Multi-epitope antigenic polypeptides derived from acinetobacter baumannii and immunotherapeutic uses thereof

Abstract

A polyclonal antibody composition specifically binds the pTonB epitope (SEQ ID NO: 11) from Acinetobacter baumannii and is elicited by immunization with a multi-epitope antigen comprising thioredoxin leader, rigid linker, and kernels including SEQ ID NOs: 6, 7, 11, 12, and 8. These antibodies enhance opsonophagocytic killing of A. baumannii Ci79 via classical complement activation and Fey receptor-mediated phagocytosis by bone marrow-derived macrophages. Absorption of pTonB-specific antibodies reduces killing by >70%, confirming epitope dominance. Passive transfer of the composition protects >60% of mice in a lethal intranasal challenge model. Pharmaceutical compositions, treatment methods (alone or with antibiotics like colistin), prophylactic uses, diagnostic kits, and polyclonal compositions are disclosed for combating multidrug-resistant A. baumannii infections.

Description

FJ ref. UTSA-P0165WO / Client ref. 2022-032-CIPMULTI-EPITOPE ANTIGENIC POLYPEPTIDES DERIVED FROM ACINETOBACTER BAUMANNII AND IM UNOTHERAPEUTIC USES THEREOFPRIORITY PARAGRAPH

[0001] This Application claims priority to US Provisional Application 63 / 717,746 filed on November 7, 2024 which is incorporated herein by reference in its entirety.STATEMENT REGARDING FEDERALLY FUNDED RESEARCH

[0002] This invention was made with government support under grant R21 Al 180267 awarded by the National Institutes of Health. The government has certain rights in the invention.REFERENCE TO SEQUENCE LISTING

[0003] The present application contains a Sequence Listing which has been submitted in electronic format via EFS-Web (or other applicable filing system) and is hereby incorporated by reference in its entirety. Said Sequence Listing, created on November 7, 2025, is named UTSA- P0165WO and is 13,351 bytes in size. The Sequence Listing complies with the requirements of WIPO Standard ST.26.FIELD OF THE INVENTION

[0004] Embodiments of the invention are generally directed to the field of microbiology and medicine, and in particular to microbial vaccines.BACKGROUND

[0005] Acinetobacter baumannii is a nonmotile, nonfermenting, Gram-negative bacterium, generally taking a rod shape in favorable conditions and a coccus shape in poor conditions, which could facilitate its persistence in a variety of environments (Houang et al., J Clin Pathol 1998, 51 :786-8). The ability to avoid desiccation and the increased propensity for multi-drug resistance (MDR) make this organism a successful nosocomial pathogen (Nordmann, Pathol Biol (Paris). 2004, 52:301-3). Patients that are critically ill, especially those within intensive care units, are at the highest risk for Acinetobacter infection, which may include pneumonia, meningitis, septicemia, urinary tract, and wound infections (Peleg et al., Clin Microbiol Rev. 2008, 21 : 538-82;FJ ref. UTSA-P0165WO / Client ref. 2022-032-CIPWeinstein et al., Clin Infect Dis. 2005, 41 :848-54; Fournier et al., Clin Infect Dis. 2006, 42:692- 9). Nosocomial infections with A. baumannii increase hospitalization costs and have a high mortality and morbidity (Gulen et al., Int J Infect Dis. 38:32-5), with reported rates as high as 52% and 10-35%, respectively (Shih et al., J Microbiol Immunol Infect. 2008, 41 :118-23). Hospital- acquired pneumonia represents the most common clinical manifestation of 4. baumannii infection, typically in patients receiving mechanical ventilation in the intensive care setting (Dijkshoom et al., Nat Rev Microbiol. 2007, 5:939-51). A. baumannii also is a common cause of bloodstream infections in ICU patients (Wisplinghoff et al. Clin Infect Dis. 2004, 39:309-17), with the most common sources associated with lower respiratory tract infections and intravascular devices (Seifert et al., Clinical features, epidemiology, and predictors of mortality. Medicine (Baltimore) 1995, 74:340-9). Pneumonia and septicemia are the leading causes of mortality by Acinetobacter (Vila and Pachon, Expert Opin Pharmacother . 2008, 9:587-99); however, gastrointestinal (GI) infection plays an important role in disease outbreaks in health care centers and in the rise of MDR strains. Although drug treatment is available for clinical use, emergence of multi-drug resistance in clinical strains renders drug treatment less effective. Currently, there is no licensed vaccine for Acinetobacter.

[0006] There is a need for additional peptide-based immunogens for prophylactic and therapeutic treatment against Acinetobacter infection, as well as formulations and methods for their use.SUMMARY

[0007] To address the deficiencies and problems described above the Inventors identified novel peptide-based immunogens that can be used for prophylactic and therapeutic treatment against Acinetobacter infection. Immunobioinformatics-based analysis of selected Acinetobacter proteins identified potential peptide antigens. These peptides can be formulated to generate vaccines for infection prevention and for use in production of polyclonal antibody compositions. These peptide antigens are also potential diagnostic antigens for Acinetobacter diseases.

[0008] Certain embodiments are directed to multi-epitope antigens having an Acinetobacter lead protein coupled to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 kernels. The leader protein can be coupled to a first kernel via a linker, such as an EAAAK linker. In certain aspects the leader protein is an A. baumannii virulence factor such as A. baumannii thioredoxin. In certain aspects each consecutiveFJ ref. UTSA-P0165WO / Client ref. 2022-032-CIP kernel is connected to the following kernel by a linker, in certain instances the linker is either a KK or GPGPG linker. The multi-epitope antigen can further comprise a carboxy terminal tag. The carboxy terminal tag can be a hexa-histidine tag for example. In other aspects, the multi-epitope antigen can have an amino acid sequence that is 85, 90, 95, 98, or 100% identical to SEQ ID NO: 1 or SEQ ID NO:2.

[0009] Other embodiments are directed to pharmaceutical compositions comprising multiepitope antigen as described herein and a pharmaceutically acceptable carrier.

[0010] Still other embodiments are directed to methods for detecting Acinetobacter haumanmi. comprising: providing a multi-epitope antigen as described herein; contacting the multi-epitope antigen with a biological sample; detecting and analyzing an immune complex formed by the multi-epitope antigen and antibodies in the biological sample to determine if Acinetobacter baumannii antibodies are present in the specimen, that is if the animal from which the biological specimen was obtained had been exposed to and mounted an immune response to Acinetobacter baumannii.

[0011] Certain embodiments are directed to diagnostic kits for detecting Acinetobacter baumannii comprising a multi-epitope antigen as described herein.

[0012] Other embodiments are directed to the use of a multi-epitope antigen as described herein for preparation of a vaccine against Acinetobacter baumannii. A vaccine composition can include at least one multi-epitope antigen described herein.

[0013] Other aspects are directed to an isolated nucleic acid encoding a multi-epitope antigen as described herein.

[0014] Certain embodiments are directed to methods of vaccinating against Acinetobacter baumannii comprising administering a multi-epitope antigen as described herein to a subject in need thereof.

[0015] Other embodiments are directed to methods of treating an Acinetobacter baumannii infection by administering a multi-epitope antigen described herein to a subject in need thereof. In certain aspects the administering of the multi-epitope antigen is in combination with one or more an antibiotics or antimicrobial agents. The one or more antibiotics can be, but are not limited to imipenem, meropenem, or colistin.FJ ref. UTSA-P0165WO / Client ref. 2022-032-CIP

[0016] Certain embodiments are directed to methods of treating colonization or infection, or treating or preventing disease by a Acinetobacter baumamiii microbe comprising administering a clinically effective dose of a multi-epitope antigen as described herein to a subject in need thereof.

[0017] Other embodiments of the invention are discussed throughout this application. Any embodiment discussed with respect to one aspect of the invention applies to other aspects of the invention as well and vice versa. Each embodiment described herein is understood to be embodiments of the invention that are applicable to all aspects of the invention. It is contemplated that any embodiment discussed herein can be implemented with respect to any method or composition of the invention, and vice versa. Furthermore, compositions and kits of the invention can be used to achieve methods of the invention.

[0018] The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and / or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.”

