,170.53,169.94,167.11,156.77,144.23,143 .70,141.32,141.23,127.67,127.63,127.17,126.95,125.20,125.13,119.93,119. 89,98.75,71.30,70.21,70.03,67.21,66.90,63.87,53.40,50.38,47.08,45.63,41.07,32.52,29.98,28.87,23.88,23.21,23.15,21.66,21.65,21.30,20.96,15.79.

[0160] HR-ESI-MS(m / z): For C 40 H 53 N3O 12 Na + (M+Na + Calculated value: 790.3521, measured value: 790.3527.

[0161] Synthesis of S-(2-((5-(5,7-di-acetamido-4,8-di-O-acetyl-1-isopropyl-α-pseudoaminooxy)pentyl)amino)-2-oxoethyl)thioacetate (15) Figure 15 ).

[0162] Add 3 mL of a 20% diethylamine solution in MeCN (v / v) to a 10 mL round-bottom flask containing 13 (27.7 mg, 0.361 mmol, 1.0 equivalent), and stir the solution at room temperature for 6 hours. Concentrate the mixture under vacuum to remove the diethylamine and redissolve the residue in 4 mL of DCM. Add acid 14 (14.5 mg, 3.0 equivalent) to the above solution, followed by EDCI (20.7 mg, 3.0 equivalent) and monitor by TLC. When complete, dilute the mixture with 20 mL of ethyl acetate, followed by washing with NaHCO3 (aqueous solution) and brine. Dry the organic phase with anhydrous Na2SO4 and remove the solvent under vacuum. Purify the residue by silica gel column chromatography using EtOAc:methanol 10:1 as the eluent. f (EtOAc:methanol 10:1) = 0.25. Product 15 was obtained as a white solid (17.4 mg, 73%).

[0163] 1 H NMR(400MHz, CDCl3)δ7.22(d,J=10.0Hz,1H,NH),6.69(d,J=9.4Hz,1H,NH),6.52(t, J=6.2Hz,1H,NH),5.24-5.15(m,2H,H-4,H-8),5.15-5.04(m,1H,CH(CH3)2),4.56(t d, J1=10.4Hz, J2=3.2Hz, 1H, H-7), 4.52 (d, J=9.4Hz, 1H, H-5), 4.02 (dd, J1=10.6Hz, J2=1.9Hz,1H,H-6),3.68(d,J=15.0Hz,1H,CH2SCOCH3),3.54(d,J=15.0Hz,1H,CH2SC OCH3),3.51-3.43(m,1H,OCH2),3.43-3.34(m,2H,1×OCH2,1×NHCH2),3.17-3.06(m, 1H,NHCH2),2.42(s,3H,CH2SCOCH3),2.13(dd,J1=13.1Hz,J2=4.4Hz,1H,H-3e),2.05 (s,3H,CH3CO),2.02(s,3H,CH3CO),1.99(s,3H,CH3CO),1.97(s,3H,CH3CO),1.78(t ,J=12.8Hz,1H,H-3a),1.67-1.41(m,6H,(CH2)3),1.35-1.26(m,9H,H-9,CH(CH3)2).

[0164] 13C NMR (100MHz, CDCl3)δ=196.03,172.24,171.25,170.88,170.44,169.15,167.18,98.64,70.79,70.17,70.15,66.87,63 .59,50.30,45.76,40.16,33.23,32.58,30.27,29.62,28.63,24.45,22.80,22.78,21.63,21.60,21.31,20.88,15.47.

[0165] HR-ESI-MS(m / z): For C 29 H 47 N3O 12 SNa + (M+Na + Calculated value: 684.2773, measured value: 684.2777.

[0166] Synthesis of 2-mercapto-N-(5-(5,7-diacetamido-α-pseudoamineoxy)pentyl)acetamide (16) Figure 16 ).