[0019] Throughout this application, the term “about” is used to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value.

[0020] The use of the term “or” in the claims is used to mean “and / or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and / or.”

[0021] As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open- ended and do not exclude additional, unrecited elements or method steps.

[0022] As used herein, the terms “comprises,” “comprising,” “includes,” “including,” “has,” “having,” “contains”, “containing,” “characterized by” or any other variation thereof, are intended to encompass a non-exclusive inclusion, subject to any limitation explicitly indicated otherwise, of the recited components. For example, a chemical composition and / or method that “comprises” a list of elements (e.g., components or features or steps) is not necessarily limited to only those elements (or components or features or steps), but may include other elements (or components or features or steps) not expressly listed or inherent to the chemical composition and / or method.FJ ref. UTSA-P0165WO / Client ref. 2022-032-CIP

[0023] As used herein, the transitional phrases “consists of’ and “consisting of’ exclude any element, step, or component not specified. For example, “consists of’ or “consisting of’ used in a claim would limit the claim to the components, materials or steps specifically recited in the claim except for impurities ordinarily associated therewith (i.e., impurities within a given component). When the phrase “consists of’ or “consisting of’ appears in a clause of the body of a claim, rather than immediately following the preamble, the phrase “consists of’ or “consisting of’ limits only the elements (or components or steps) set forth in that clause; other elements (or components) are not excluded from the claim as a whole.

[0024] As used herein, the transitional phrases “consists essentially of’ and “consisting essentially of’ are used to define a chemical composition and / or method that includes materials, steps, features, components, or elements, in addition to those literally disclosed, provided that these additional materials, steps, features, components, or elements do not materially affect the basic and novel characteristic(s) of the claimed invention. The term “consisting essentially of’ occupies a middle ground between “comprising” and “consisting of’.

[0025] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

[0026] The following terms, as used throughout the specification and claims, are assigned the meanings set forth below unless otherwise indicated or clearly contradicted by context. These definitions are provided to aid in describing particular embodiments and are not intended to limit the claimed invention. To the extent a term is not specifically defined herein, that term should be given its ordinary meaning in the art to which the invention pertains. Unless otherwise noted, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art of immunology, vaccinology, and microbiology.

[0027] Acinetobacter baumannii - Refers to the Gram-negative, non-motile, oxidase-negative, coccobacillary bacterium of the genus Acinetobacter (italicized per taxonomic convention) and includes all clinical isolates, multi drug-resistant (MDR) strains, and the type strain ATCC 19606.FJ ref. UTSA-P0165WO / Client ref. 2022-032-CIP

[0028] Administering / Administration - The delivery of a composition of the invention to a subject by any route, including but not limited to subcutaneous, intramuscular, intranasal, intravenous, intraperitoneal, intradermal, or oral administration.

[0029] Antibody - An immunoglobulin molecule (including IgG, IgM, IgA, IgD, IgE) or antigen-binding fragment thereof (e.g., Fab, F(ab')2, scFv, nanobody) capable of specifically binding an epitope. The term encompasses polyclonal, chimeric, humanized, and fully human antibodies.

[0030] The term “polyclonal antibody composition” as used herein refers to a composition comprising a heterogeneous population of antibodies that recognize and bind to a plurality of distinct epitopes on one or more target antigens, e.g., pTonB. The antibodies are produced by multiple B-cell clones in a host organism or in vitro system and comprise a mixture of immunoglobulin molecules (typically IgG, but also including IgM, IgA, IgE, or IgD isotypes, singly or in combination) exhibiting diversity in their variable regions, including heavy-chain and light-chain complementarity-determining regions (CDRs). The polyclonal antibody composition may be: (a) purified or partially purified from serum, plasma, colostrum, milk, egg yolk, or other biological fluids of an immunized animal (including but not limited to mammals, avians, or reptiles); (b) derived from in vitro immunization of isolated B lymphocytes or immortalized cell lines; (c) produced by recombinant DNA technology in which a library of antibody variable region genes is expressed in a host cell population; or (d) any combination thereof.

[0031] Clinically effective dose - An amount of an antibody composition, multi -epitope antigen, or vaccine sufficient to reduce bacterial load, ameliorate symptoms, prevent colonization, or increase survival in a subject infected with or at risk of A. baumannii infection, as determined by standard clinical endpoints.

[0032] EAAAK linker - A rigid a-helical peptide linker consisting of one or more repeats of the pentapeptide EAAAK used to spatially separate a leader protein from the first immunogenic kernel.

[0033] Functional variant - A polypeptide that differs from a reference sequence (e.g., SEQ ID NO: 1, SEQ ID NO: 2, or any immunogenic kernel) by substitutions, deletions, or insertions, yet retains at least 80 % of the immunogenic activity of the reference sequence as measured by ELISA endpoint titer, opsonophagocytic killing assay (OPKA), or in vivo protection in a murineFJ ref. UTSA-P0165WO / Client ref. 2022-032-CIP model. Functional variants include sequences that are 85 %, 90 %, 95 %, 98 %, or 99 % identical to the reference sequence over its entire length.

[0034] GPGPG linker - A flexible glycine-rich linker consisting of one or more pentapeptide GPGPG (SEQ ID NO: 15) used to connect consecutive immunogenic kernels while minimizing junctional epitopes.

[0035] Immunogenic kernel - A linear peptide derived from an A. baumannii virulence factor, predicted by immunoinformatics to contain B-cell epitopes and MHC-I / MHC-II binding motifs, and bounded by cathepsin cleavage sites. Exemplary kernels are listed in Table 1 and assigned SEQ ID NOs: 3-12.

[0036] KK linker - A short cationic linker consisting of two lysine residues (KK) used to separate immunogenic kernels and enhance solubility.

[0037] Leader protein - A polypeptide fused N-terminally to the first immunogenic kernel to improve expression, folding, or immunogenicity. In preferred embodiments, the leader protein is A. baumannii thioredoxin (SEQ ID NO: 13) or another baumannii virulence factor.

[0038] Multi-epitope antigen - A recombinant fusion protein comprising a leader protein; 1 to 10 immunogenic kernels (preferably 5) connected by KK, GPGPG, or a combination thereof; an optional C-terminal hexa-histidine tag (6 / His); and an EAAAK linker between the leader protein and the first kernel. Examples of multi -epitope antigens are AMEV1 (SEQ ID NO: 1) and AMEV2 (SEQ ID NO: 2).

[0039] Pharmaceutically acceptable carrier - Any inert substance (solid, semi-solid, or liquid) suitable for formulating the antibody composition or multi-epitope antigen for administration, including but not limited to saline, buffered saline, dextrose, water, glycerol, ethanol, sterile isotonic aqueous buffers, and combinations thereof.

[0040] Polyclonal antibodies that specifically bind TonB - A heterogeneous population of antibodies raised against the pTonB immunogenic kernel (SEQ ID NO: 11) or a polypeptide comprising SEQ ID NO: 11, wherein the antibody population demonstrates specific binding to SEQ ID NO: 11 in an ELISA with an endpoint titer > 1 :5000.

[0041] pTonB - The immunogenic kernel derived from the A. baumannii TonB-dependent receptor having the amino acid sequenceEDNQNPEREGNYLANTSKNTGNLFVRYLPTEQWYTEVGVTYVGSYY (SEQ ID NO: 11).FJ ref. UTSA-P0165WO / Client ref. 2022-032-CIP

[0042] SEQ ID NO: 1 (AMEV1) - The full-length amino acid sequence of the AMEV1 multiepitope antigen, comprising A. baumannii thioredoxin, EAAAK linker, immunogenic kernels pOmpA - pPLcl - pBamA - pBauA - pBlp2, KK / GPGPG linkers, and a C-terminal 6><His tag.

[0043] SEQ ID NO: 2 (AMEV2) - The full-length amino acid sequence of the AMEV2 multiepitope antigen, comprising A. baumannii thioredoxin, EAAAK linker, immunogenic kernels pNlpE - pNucAB - pTonB - pZnuD - pOmp38, KK / GPGPG linkers, and a C-terminal 6*His tag.

[0044] Subject - A mammal, preferably a human, at risk of or suffering from A. baumannii infection, including immunocompromised patients, ICU patients, or individuals with indwelling medical devices.

[0045] Treating / Treatment - Any administration that partially or completely alleviates, ameliorates, relieves, delays onset of, inhibits progression of, reduces severity of, or reduces incidence of one or more symptoms or features of A. baumannii infection (e.g., pneumonia, bacteremia, wound infection).

[0046] Vector or expression vector - A recombinant nucleic acid containing a desired coding sequence and appropriate nucleic acid sequences necessary for the expression of the operably linked coding sequence in a particular host organism or expression system, e.g., cellular or cell- free. Nucleic acid sequences necessary for expression in prokaryotes usually include a promoter, an operator (optional), and a ribosome binding site, often along with other sequences. Eukaryotic cells are known to utilize promoters, enhancers, and termination and polyadenylation signals.

[0047] Prevent - Reducing the chance that a subject develops an infection.

[0048] Sample or biological sample -Is a fluid, tissue, or organ from an animal. A sample or biological sample includes, but is not limited to, blood, blood fractions, urine, stool, saliva, tears, or bile.DESCRIPTION OF THE DRAWINGS

[0049] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of the specification embodiments presented herein.FJ ref. UTSA-P0165WO / Client ref. 2022-032-CIP

[0050] FIG. 1 A - IB. Schematic structures of multi-epitope antigens (A) AMEV1 and (B) AMEV2, each comprising A. baumannii thioredoxin, an EAAAK linker, five immunogenic kernels connected by KK and GPGPG linkers, and a C-terminal 6><His tag.

[0051] FIG. 2. Coomassie-stained SDS-PAGE gel showing purified AMEV1 (-38.3 kDa) and AMEV2 (-41.5 kDa) proteins.

[0052] FIG. 3A - 3B. Indirect ELISA endpoint titers of mouse sera against (A) AMEV1 and (B) AMEV2 whole proteins.

[0053] FIG. 4A - 4B. Indirect ELISA reactivity of mouse sera against individual peptide components of (A) AMEV1 and (B) AMEV2.

[0054] FIG. 5A - 5B. (A) Vaccination and challenge schedule; (B) 30-day survival of mice challenged intraperitoneally with A. baumannii AB5075.

[0055] FIG. 6A - 6B. (A) Vaccination and challenge schedule; (B) 30-day survival of mice challenged intranasally with A. baumannii Ci79.

[0056] FIG. 7A-7B. (A) Reactivity of sera against UV-inactivated E. coli and A. baumannii strains; (B) complement-mediated killing assay.

[0057] FIG. 8A-8B. (A) Opsonophagocytic killing (OPK) of A. baumannii Ci79 with or without BMDMs; (B) macrophage bacterial uptake.

[0058] FIG. 9A-9B. OPK assays (A) with or without complement and (B) using WT or FcyR / BMDMs.

[0059] FIG. 10. 30-day survival of mice passively immunized with mock or AMEV2 sera prior to intranasal challenge.

[0060] FIG. 11 A-l IE. (A) ELISpot readouts; (B) ZFNy, IL-4, IL-5 SFUs; (C) pTonB-specific antibody levels in protected vs. unprotected mice; (D) pTonB antibody titer post-absorption; (E) OPK after pTonB absorption.DESCRIPTION

[0061] The following discussion is directed to various embodiments of the invention. The term “invention” is not intended to refer to any particular embodiment. Although one or more of these embodiments may be preferred, the embodiments disclosed should not be interpreted as limiting the scope of the disclosure, including the claims. In addition, one skilled in the art will understand that the following description has broad application, and the discussion of anyFJ ref. UTSA-P0165WO / Client ref. 2022-032-CIP embodiment is meant only to be a representative example of that embodiment, and not intended to imply that the scope of the disclosure, including the claims, is limited to that embodiment.I. Acinetobacter

[0062] The genus Acinetobacter are aerobic non-fermentative Gram-negative bacilli. They typically show coccobacillary morphology on nonselective agar. Rods predominate in fluid media, especially during early growth. Most strains of Acinetobacter, except some of the A Iwoffii strains, grow well on MacConkey agar (without salt). Although officially classified as nonlactose- fermenting, they are often partially lactose-fermenting when grown on MacConkey agar. They are oxidase-negative, nonmotile, and usually nitrate negative. Bacteria of the genus Acinetobacter are known to form intracellular inclusions of polyhydroxyalkanoates under certain environmental conditions.A. Antigens or Epitopes

[0063] Antigens described herein can have a leader protein linked to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more predicted immunogenic kernels, wherein the leader protein is linked to a first immunogenic kernel by a linker. In certain aspects each immunogenic kernel is coupled by a linker. In certain aspects the linker is an (EAAK)n, (KK)n, or (GPGPG)n linker where n is 1, 2, 3, 4, 5, 6, or more. In certain aspects the antigen has a carboxy terminal tag (e.g., hexa-histidine tag). In certain embodiments AMEV1 and AMEV2 antigens include, consist essentially of, or consist of A. baumannii thioredoxin leader protein(MSATIVNTTDDNFQADVLDAETPVLVDFWAGWCAPCKAIAPVLEDLSSEYAGKVKIVK VDVTS CEETAVKYNIRNIPALLLFKNGEVVAQQIGAVPRSKLVSFIDENV SEQ ID NO: 13) linked to five predicted immunogenic kernels followed by a hexa-histidine tag. In certain embodiments a thioredoxin leader protein is linked to the first peptide by a rigid EAAK linker or functionally equivalent linker while the peptides can be linked by KK and GPGPG linkers or functionally similar linkers to ensure epitope separation. In certain aspects the immunogenic kernels are selected from pBamA (SEQ ID NO:3), pBauA (SEQ ID NO:4), pBlp2 (SEQ ID NO:5), pNlpE (SEQ ID NO: 6), pNucAB (SEQ ID NO: 7), pOmp38 (SEQ ID NO: 8), pOmpA (SEQ ID NO:9), pPlcl (SEQ ID NO: 10), pTonB (SEQ ID NO: 11), or pZnuD (SEQ ID NO: 12). An antigen of the invention can include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more immunogenic kernels in any orderFJ ref. UTSA-P0165WO / Client ref. 2022-032-CIP and with immunogenic kernels appearing 1, 2, 3, 4, 5, or more times in the antigen. In certain aspects the antigen includes, consists essentially of, or consists of immunogenic kernels pOmpA, pPLcl, pBamA, pBauA, and pBlp2 from amino terminus to carboxy terminus. In certain aspects the antigen includes, consists essentially of, or consists of immunogenic kernels pNlpE, pNucAB, pTonB, pZnuD, and pOmp38 from amino terminus to carboxy terminus.

[0064] The present invention also encompasses variants and functional equivalents of the antigens disclosed herein. Functional equivalence can be established by similar binding affinities to HLA class I molecules, or similar potency demonstrated by the ELISPOT assay. Functional equivalents or variants of an Acinetobacter antigen as described herein will be understood to exhibit amino acid sequences differing from the provided sequences. This difference may be measured as a reduction in identity between a provided sequence and the Acinetobacter antigen variant or Acinetobacter antigen (AMEV1 or AMEV2).

[0065] The identity between amino acid sequences may be calculated using algorithms well known in the art. Fragments sharing homology with fragments comprising or consisting of consecutive Acinetobacter antigen amino acid residues are to be considered as falling within the scope of the present invention when they are, over the entire length at least 75% identical, at least 80% identical, at least 85% identical, at least 88% identical, at least 90% identical, at least 94% identical, including 95%, 96%, 97%, 98% or 99% identical with a provided antigen sequences.