[0167] Lithium hydroxide monohydrate (11.0 mg, 0.263 mmol, 10.0 equivalent) was dissolved in a mixture of THF, MeOH, and H₂O (3 mL + 0.75 mL + 0.75 mL) to obtain a 0.5 mM LiOH solution. This solution was added to a 10 mL round-bottom flask containing compound S5 (17.4 mg, 0.0263 mmol, 1.0 equivalent), and the mixture was stirred at room temperature for 48 hours. The mixture was then passed through a Dowex 50H... + The resin was neutralized and filtered. The filtrate was evaporated under vacuum and purified into an aqueous solution by RP HPLC: t R = 9.5 minutes (Column: Vydac 218TP C18) Column (Grace Davison Discovery Sciences); Eluent A: MeCN and B H2O; Gradient: Sample run at 10 mL / min, gradient 5-35% A, duration 35 min; Detection: UV 200 nm. Compound S6 (9.4 mg, 72%) was obtained as a mixture of thiols and disulfides.

[0168] 1H NMR (400MHz, D2O) δ = 4.02-4.16 (m, 4H), 3.70 (d, J = 10.1Hz, 1H), 3.23 (t, J = 6.2Hz, 2H), 3.06-3.16 (m, 3H), 1.94-2.02(m,1H),1.88(s,3H),1.87(s,3H),1.38-1.59(m,5H),1.20-1.36(m,2H),1.03(d,J=6.5Hz,3H).

[0169] 13 C NMR(100MHz,D2O)δ=175.0,174.6,1737,173.5,163.1,162.8,120.6,117.7,114.8,111.9,100.1 ,70.4,68.6,66.9,65.0,63.6,53.6,48.7,39.5,35.3,28.4,28.1,27.2,22.7,22.0,21.9,15.6.

[0170] HR-ESI-MS(m / z): For C 20 H 35 N3O9SNa + (M+Na + Calculated value: 516.1986, measured value: 516.1991.

[0171] Preparation of CRM197-Pse conjugates 1-3

[0172] OPA-modified pseudoamine 10 (20.0, 30.0, or 50.0 equivalents) was dissolved in 200 μL of 0.1 M phosphate-buffered saline (PBS) at pH 7.4 and added to a lyophilized solution of CRM197 (1.0 mg, 17.1 nmol, 1.0 equivalent) in 1.5 mL of 0.1 M PBS at pH 7.4. The mixture was incubated at room temperature for 6 hours, then diluted to 5 mL with sterile water and dialyzed at 4 °C using a centrifugal filter (10 kDa MWCO, Milipore, Amicon Ultra). The protein solution was concentrated to 500 μL and diluted to 5 mL with sterile water. This process was repeated three times, with a final concentration of 300 μL. 20 μL was used for analysis, and the protein solution was reburied in 0.1 M PBS at pH 7.4 to 5 mL, concentrated to 500 μL, and stored at -40 °C before immunization. CRM197-Pse1, 2 and 3 refer to conjugations with 20, 30 and 50 equivalent Pse, respectively.

[0173] Preparation of Pse-BSA conjugate 17

[0174] At room temperature, a solution of N-(ε-maleimide hexoxy)sulfosuccinimide ester (sulfon-EMCS) (2.1 mg, 5.2 μmol) in 200 μL of 0.1 M PBS buffer at pH 7.4 was added to a stirred solution of bovine serum albumin (BSA, 3 mg, 51.7 nmol) in 1.5 mL of 0.1 M PBS buffer at pH 7.4. The mixture was stirred for 2 hours, then diluted to 5 mL with sterile water and dialyzed at 4 °C using a centrifugal filter (10 kDa MWCO, Milipore, Amicon Ultra). This process was repeated three times, and the final concentration was 300 μL. 20 μL was used for analysis, and the protein solution was reburied in 5 mL of 0.1 M PBS buffer at pH 7.4 for the next step.