[0066] The vaccine composition according to the invention comprise one or more Acinetobacter antigens or Acinetobacter antigen variants, wherein the sequence of the peptide variant, over the entire length, is at least 85%, 90%, 91%, 92%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a consecutive amino acid sequence of SEQ ID NO: 1 or SEQ ID NO:2 or includes 1, 2, 3, 4, 5, 6, 7, 8, 10, 20, 30, 40, 50 or more amino acid substitutions while maintaining immunogenicity.B. Nucleic Acid Vaccines

[0067] Compositions of the invention may comprise a nucleic acid encoding an Acinetobacter antigen or Acinetobacter antigen variant. The nucleic acid may thus encode any of the antigens described herein. The nucleic acid may for example be DNA, RNA, LNA, HNA, PNA, preferably the nucleic acid is DNA or RNA (e.g., mRNA).FJ ref. UTSA-P0165WO / Client ref. 2022-032-CIP

[0068] The nucleic acids of the invention may be comprised within any suitable vector, such as an expression vector. Numerous vectors are available that can be selected as a useful vector for specific purposes. The appropriate nucleic acid sequence may be inserted into the vector by a variety of established procedures. The vector can further comprise one or more signal sequence, translational start, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence. The vector can be an expression vector, comprising the nucleic acid operably linked to a regulatory nucleic acid sequence directing expression thereof in a suitable cell. Within the scope of the present invention said regulatory nucleic acid sequence should in general be capable of directing expression in a mammalian cell, preferably a human cell, more preferably in an antigen presenting cell.II. Vaccine Compositions

[0069] Embodiments of the invention relate to vaccine compositions comprising one or more multi-epitope Acinetobacter antigens or multi-epitope Acinetobacter antigen variants. Vaccine composition described herein can be formulated according to known methods such as by the admixture of one or more pharmaceutically acceptable excipients or carriers with the active agent, preferably acceptable for administration to humans. Examples of such excipients, carriers and methods of formulation may be found e.g. in Remington's Pharmaceutical Sciences (Maack Publishing Co, Easton, Pa). To formulate a pharmaceutically acceptable composition suitable for effective administration, such compositions will contain an effective amount of a multi-epitope antigen as described herein.

[0070] Vaccine compositions according to the invention may be administered to a subject in therapeutically effective amounts. The effective amount may vary according to a variety of factors such as the individual's condition, weight, sex and age. Other factors include the mode of administration. Vaccine compositions encompass compositions useful for therapeutic use, including stimulating an immune response in a patient, such as a strong specific cytotoxic T cell response upon administration of said composition.

[0071] To obtain vaccines or immunogenic compositions it may be required to combine the multi-epitope antigen(s) described herein with various materials such as adjuvants, immunostimulatory components, and / or carriers. Adjuvants are included in the vaccine composition to enhance the specific immune response.FJ ref. UTSA-P0165WO / Client ref. 2022-032-CIPA. Adjuvants

[0072] A number of adjuvants have been described and used for the generation of antibodies in laboratory animals, such as mice, rats, and rabbits. In such setting the tolerance of side effect is rather high as the main aim is to obtain a strong antibody response. For use and for approval for use in pharmaceuticals it is required that the components of the vaccine composition are well characterized. In a preferred embodiment the vaccine composition is suitable for administration to a human subject. The choice of adjuvant may be selected by its ability to stimulate the type of immune response desired, B-cell or / and T-cell activation and the vaccine composition may be formulated to optimize distribution and presentation to the relevant lymphatic tissues.

[0073] Adjuvants useful in therapeutic vaccines may be mineral salts, such as aluminium hydroxide, aluminium or calcium phosphates gels, oil emulsions and surfactant based formulations such as MF59 (microfluidized detergent stabilized oil in water emulsion), QS21 (purified saponin), AS02 (SBAS2, oil-in-water emulsion+monophosphoryl lipid A (MPL)+QS21), Montanide ISA 51 and ISA-720 (stabilised water in oil emulsion), Adjuvant 65 (containing peanut oil, mannide monooleate and aluminum monostearate), RIBI ImmunoChem Research Inc., Hamilton, Utah), particulate adjuvants, such as virosomes (unilamellar liposomal cehicles incorporating influenza haemagglutinin), AS04 (Al salt with MPL), ISCOMS (structured complex of saponins and lipids (such as cholesterol), polyactide co-glycolide (PLG), microbial derivatives (natural and synthetic) such as monophosphoryl lipid A (MPL), Detox (MPL+A7. Phlei cell wall skeleton), AGP (RC-529 (synthetic acylated monosaccharide)), DC chol (lipoidal immunostimulators able to self organise into liposomes), OM-174 (lipid A derivative), CpG motifs (synthetic oligonucleotides containing immunostimulatory CpG motifs), modified bacterial toxins, LT and CT, with non-toxic adjuvant effects, Endogenous human immunomodulators, e.g., hGM-CSF or hIL-12 or Immudaptin (C3d tandem array), inert vehicles such as gold particles.

[0074] In one embodiment the vaccine composition comprises an adjuvant and one or more multi-epitope antigen as described. In some embodiments, the vaccine composition may further comprise one or more additional immunostimulatory components - including without limitation muramyldipeptide (MDP), e.g. N-acetyl-muramyl-L-alanyl-D-isoglutamine (ala-MDP), N-acetyl- muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-nor-muramyl-L-alanyl-D- isoglutamine (CGP 11637, nor-MDP) and N-acetyl-muramyl-L-alanyl-D-isoglutaminyl-L- alanine-2-(l-2'-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (CGP 19835 A,FJ ref. UTSA-P0165WO / Client ref. 2022-032-CIPMTP-PE); dimethylglycine; tuftsin; trehalose dimycolate monophosphoryl -lipid A (MPL); and / or formyl-methionine containing tri-peptides such as N-formyl-Met-Leu-Phe. Such compounds are commercially available from Sigma Chemical Co. (St. Louis, Mo.) and RIBI ImmunoChem Research, Inc. (Hamilton, Mont.), for example.

[0075] A carrier may be present independently of an adjuvant. The function of a carrier can be to increase the molecular weight of peptides in order to increase their activity or immunogenicity, to confer stability, to increase the biological activity, or to increase serum half-life. The carrier may be any suitable carrier known to the person skilled in the art. A carrier protein could be but is not limited to keyhole limpet hemocyanin, serum proteins such as transferrin, bovine serum albumin, human serum albumin, thyroglobulin or ovalbumin, immunoglobulins, or hormones, such as insulin or palmitic acid.

[0076] Oil emulsions and surfactant based vaccines may be grouped as water in oil, oil in water and water in oil in water formulations. The adjuvant used for oil in water formulation may be a mineral oil or / and a non-mineral oil and a surfactant / emulsifier. The adjuvant is mixed with the aqueous antigen composition providing the vaccine formulation.B. Dose

[0077] It is contemplated that useful vaccine compositions comprise an immunologically effective amount of the multi-epitope Acinetobacter antigen(s) or variants thereof. The amount of the multi-epitope Acinetobacter antigen(s) in the vaccine composition may vary, depending on the particular application. However, a single dose of the immunogen is anywhere from about 10 pg to about 5000 pg more, from about 25 pg to about 2500 pg, or from about 50 pg to about 1000 pg, or from about 50 pg to about 500 pg, or from about 50 pg to about 250 pg, or from about 50 pg to about 200 pg, or from about 75 pg to about 150 pg. In certain aspects a dose of the immunogen is from about 75 pg to about 150 pg. In other aspects a dose is about 100 pg.C. Administration

[0078] Modes of administration include intradermal, subcutaneous, and / or intravenous administration - including implantation in the form of a time release formulation. Any and all forms of administration known to the art are encompassed herein. Subcutaneous administration is preferred, in certain instances deep subcutaneous administration. Any and all conventional dosageFJ ref. UTSA-P0165WO / Client ref. 2022-032-CIP forms that are known in the art can be appropriate for formulating injectable immunogenic peptide compositions, such as lyophilized forms and solutions, suspensions or emulsion forms containing conventional pharmaceutically acceptable carriers, diluents, preservatives, adjuvants, buffer components, etc.

[0079] The immunologic effect of the composition of the invention can be determined using several approaches as know by a person skilled in the art. A successful immune response may also be determined by the detection of antibodies specifically recognizing the peptide(s) of the vaccine composition. In certain embodiments, the immunogenic composition or vaccine is capable of eliciting an immune response to Acinetobacter or a species or strain thereof. As used herein, the expression “immunogenic composition or vaccine” refers to a composition eliciting at least one type of immune response directed against a bacterium.III. Antibodies

[0080] Certain aspects are directed to antibodies generated against one or more antigens described herein, in a particular aspect a polyclonal antibody composition that binds the TonB peptide EDNQNPEREGNYLANTSKNTGNLFVRYLPTEQWYTEVGVTYVGSYY (SEQ ID NO:11).

[0081] In some embodiments, once the antigen is expressed and purified, it is prepared as an immunogen for delivery to a host for eliciting an immune response. The host can be any animal capable of producing recoverable antibodies when administered an immunogen, such as but not limited to, rabbits, mice, rats, hamsters, goats, horses, monkeys, baboons, and humans. In one aspect, the host is a non-human transgenic that produces human antibodies, e.g., a mouse expressing the human antibody repertoire, thereby greatly facilitating the development of a human therapeutic.

[0082] An intact “antibody” comprises at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CHi, CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as LCVR or Vi.) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The Vuand VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR),FJ ref. UTSA-P0165WO / Client ref. 2022-032-CIP interspersed with regions that are more conserved, termed framework regions (FR). Each Vnand Vtis composed of three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. The term antibody includes antigen-binding portions of an intact antibody that retain capacity to bind. Examples of binding include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., Nature, 341 :544- 546 (1989)), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR).

[0083] An “antibody fragment” is an incomplete or isolated portion of the full sequence of the antibody which retains the antigen binding function of the parent antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

[0084] Single chain antibodies or single chain Fv (scFv) refers to an antibody fusion molecule of the two domains of the Fv fragment, VL and VH. Although the two domains of the Fv fragment, Vr and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see, e.g., Bird et al., Science, 242:423-426 (1988); and Huston et al., Proc Natl Acad Sci USA, 85:5879-5883 (1988)). Such single chain antibodies are included by reference to the term “antibody” fragments can be prepared by recombinant techniques or enzymatic or chemical cleavage of intact antibodies.

[0085] A “human sequence antibody” includes antibodies having variable and constant regions (if present) derived from human germline immunoglobulin sequences. The human sequence antibodies of the invention can include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). Such antibodies can be generated in non-human transgenicFJ ref. UTSA-P0165WO / Client ref. 2022-032-CIP animals, e g., as described in PCT App. Pub. Nos. WO 01 / 14424 and WO 00 / 37504. However, the term “human sequence antibody”, as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences (e.g., humanized antibodies).

[0086] Also, recombinant immunoglobulins can be produced. See, Cabilly, U.S. Pat. No. 4,816,567, incorporated herein by reference in its entirety and for all purposes; and Queen et al., Proc Natl Acad Sci USA, 86: 10029-10033 (1989).

[0087] An “antigen” is a substance that prompts the generation of antibodies and can cause an immune response. It can be used interchangeably in the present disclosure with the term “immunogen”. In the strict sense, immunogens are those substances that elicit a response from the immune system, whereas antigens are defined as substances that bind to specific antibodies. An antigen or fragment thereof can be a molecule (i.e., an epitope) that makes contact with a particular antibody. When a protein or a fragment of a protein is used to immunize a host animal, numerous regions of the protein can induce the production of antibodies (i.e., elicit the immune response), which bind specifically to the antigen (given regions or three-dimensional structures on the protein).

[0088] As used herein, the term “humanized antibody,” refers to at least one antibody molecule in which the amino acid sequence in the non-antigen binding regions and / or the antigen-binding regions has been altered so that the antibody more closely resembles a human antibody, and still retains its original binding ability.

[0089] In addition, techniques developed for the production of “chimeric antibodies” (Morrison, et al., Proc Natl Acad Sci, 81 :6851-6855 (1984), incorporated herein by reference in their entirety) by splicing the genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity can be used. For example, the genes from a mouse antibody molecule specific for an autoinducer can be spliced together with genes from a human antibody molecule of appropriate biological activity. A chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine mAb and a human immunoglobulin constant region.

[0090] In addition, techniques have been developed for the production of humanized antibodies (see, e.g., U.S. Pat. No. 5,585,089 and U.S. Pat. No. 5,225,539, which are incorporatedFJ ref. UTSA-P0165WO / Client ref. 2022-032-CIP herein by reference in their entirety). An immunoglobulin light or heavy chain variable region consists of a “framework” region interrupted by three hypervariable regions, referred to as complementarity determining regions (CDRs). Briefly, humanized antibodies are antibody molecules from non-human species having one or more CDRs from the non-human species and a framework region from a human immunoglobulin molecule.

[0091] Alternatively, techniques described for the production of single chain antibodies can be adapted to produce single chain antibodies against an immunogenic conjugate of the present disclosure. Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single chain polypeptide. Fab and F(ab')2 portions of antibody molecules can be prepared by the proteolytic reaction of papain and pepsin, respectively, on substantially intact antibody molecules by methods that are well-known. See e.g., U.S. Pat. No. 4,342,566. Fab1antibody molecule portions are also well-known and are produced from F(ab')2 portions followed by reduction of the disulfide bonds linking the two heavy chain portions as with mercaptoethanol, and followed by alkylation of the resulting protein mercaptan with a reagent such as iodoacetamide.

[0092] “Passive immunity” refers generally to the transfer of active humoral immunity in the form of pre-made antibodies. Thus, passive immunity is a form of short-term immunization that can be achieved by the transfer of antibodies, which can be administered in several possible forms, for example, as human or animal blood plasma or serum, as pooled animal or human immunoglobulin for intravenous (IVIG) or intramuscular (IG) use, as high-titer animal or human IVIG or IG from immunized subjects or from donors recovering from a disease, and as polyclonal antibodies. Passive transfer can be used prophylactically for the prevention of disease onset, as well as, in the treatment of several types of acute infection. Typically, immunity derived from passive immunization lasts for only a short period of time, and provides immediate protection, but the body does not develop memory, therefore the patient is at risk of being infected by the same pathogen later.IV. Examples

[0093] The following examples as well as the figures are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples or figures represent techniques discovered by the inventorsFJ ref. UTSA-P0165WO / Client ref. 2022-032-CIP to function well in the practice of the invention and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.EXAMPLE 1IDENTIFICATION OF NOVEL ACINETOBACTER BAUMANNII ANTIGEN PEPTIDESA. Material and methods

[0094] Selected A. baumannii virulence factors important for bacterial pathogenesis were subjected to immunobioinformatic analysis using EigenBio’s proprietary epitope prediction software to identify putative B and T cell epitopes. Initial analysis was to identify short peptides (approximately 30-60 amino acids) each bounded by cathepsin cleavage sites and comprising putative B-cell linear epitopes, MHC-I and MHC-II binding peptides. These identified “immunologic kernels” were further down selected based on conserved homology among A. baumannii strains and other bacteria for potential broad treatment application and minimize autoimmune response in humans and mice, which are used as the animal model of Acinetobacter infection.B. ResultsTABLE 1. Peptides identified to be potentially immunogenic by EigenBio’s epitope predictionFJ ref. UTSA-P0165WO / Client ref. 2022-032-CIPEXAMPLE 2FORMULATION OF NOVEL MULTI-PEPTIDE VACCINES, AMEV1 AND AMEV2, AGAINST ACINETOBACTER BAUMANNIIA. Materials and Methods

[0095] AMEV1 and AMEV2 Construction. Ten identified “immunologic kernels” were linked in tandem to construct the novel multi-epitope vaccines. Specifically, each consisted of A. baumannii thioredoxin, a known virulence factor, linked via a rigid EAAAK linker followed by five of the kernels connected by either KK or GPGPG linkers. Additionally, the protein included a terminal 6-histidine tag to aid in protein purification.