[0175] Pse-thiol substance 16 (2.6 mg, 5.2 μmol corresponding to the monomer, thiol to disulfide ratio assessed by UPLC) in 0.1 M PBS buffer (0.2 mL) at pH 7.4 was treated with tris(2-carboxyethyl)phosphine (TCEP, 25 μL 100 mM stock solution, pH 7.4) and incubated at this temperature under argon atmosphere for 1 hour. This was then added to the solution of the activated protein. The mixture was stirred at room temperature for 16 hours. The glycoconjugate was then treated with L-cysteine ​​(625 μg, 5.1 μmol) in 100 μL sterile water at room temperature and incubated for 1 hour. The mixture was then diluted to 5 mL with sterile water and dialyzed at 4 °C using a centrifugal filter (10 kDa MWCO, Milipore, Amicon Ultra). This process was repeated three times, and the final volume was concentrated to 300 μL. Take 20 μL for analysis, and rebuffer the protein solution in 5 mL of 0.1 M PBS buffer at pH 7.4, concentrate to 500 μL and store at -40 °C.

[0176] Characterization of synthesized Pse-CRM197 conjugates 1-3 and Pse-BSA conjugate 17

[0177] Conjugates were prepared in 1X SDS-PAGE samples loaded with dye and resolved on 10% SDS-PAGE. Electrophoresis was performed in electrode buffer at 80 V for 30 min, followed by electrophoresis at 160 V for 30 min, and the gel was stained with Coomassie Brilliant Blue. The average molecular size of the glycoconjugates was determined by matrix-assisted laser desorption / ionization (MALDI) analysis using 2,5-dihydroxybenzoic acid (DHB) as the matrix on a Bruker ultrafleXtreme mass spectrometer. Recombinant CRM197 protein (Pfenex Inc, San Diego) and BSA (Sangon Biotech, Shanghai) were used as standards.

[0178] Table 1. Antigen loading calculation of CRM197-Pse conjugates. The MW of sugar is 334 (697 with spacer regions).

[0179]

[0180] Example 2 – Antibody Response to Pse-CRM197 Vaccine

[0181] The immunogenicity of the Pse-CRM197 conjugate was assessed by immunizing male C57BL / 6J mice mixed with aluminum hydroxide in a primary booster strategy. Figure 1A The control group received CRM197 mixed with aluminum hydroxide in PBS. Post-immunization serum Pse-specific antibody responses were characterized by ELISA. Mice receiving the first dose of all three Pse vaccines produced a small antibody response to BSA-Pse, while no humoral immune response was observed in the CRM197 control mice. Figure 1B On day 21, one week after receiving the second dose of all three Pse-CRM197 vaccines, antibody titers significantly increased. Figure 1C The antibody response to Pse-CRM1973 was found to be slightly lower compared to that of Pse-CRM1971 and Pse-CRM1972, possibly due to the high sugar content. On day 35, one week after the third dose, antibody titers for all three Pse vaccines remained stable. Figure 1D Furthermore, all three Pse vaccines maintain an antibody response for up to one month, that is, five weeks after the last immunization. Figure 1E The titer of Pse-CRM197 3 eventually reached the levels of Pse-CRM197 1 and Pse-CRM197 2. These data demonstrate that Pse vaccination induces significant levels of anti-Pse-BSA IgG, while control mice showed no detectable antigen-specific IgG.

[0182] To identify the immune response during immunization with the Pse-CRM197 vaccine, isotypes of Pse-specific antibodies in serum were determined by ELISA on day 35 after immunization. No formulation induced detectable levels of IgA antibodies. Figure 2A For the three Pse-CRM197 vaccines, IgG1, IgG2b, and IgG3 contributed the majority of the Pse-specific IgG titers, while a weak IgG2c response was observed, demonstrating that immunization with the Pse-CRM197 vaccine produced antibodies against all three subtypes. Figure 2C-2F Conversely, mice vaccinated with CRM197 failed to elicit Pse-specific IgG. All detected Pse-specific antibodies carried the κ light chain. These data indicate that vaccination with all three Pse vaccines elicited a humoral immune response and produced sustained and significant levels of IgG-type Pse-specific antibodies.