[0096] Expression and purification of AMEV1 and AMEV2. The AMEV1 and AMEV2 encoding nucleotide sequences were codon-optimized for protein expression in E. coli. The synthesized AMEV1 and AMEV2 DNA fragments were separately cloned into the pET-23a plasmid. Each resulting plasmid was transformed into E. coli BL21(DE3). Protein expression was induced with 1 mM isopropyl 0-D-1 -thiogalactopyranoside (LPTG) overnight at 37°C. Each recombinant protein was purified with cobalt affinity chromatography under denaturing conditions in the presence of 8 M urea. Cobalt column bound proteins were eluted with an elution buffer (8 M Urea, 50 mM sodium phosphate, 300 mM sodium chloride, 150 mM imidazole, pH 7.4) and dialyzed against PBS (pH 7.4) to remove the imidazole and urea. Protein concentrations were determined using the Pierce BCA Protein Assay kit (Thermo Scientific). The purified protein sequence was confirmed using trypsin digestion followed by peptide sequence analysis using liquid chromatography tandem mass spectrometry (LC-MS / MS).FJ ref. UTSA-P0165WO / Client ref. 2022-032-CIPB. Results

[0097] Both AMEV1 and AMEV2 constructs (Fig. 1.) encoded in the pET-23a plasmid were transformed into E. coll BL21 (DE3) and expressed. Both proteins were purified in the insoluble fraction under denaturing condition with cobalt affinity chromatography (Fig. 2.) to a high purity. The identity of each protein was confirmed by trypsin digestion and LC-MS / MS proteomic analysis (Table 2).TABLE 2. Amino acid sequences of peptides derived from trypsin digestion of purified AMEV1 and AMEV2 protein sequenced by LC-MS / MS. The matched peptides are in bold in the sequence of AMEV1 and AMEV2.EXAMPLE 3EVALUATION OF AMEV 1 AND AMEV2 IMMUNOGENICITYFJ ref. UTSA-P0165WO / Client ref. 2022-032-CIPA. Materials and Methods

[0098] Whole Protein ELISA. The antigenicity of AMEV1 and AMEV2 was assessed using enzyme-linked immunosorbent assay. Purified AMEV1 or AMEV2 protein was coated onto a 96- well microplate with 500 ng / 100 pL per well in carbonate buffer and incubated overnight at 4°C. Plates were washed once with PBS containing 0.05% Tween 20 (PBST) the following day and blocked with blocking buffer (PBS / 10% FBS) for 1 hour at room temperature. The plates were washed once, and vaccinated mouse serum was serially diluted in blocking buffer and incubated for 2 hours at room temperature. The plates were washed three times with PBST and then incubated with 100 pL of secondary antibody (goat anti-mouse Ig conjugated to horseradish peroxidase) diluted 1 :4000 in blocking buffer for one hour. After washing the plates three times with PBST, 100 pL of TMB substrate was added to the wells. The wells were developed for 10 minutes and then quenched with 50 pL of 2 M H2SO4. The absorbance of each well was measured spectroscopically at a wavelength of 450nm.

[0099] Peptide ELISA. The antigenicity of AMEV1 and AMEV2’s peptide components was assessed using enzyme-linked immunosorbent assay. Commercially synthesized peptides were coated onto a 96-well microplate with 1 pM / 100 pL per well in carbonate buffer and incubated overnight at 4°C. Plates were washed once with PBS containing 0.05% Tween 20 (PBST) the following day and blocked with blocking buffer (PBS / 10% FBS) for 1 hour at room temperature. The plates were washed once and 100 pL of vaccinated mouse sera was added to the wells (1 :500 dilution in blocking buffer). The plates were washed three times with PBST and then incubated with 100 pL of secondary antibody (goat anti-mouse Ig conjugated to horseradish peroxidase) diluted 1 :4000 in blocking buffer for one hour. After washing the plates three times with PBST, 100 pL of TMB substrate was added to the wells. The wells were developed for 10 minutes and then quenched with 50 pL of 2 M H2SO4. The absorbance of each well was measured spectroscopically at a wavelength of 450nm.B. Results

[0100] Mice (C57BL / 6) were primed (Day 0) with either PBS (Mock), AMEV1, or AMEV2 and boosted twice (days 14 and 28). Sera were obtained at Days 0, 11, 25, and 53 to measure vaccination induced antigen-specific antibody production. ELISA results demonstrate that miceFJ ref. UTSA-P0165WO / Client ref. 2022-032-CIP vaccinated with TiterMax Gold adjuvanted AMEV1 and AMEV2 elicited a robust antibody response (Fig. 3). Both vaccines generated high titers after the first vaccination and subsequent boosts. Additionally, the peptide ELISA demonstrate their ability to generate antibodies (Day 53) specific to the peptide components of each protein (Fig. 4). All five peptides in AMEV1 and four in AMEV2 generated high specific antibodies.EXAMPLE 4AMEV 1 AND AMEV2 ARE EFFICACIOUS VACCINES AGAINST ACINETOBACTER IN A MOUSE MODEL OF SYSTEMIC INFECTIONA. Materials and Methods

[0101] Vaccinations. C57BL / 6 mice between the ages of 6 and 8 weeks were purchased from Jackson Laboratories and randomly divided into three groups: PBS Mock, 5 pg AMEV1, or 5 pg AMEV2. Each protein or PBS was mixed 1 : 1 with TiterMax Gold adjuvant into an emulsion and 100 pL was injected subcutaneously into the mouse on day 0. The mice were boosted with a half dose (50 pL) on days 14 and 28.

[0102] Bacterial Challenge. Frozen AB5075 bacteria stocks were grown overnight in Luria- Bertani (LB) broth and subcultured (1 : 100) in fresh LB broth for 3 hours until mid-log phase. Bacteria was washed with PBS and centrifuged (5000 g, 5 min). The resulting pellet was resuspended in PBS and diluted to an ODeoonm = 0.3. This is further diluted to a concentration of 2 x 107CFU / mL. 100 pL of this inoculum was injected intraperitoneal into each mouse and the inoculum was serially diluted and plated for accurate CFU enumeration.B. Results

[0103] Following initial vaccination and two boosts, the mice were rested for nearly 5 weeks before bacterial challenge (FIG. 5 A). Both AMEV1 and AMEV2 were efficacious and provided partial protection against hypervirulent AB5075 systemic infection via intraperitoneal injection (FIG. 5B). All PBS mock vaccinated mice succumbed to the acute infection from 2.07 x 106CFUs (~ 4 x LD50) within two days while AMEV1 and AMEV2 vaccination achieved a 60% and 80% survival rate, respectively.

[0104] In a second AMEV2 protection study (see FIG. 6A-6B). C57BL / 6 mice (n=10) were randomly divided into two groups and vaccinated with AMEV2+Adjuvant or PBS+Adjuvant inFJ ref. UTSA-P0165WO / Client ref. 2022-032-CIP two-week intervals and then rested for nearly 5 weeks following the second boost. Mock and AMEV2 vaccinated mice were intranasally challenged with 108CFUs of Acinetobacter baumannii Ci79 strain on day 56. Mice were monitored for 30 days.

[0105] AMEV2-vaccinated mice that were protected from A. baumannii challenge had significantly more pTonB-specific antibodies compared to those that were unprotected. To evaluate the importance of pTonB-specific antibodies in vitro, we absorbed AMEV2 antisera with pTonB to reduce the pTonB-specific antibody titer eightfold from 1 : 16000 to 1 :2000. The pTonB- absorbed antisera used in the OPKA assay showed significantly reduced killing compared to unabsorbed and nonspecifically absorbed sera resulting in no significant difference in killing between mock and pTonB-absorbed sera. These data indicate that the pTonB peptide contains epitopes important for antibody-mediated protection against Acinetobacter baumannii.EXAMPLE 5AN IMMUNOINFORMATICS-B SED MULTI-PEPTIDE VACCINE PROVIDES ANTIBODY- MEDIATED PROTECTION AGAINST ACINETOBACTER L J ANNII INFECTION.