[0183] Example 3 – Flow cytometry analysis of post-immunization serum against Acinetobacter baumannii strain Ab2. The binding affinity of post-immunization serum to Pse-producing Acinetobacter baumannii strain Ab2 was further determined by flow cytometry. Bacteria incubated with post-immunization serum treated with three Pse vaccines showed similar distributions, while the CRM-197 control resembled the negative control. Figure 3 The significant difference in fluorescence intensity indicates that the glycoconjugate-enhanced serum can recognize bacteria carrying Pse on the surface.

[0184] Example 4 – Immunization of mice with Pse-CRM197 vaccine to protect them from Acinetobacter baumannii infection.

[0185] To characterize the efficacy of the Pse vaccine, mice and control mice were challenged with Acinetobacter baumannii strain Ab2 using a mouse sepsis model. The LD50 of strain Ab2 was determined by infecting mice with different doses of the bacteria. The LD50 was 5.9 × 10⁻⁶. 6 1×10 7 and 5.9×10 7 CFU strain Ab191 infection in mice resulted in mortality rates of 20%, 50%, and 100%, respectively. Figure 4A Next, it was determined whether the response to the Pse vaccine was sufficient to provide protection against Acinetobacter baumannii infection. Two weeks after the last immunization, mice immunized as described above were challenged with Acinetobacter baumannii strain Ab2 at weeks 0, 2, and 4, and survival was monitored for 7 days (n = 4 mice / group). A 2.0 × 10⁻⁶ vaccine was used. 7Mice challenged with CFU (2×LD50) Acinetobacter baumannii strain Ab2 were completely immune to the attack, while all control and negative control mice receiving CRM-197 died within 36 hours. Figure 4B When using 5.0×10 7 When challenged with strain Ab2 containing CFU (5×LD50), a 25% mortality rate was recorded in mice receiving Pse-CRM197 1 and Pse-CRM197 3, a 0% mortality rate in mice receiving Pse-CRM197 2, and a 100% mortality rate in control mice receiving CRM-197 and negative control mice. Figure 4C These data indicate that these Pse vaccines all provide protection against infections caused by Pse-carrying Acinetobacter baumannii strains.

[0186] Example 5 – Immunization with Pse-CRM197 vaccine reduces post-infectious bacterial load

[0187] Using the Acinetobacter baumannii sepsis model, at 5.0 × 10⁻⁶... 7 The effect of vaccination on bacterial load in infected tissues was determined 12 hours after infection with CFU (5×LD50) strain Ab2 by measuring the amount of viable bacteria in the blood and tissues of vaccinated and control mice (n=4 mice / group). For all tested tissues, vaccination resulted in a 10% reduction in tissue bacterial load compared to those in control mice. 3 -10 5 It is twice as effective, and it reduces blood volume by 10 times. 6 times ( Figures 5A-5F Next, serum levels of pro-inflammatory cytokines IL-1β, IL-6, and TNF-α were measured to determine whether immunization with these Pse-CRM197 vaccines could protect infected mice from the release of these cytokines produced during bacterial sepsis. It was found that levels of all three cytokines were significantly lower in vaccinated mice than in control mice, indicating that vaccinated mice did not experience the release of pro-inflammatory cytokines associated with the development of septic shock. Figures 6A-6C ).

[0188] It should be understood that the embodiments and implementations described herein are for illustrative purposes only, and various modifications or changes thereto will be suggested to those skilled in the art and will be included within the spirit and scope of this application. Furthermore, any element or limitation of any invention or implementation thereof disclosed herein may be combined with any and / or all other elements or limitations disclosed herein (alone or in any combination) or any other invention or implementation thereof, and all such combinations are covered within the scope of this invention but are not limited thereto.

[0189] References

[0190] 1. Howard, A.; O'Donoghue, M.; Feeney, A.; Sleator, R.D., Acinetobacter baumannii: an emerging opportunistic pathogen. Virulence 2012, 3(3), 243-250.

[0191] 2. Morris, F.C.; Dexter, C.; Kostoulias, X.; Uddin, M.I.; Peleg, A.Y., The Mechanisms of Disease Caused by Acinetobacter baumannii. Front Microbiol 2019, 10, 1601.