[0106] Acinetobacter baumannii is an opportunistic nosocomial pathogen characterized by its multidrug-resistant (MDR) phenotype, increasing patient mortality and healthcare costs as a result. Previously, we evaluated an immunoinformatics-based Acinetobacter Multi Epitope Vaccine (AMEV2) candidate against a pathogen with few remaining effective antimicrobials that can be used against it. In this study, we demonstrate the antibody -mediated protection against Acinetobacter infection induced by AMEV2 vaccination. Although AMEV2 vaccinated sera reacts with bacterial antigens, it is not bactericidal on its own and does not enhance complement- mediated direct killing of A. baumannii. However, in vitro opsonophagocytic killing assays (OPKA) demonstrate AMEV2 sera enhancement of the killing of A. baumannii in the presence of primary bone marrow-derived macrophages. This killing occurs via complement and Fc gamma receptor-mediated phagocytosis. Humoral protection was evaluated in vivo with passive transfer of AMEV2 vaccinated sera to naive mice which afforded 67 % protection in a pulmonary challenge mouse model. A highly immunogenic AMEV2 component peptide, pTonB, elicits pTonB-specific antibodies and protection in vivo. Antibody absorption of pTonB antibodies from AMEV2 vaccinated sera significantly reduced opsonophagocytic killing of A. baumannii in vitro. DataFJ ref. UTSA-P0165WO / Client ref. 2022-032-CIP presented here expands further the in vivo evaluation of in silico-&e \ ne& vaccines as a viable alternative to combat the current global MDR pathogen health crisis.A. Results

[0107] In vitro reactivity of mock and AMEV2 vaccinated sera was screened against two strains of A. baumannii by indirect ELISA (FIG. 7A). AMEV2 antisera exhibited significant reactivity to the clinical isolate (Ci79) and ATCC type strain (19606) while mock sera did not. E. coli (BL21), a nonspecific Gram-negative bacterium used in expression and purification of AMEV2 was included as a specificity control, and showed minimal reactivity with both mock and AMEV2 sera. These results indicated the presence of m \-Acinelobacler specific antibody in the AMEV2 vaccinated sera. We then evaluated the ability of sera to inhibit bacterial growth via complement-mediation. A. baumannii Ci79 (a complement-resistant strain) were incubated with PBS, pooled mock sera, or pooled AMEV2 sera in the presence of either freshly reconstituted baby rabbit sera with intact complement or heat-inactivated sera. As shown in FIG. IB, Ci79 replicated in the sera without functional complement. The complement-resistant A. baumannii Ci79 strain exhibited no increase in killing when incubated with PBS, mock sera, or AMEV2 sera in the presence of intact complement. However, in the absence of complement, PBS and AMEV2 sera treated wells showed a significant reduction in growth. A slight but not statistically significant reduction in Ci79 growth was observed when incubated with AMEV2 sera compared to treatment with mock sera. In the presence of complement, there was no enhancement of killing, and AMEV2 sera did not abrogate complement resistance in the Ci79 strain. Thus, the sera’s protective mechanism is not bactericidal -based.

[0108] Since AMEV2 sera demonstrated no enhancement of complement-mediated killing, we investigated whether the sera could opsonize the pathogen, and subsequently enhance phagocytic uptake using primary bone marrow-derived macrophages (BMDM). An opsonophagocytic killing assay (OPKA) was employed to determine if in vitro antibody reactivity with AMEV2 antisera resulted in opsonization and enhanced killing when incubated with the macrophages (FIG. 8A). Bacteria that had been opsonized with AMEV2 sera exhibited about 50% reduction in viable Ci79 compared to naive serum. In contrast, mock serum opsonized bacteria exhibited no difference in killing. When both opsonized bacteria were incubated in wells without macrophages, no increase in killing was observed indicating no serum bactericidal activity. Following incubation,FJ ref. UTSA-P0165WO / Client ref. 2022-032-CIP macrophages were lysed and bacteria taken up were enumerated (FIG. 8B). Macrophage incubated with AMEV2 opsonized bacteria exhibited a significant increase in bacterial uptake compared to that of the mock opsonized control. Thus, AMEV2 antisera enhances opsonophagocytic killing of A. baumannii by enhancement of primary bone marrow-derived macrophage recognition and uptake of the pathogen in vitro.

[0109] Enhancement of opsonophagocytic killing by AMEV2 antisera appears to be the primary mechanism of antibody-mediated protection in AMEV2-vaccinated mice. Therefore, we investigated whether this killing was facilitated by activation of the classical complement pathway or primarily Fc receptor mediated. An identical OPKA experiment was performed using bacteria opsonized in the presence of either an intact complement source or a heat-inactivated complement source (FIG. 9A). A significant amount of bacterial killing observed in wells with AMEV2 sera opsonized bacteria and macrophages was associated with a significant reduction of killing in the absence of intact complement. Consistent with the lack of complemented-mediated bacterial killing previously observed supports AMEV2 antibody activation of the classic complement pathway enhancing macrophage pathogen recognition. Additionally, the OPKA assays were carried out on wells containing either WT BMDMs or BMDMs from B6.129P2 -Feer lgtmlRa'TA 12 mice, which are deficient in the gamma chain subunit of the FcyRl, FcyRIII, and FceRl receptors (FIG. 9B). In this experiment, the complement source and vaccinated sera were heat-inactivated to negate complement-mediated phagocytosis. WT BMDMs continued to demonstrate significant killing of bacteria opsonized with AMEV2 serum. In contrast, killing was significantly reduced and / or absent when the AMEV2 opsonized bacteria was incubated with BMDMs deficient in FCyRs. These data suggest that AMEV2 antibodies opsonize the bacteria, thus enhancing killing through complement and Fey receptor-mediated phagocytosis.

[0110] Having demonstrated robust humoral immunogenicity generated in AMEV2- vaccinated mice and in vitro opsonization, we then determined if antibodies alone could protect against A. baumannii infection in vivo. Naive C57BL / 6 mice (n = 6 per group) were injected intraperitoneally with 100 pL mock or AMEV2 vaccinated serum. Mice were anesthetized 24 hours post injection and challenged intranasally with 50 pL (108CFUs) PBS containing Ci79 (FIG. 10). Four of six mice passively vaccinated with AMEV2 antiserum survived the pulmonary challenge, while only one of the mock-inoculated mice survived. Thus, passive vaccination ofFJ ref. UTSA-P0165WO / Client ref. 2022-032-CIP naive mice with AMEV2 antiserum provides antibody -mediated protection against Acinetobacter baumannii infection in vivo.

[0111] The AMEV2 multi-peptide construct allows investigation of which of the five component peptides are necessary for antibody-mediated protection. Previously, we demonstrated that vaccination with AMEV2 resulted in increased IL-4 secreting splenocytes upon restimulation with the AMEV2 peptide construct. The identical T-cell ELISpot recall assay was used to determine peptide-specific reactivity when AMEV2-vaccinated splenocytes were restimulated with the respective peptides (FIG. 11). AMEV2- vaccinated splenocytes demonstrate significant levels of IL-4 and IL-5 but minimal IFNy secretion when restimulated with AMEV2 constituent peptides. Splenocytes restimulated with pNLPE, pTonB, and pOmp38 peptides showed increased IL-5 secretion, while pNucAB and pTonB peptide recall exhibited IL-4 secretion. Additionally, no recall to the pNspec peptide was showed. pNspec is a peptide of similar length to AMEV2 peptides but used in Coccidioides posadassii studies in our laboratory, and was included as a specificity control. Mock- vaccinated mice exhibited no recall response to any of the peptides. Of all the peptides, pTonB showed the highest frequency of Th2 T cell reactivity. The variability of peptide-specific antibody generation in each mouse prompted our investigating the in vivo importance of each peptide across two protection studies (n = 20 mice) (FIG. 11C). AMEV2- vaccinated mice that were protected from A. baumannii challenge had significantly more pTonB- specific antibodies compared to those that were unprotected. pOmp38-specific antibody also showed an increase in protected mice, but not as striking as that observed for the pTonB antibody. To evaluate the importance of pTonB-specific antibodies in vitro, we absorbed AMEV2 antisera with pTonB to reduce the pTonB-specific antibody titer eightfold from 1 : 16000 to 1 :2000 (FIG. 1 ID). The pTonB-absorbed antisera used in the OPKA assay showed significantly reduced killing compared to unabsorbed and nonspecifically absorbed sera resulting in no significant difference in killing between mock and pTonB-absorbed sera. These data indicate that the pTonB peptide contains epitopes important for antibody-mediated protection against Acinetobacter baumannii.

[0112] Given the urgent need for developing new antimicrobials to combat the rise of MDR pathogens, researchers have begun to focus on immunotherapeutics as a viable solution to this global health problem. Previously, we demonstrated the in vivo effectiveness of an immunoinformatics-based multi-peptide vaccine for protection against Acinetobacter baumannii infection. Data presented here show this protection is antibody-mediated and that the antibody-FJ ref. UTSA-P0165WO / Client ref. 2022-032-CIP eliciting peptide is worthy of further investigation for future immunotherapeutic improvement and use against this pathogen.