[0192] 3. Antunes, L.C.S.; Visca, P.; Towner, K.J., Acinetobacter baumannii: evolution of a global pathogen. Pathogens and Disease 2014, 71(3), 292-301.

[0193] 4. Butler, D.A.; Biagi, M.; Tan, X.; Qasmieh, S.; Bulman, Z.P.; Wenzler, E., Multidrug Resistant Acinetobacter baumannii: Resistance by Any Other Name Would Still be Hard to Treat. Curr Infect Dis Rep 2019, 21(12), 46.

[0194] 5. Dijkshoorn, L.; Nemec, A.; Seifert, H., An increasing threat in hospitals: multidrug-resistant Acinetobacter baumannii. Nat Rev Microbiol 2007, 5(12), 939-51.

[0195] 6.Poirel,L.;Nordmann,P.,Carbapenem resistance in Acinetobacterbaumannii:mechanisms and epidemiology.Clin Microbiol Infect 2006,12(9),826-36.

[0196] 7.Qureshi,Z.A.;Hittle,L.E.;O'Hara,J.A.;Rivera,J.I.;Syed,A.;Shields,R.K.;Pasculle,A.W.;Ernst,R.K.;Doi,Y.,Colistin-resistant Acinetobacterbaumannii:beyond carbapenem resistance.Clin Infect Dis 2015,60(9),1295-303.

[0197] 8.He,T.;Wang,R.;Liu,D.;Walsh,T.R.;Zhang,R.;Lv,Y.;Ke,Y.;Ji,Q.;Wei,R.;Liu,Z.;Shen,Y.;Wang,G.;Sun,L.;Lei,L.;Lv,Z.;Li,Y.;Pang,M.;Wang,L.;Sun,Q.;Fu,Y.;Song,H.;Hao,Y.;Shen,Z.;Wang,S.;Chen,G.;Wu,C.;Shen,J.;Wang,Y.,Emergence ofplasmid-mediated high-level tigecycline resistance genes in animals andhumans.Nat Microbiol 2019,4(9),1450-1456.

[0198] 9.Global Priority List of Antibiotic-resistant Bacteria to GuideResearch,Discovery,and Development of New Antibiotics.The World HealthOrganization:2017.

[0199] 10.Zeng,P.;Yi,L.;Xu,J.;Gao,W.;Xu,C.;Chen,S.;Chan,K.F.;Wong,K.Y.,Investigation of antibiofilm activity,antibacterial activity,and mechanisticstudies of an amphiphilic peptide against Acinetobacter baumannii.BiochimBiophys Acta Biomembr 2021,1863(6),183600.

[0200] 11.Garcia-Quintanilla,M.;Pulido,M.R.;Lopez-Rojas,R.;Pachon,J.;McConnell,M.J.,Emerging therapies for multidrug resistant Acinetobacterbaumannii.Trends Microbiol 2013,21(3),157-63.

[0201] 12.Gellings,P.S.;Wilkins,A.A.;Morici,L.A.,Recent Advances in thePursuit of an Effective Acinetobacter baumannii Vaccine.Pathogens 2020,9(12),1066.

[0202] 13.Micoli,F.;Costantino,P.;Adamo,R.,Potential targets for nextgeneration antimicrobial glycoconjugate vaccines.FEMS Microbiol Rev 2018,42(3),388-423.

[0203] 14.Vella,M.;Pace,D.,Glycoconjugate vaccines:an update.Expert Opinionon Biological Therapy 2015,15(4),529-546.

[0204] 15.Luo,F.J.;Shi,N.M.;Li,L.;Zhang,Z.;Ai,X.;Yang,L.Q.;Bai,Y.H.;Wang,Z.Y.;Lu,Q.,Safety observation study on Haemophilus influenza type B conjugatevaccines injected at different sites in Chinese infants.Biomed Environ Sci2013,26(8),693-6.

[0205] 16.Vila-Corcoles,A.;Ochoa-Gondar,O.,Preventing pneumococcal diseasein the elderly:recent advances in vaccines and implications for clinicalpractice.Drugs Aging 2013,30(5),263-76.

[0206] 17.Dretler,A.W.;Rouphael,N.G.;Stephens,D.S.,Progress toward theglobal control of Neisseria meningitidis:21st century vaccines,currentguidelines,and challenges for future vaccine development.Hum VaccinImmunother 2018,14(5),1146-1160.

[0207] 18.Jin,C.;Gibani,M.M.;Moore,M.;Juel,H.B.;Jones,E.;Meiring,J.;Harris,V.;Gardner,J.;Nebykova,A.;Kerridge,S.A.;Hill,J.;Thomaides-Brears,H.;Blohmke,C.J.;Yu,L.M.;Angus,B.;Pollard,A.J.,Efficacy and immunogenicity of a Vi-tetanus toxoid conjugate vaccine in the prevention of typhoid fever using acontrolled human infection model of Salmonella Typhi:a randomised controlled,phase 2b trial.Lancet 2017,390(10111),2472-2480.

[0208] 19.Tomczyk,S.;Bennett,N.M.;Stoecker,C.;Gierke,R.;Moore,M.R.;Whitney,C.G.;Hadler,S.;Pilishvili,T.,Use of 13-valent pneumococcal conjugate vaccineand 23-valent pneumococcal polysaccharide vaccine among adults aged≥65years:recommendations of the Advisory Committee on Immunization Practices(ACIP).MMWR Morb Mortal Wkly Rep 2014,63(37),822-5.

[0209] 20. G.;Toledo,M.E.;Baly,A.;Fernandez-Santana,V.;Rodriguez,F.;Alvarez,Y.;Serrano,T.;Musachio,A.;Hernandez,I.;Hardy,E.;Rodríguez,A.;Hernandez,H.;Aguilar,A.;Sánchez,R.;Diaz,M.;Muzio,V.;Dfana,J.;Rodríguez,M.C.;Heynngnezz,L.;Verez-Bencomo,V.,Phase I clinical evaluation of a syntheticoligosaccharide-protein conjugate vaccine against Haemophilus influenzae typeb in human adult volunteers.Clin Vaccine Immunol 2006,13(9),1052-1056.

[0210] 21.Mettu,R.;Chen,C.Y.;Wu,C.Y.,Synthetic carbohydrate-based vaccines:challenges and opportunities.J Biomed Sci 2020,27(1),9.

[0211] 22.Schirm,M.;Soo,E.C.;Aubry,A.J.;Austin,J.;Thibault,P.;Logan,S.M.,Structural,genetic and functional characterization of the flagellinglycosylation process in Helicobacter pylori.Mol Microbiol 2003,48(6),1579-92.

[0212] 23.Knirel,Y.A.;Vinogradov,E.V.;L'Vov V,L.;Kocharova,N.A.;Shashkov,A.S.;Dmitriev,B.A.;Kochetkov,N.K.,Sialic acids of a new type from thelipopolysaccharides of Pseudomonas aeruginosa and Shigella boydii.CarbohydrRes 1984,133(2),C5-8.

[0213] 24.Logan,S.M.,Flagellar glycosylation-a new component of the motilityrepertoire?Microbiology(Reading)2006,152(Pt 5),1249-62.

[0214] 25.Senchenkova,S.N.;Popova,A.V.;Shashkov,A.S.;Shneider,M.M.;Mei,Z.;Arbatsky,N.P.;Liu,B.;Miroshnikov,K.A.;Volozhantsev,N.V.;Knirel,Y.A.,Structureof a new pseudaminic acid-containing capsular polysaccharide of Acinetobacterbaumannii LUH5550 having the KL42 capsule biosynthesis locus.Carbohydr Res2015,407,154-7.

[0215] 26.Vinogradov,E.;Wilde,C.;Anderson,E.M.;Nakhamchik,A.;Lam,J.S.;Rowe-Magnus,D.A.,Structure of the lipopolysaccharide core of Vibrio vulnificustype strain 27562.Carbohydr Res 2009,344(4),484-90.

[0216] 27.Goon,S.;Kelly,J.F.;Logan,S.M.;Ewing,C.P.;Guerry,P.,Pseudaminicacid,the major modification on Campylobacter flagellin,is synthesized via theCj1293 gene.Mol Microbiol 2003,50(2),659-71.

[0217] 28.Lee,I.M.;Yang,F.L.;Chen,T.L.;Liao,K.S.;Ren,C.T.;Lin,N.T.;Chang,Y.P.;Wu,C.Y.;Wu,S.H.,Pseudaminic Acid on Exopolysaccharide of Acinetobacterbaumannii Plays a Critical Role in Phage-Assisted Preparation ofGlycoconjugate Vaccine with High Antigenicity.J Am Chem Soc 2018,140(28),8639-8643.

[0218] 29.Andolina,G.;Wei,R.;Liu,H.;Zhang,Q.;Yang,X.;Cao,H.;Chen,S.;Yan,A.;Li,X.D.;Li,X.,Metabolic Labeling of Pseudaminic Acid-Containing Glycans onBacterial Surfaces.ACS Chemical Biology 2018,13(10),3030-3037.

[0219] 30.Liu,H.;Zhang,Y.;Wei,R.;Andolina,G.;Li,X.,Total Synthesis ofPseudomonas aeruginosa 1244 Pilin Glycan via de Novo Synthesis of PseudaminicAcid.J Am Chem Soc 2017,139(38),13420-13428.

[0220] 31.Wei,R.;Liu,H.;Tang,A.H.;Payne,R.J.;Li,X.,A Solution to ChemicalPseudaminylation via a Bimodal Glycosyl Donor for Highly Stereocontrolledα-and β-Glycosylation.Org Lett 2019,21(10),3584-3588.

[0221] 32.McConnell,M.J.;Actis,L.;Pachón,J.,Acinetobacter baumannii:humaninfections,factors contributing to pathogenesis and animal models.FEMSMicrobiol Rev 2013,37(2),130-155.

Claims

1. A compound of formula (IV) comprising at least one pseudoamine (Pse) moiety conjugated to a carrier protein, wherein the pseudoamine moiety is linked to the carrier protein via a linker and a linker, the linker being a cyclic lactam structure generated by a linking reaction between an orthophthalaldehyde (OPA) moiety and a lysine side chain, wherein the carrier protein is CRM197 or bovine serum albumin (BSA): Formula (IV): Where n is 4 to 20, m is 0 to 5, and R 1 The group is acetyl, formyl, or (R)-3-hydroxybutyryl, R 2 The group is acetyl, formyl, or (R)-3-hydroxybutyryl.

2. The compound of claim 1, wherein 5 to 20 Pse compounds are conjugated to a carrier protein.

3. The compound of claim 1, wherein the connection between the pseudoamine moiety and the linker is a glycosidic bond.

4. The compound of claim 3, wherein the glycosidic bond is α or β.

5. The compound of claim 1, wherein the structure of the connector is -(CH2CH2O). p -CH2CH2-, where p is 1 to 5.

6. The compound of claim 1, wherein the linker has the structure -CH2-(CH2). q -CH2, where q is 0 to 8.

7. The compound of claim 1, wherein the compound is selected from Pse-CRM197 1 (Formula (IX)), Pse-CRM1972 (Formula (X)), and Pse-CRM197 3 (Formula (XI)): Formula (IX): Formula (X): Formula (XI): 。 8. A composition comprising the compound of claim 1.

9. The composition of claim 8, further comprising an adjuvant, a carrier, an excipient, or a buffer.

10. Use of the composition of claim 8 in the preparation of a medicament for generating an immune response in a subject, wherein the medicament is a medicament for inhibiting the growth of bacteria in the subject, said bacteria being Acinetobacter baumannii.

11. Use of the composition of claim 8 in the preparation of a medicament for inhibiting bacterial growth in a subject, wherein the bacteria synthesize Pse and bacterial growth is inhibited, said bacteria being Acinetobacter baumannii.

12. The use according to claim 11, wherein the Acinetobacter baumannii is Acinetobacter baumannii strain Ab2.

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