[0113] Due to the acute nature of the experimental challenge model of A. baumannii and previous vaccine research germane to this pathogen, it was hypothesized that the protective mechanism of AMEV2 was primarily antibody mediated. The robust humoral immune response demonstrated in our previous study was tested for reactivity against UV-inactivated whole A. baumannii bacteria using AMEV2 vaccinated mouse serum. Here, we showed that vaccination successfully generates antibodies that recognize antigens on both the clinical isolate strain used in our protection studies as well as the ATCC type strain. In vitro assays were utilized to delineate whether AMEV2 antibodies function to neutralize or opsonize baumannii.

[0114] The complement system bridges the innate and adaptive immune systems in defense against extracellular pathogens. Variation in complement susceptibility has been well characterized in A. baumannii isolates throughout the years (Kamuyu et al., 2022, Front Immunol 13:853690). Early isolates showed susceptibility while most recent clinical isolates are complement resistant likely due to their capsule (Talyansky et al., 2021, PLoS Pathog 17:el009291). When AMEV2 antiserum was incubated with intact complement, it did not inhibit Ci79 complement resistance. Additionally, no difference in bacteria growth in the absence of complement was observed, indicating no inhibition of bacterial growth in the presence of these antibodies. Additionally, antibodies do not enhance formation of the membrane attack complex (MAC) and subsequent lysis of A. baumannii. The lack of bactericidal activity of vaccinated sera against A. baumannii is consistent with other studies (Luo et al., 2012, PLoS One 7:e29446). Despite exhibiting no bactericidal activity against the pathogen, AMEV2 antisera enhances the killing of A. baumannii when incubated with primary bone marrow-derived macrophages (BMDM) in vitro. This killing is diminished albeit still significant when complement is removed from the assay, indicating that despite the pathogen’s inhibition of MAC formation, AMEV2 antibodies recognize the pathogen and activate the classical complement pathway leading to complement-mediated phagocytosis. Reduction of opsonophagocytic killing in the absence of complement has been observed in other A. baumannii vaccines (Huang et al., 2015, Vaccine 33:4479-85; Huang et al., 2016, Sci Rep 6:20724; Huang et al., 2014, PLoS One 9:el00727). The use of primary BMDMs generated from mice deficient in FC gamma receptors revealed killing of the bacterium to be facilitated through recognition of the FC region of bound AMEV2 antibodiesFJ ref. UTSA-P0165WO / Client ref. 2022-032-CIP by the FCyR of BMDM. Tn the absence of complement, WT BMDM still demonstrated significant killing of bacteria when opsonized with AMEV2 antisera. However, killing wasn’t observed when incubated with FCyR "" BMDM. Thus, we are one of the first to demonstrate the specificity of opsonophagocytic killing in vitro. However, one whole-cell intranasal vaccination study used FCyR" " mice in vivo to elucidate its protective mechanism (KuoLee et al., Vaccine 33:260-7). To their surprise, FCyR’ ’ vaccinated mice were protected in an intranasal challenge, indicating that FC gamma receptor-mediated opsonophagocytosis is unnecessary in the mucosal vaccination regimen. Complement-mediated opsonophagocytosis alone may be sufficient for antibody- mediated protection against A. baumannii.

[0115] The success of passive immunization against A baumannii has demonstrated antibody- mediated protection to be of paramount importance in controlling infections of this extracellular pathogen (Bentancor et al., 2012, Infect Immun 80:3381-8; McConnell and Pachon, 2010 Vaccine 29: 1-5; Russo et al., 2013, Infect Immun 81 :915-22; Cabral et al., 2017, Nat Commun 8: 15480; Nielsen et al., 2021, Infect Immun 89:e0016221). Congruent with this thinking, we passively transferred mock and AMEV2 immunized sera to naive mice before intranasal challenge with A. baumannii. Mice that received AMEV2 antiserum 24 hours prior to challenge received partial protection (67 % survival) compared to the passively immunized mock mice (17% survival). Previously one vaccine was shown not to afford protection via passive immunization (KuoLee et al., Vaccine 33:260-7). KuoLee et al. showed that intranasal vaccination with formalin-killed A. baumannii protected against intranasal challenge; however, when passively transferred to naive mice, they were unprotected. This suggests that a systemic vaccination likely generates a better antibody response for passive transfer than one that solely stimulates mucosal immunity.

[0116] Of the five peptides contained in AMEV2, data presented here indicate antibodies generated against the pTonB peptide significantly enhance opsonophagocytic killing of A. baumannii in vitro. When evaluating AMEV2 peptide-specific immunogenicity by ELISpot, we observed significant IL-4 and IL-5 recall response in AMEV2 splenocytes reexposed to the pTonB peptide. This strong Th2 response results in pTonB-specific IgGl antibodies which are ideal for identification of an extracellular pathogen though enhanced binding to Clq and Fey receptors (Vidarsson et al., 2014, Front Immunol 5:520). Along with the high immunogenicity of this peptide, our in vivo data also demonstrate a correlation between protection, and the amount of pTonB-specific antibody generated in AMEV2 vaccinated mice. The acute nature of this infectionFJ ref. UTSA-P0165WO / Client ref. 2022-032-CIP indicates how successful this pathogen is at growing within the host in which a significant part involves siderophore receptors and iron acquisition (Cook-Libin et al., 2022, Infect Immnti 90:e0022322). Therefore, antibodies targeting this essential function result in protection in an acute model.B. Materials and Methods

[0117] Mouse. Animal experiments were performed using 7-8-week-old C57BL / 6 mice purchased from Jackson Laboratories (Bar Harbor, ME). 7-8-week-old B6.129P2-Fcer / gft”7AavN12 mice were purchased from Taconic Biosciences (Germantown, NY). The mice were housed at the University of Texas at San Antonio in an AAALAC-accredited animal facility. All animal experiments were performed in accordance with Institutional Animal Care and Use Committee Protocol MU070.

[0118] Bacteria. Acinetobacter haumannii type strain ATCC 19606 was purchased from American Type Culture Collection (ATCC, USA). Acinetobacter baumannii clinical isolate 79 (Ci79) obtained from the San Antonio Military Medical Center (SAMMC; Fort Sam Houston, San Antonio, TX, USA) was provided by Dr. James Jorgensen (University of Texas Health Science Center at San Antonio, San Antonio, TX, USA) (Ketter et al., 2014, Genome Announc 2; Ketter et al., 2014, Infect Immun 82:3910-8). Bacteria were streak-plated on Luria-Bertani (LB) agar plates supplemented with ampicillin (100 pg / mL) from a frozen stock. An overnight culture was prepared from a single colony, incubated overnight at 37 °C, subcultured the following day to an ODeoonm = 0.03 in fresh LB broth, and grown for 3.5 h until mid-log phase. Subcultures were centrifuged at 5000 rpm for 5 min, and bacteria pellets were resuspended and washed in phosphate-buffered saline (PBS). Following the first PBS wash, bacteria were diluted to an ODeoonm = 0.5 («2 x io8CFUs / mL). This inoculum was centrifuged and concentrated for the intranasal (i. n.) challenge. Bacterial inoculum CFU / mL was determined by serial dilution and plating.

[0119] Complement Killing Assay. Mock and AMEV2 serum pools were heat-inactivated at 56 °C for 30 minutes to remove complement. Heat-inactivated serum pools were diluted 1 to 10 with PBS, and 50 pL was added to a 96-well plate in quadruplicate. 25 pL of either freshly reconstituted baby rabbit serum or heat-inactivated baby rabbit serum was added to corresponding wells. 15 pL of PBS was added to all wells to make the final reaction volume 100 pL. Lastly, Ci79 strain of A. baumannii wase grown and diluted to approximately 5 x 104CFUs with PBS. 10 pLFJ ref. UTSA-P0165WO / Client ref. 2022-032-CIP(500 CFUs) of each strain was added to the wells. The prepared plate was incubated at 37 °C with 200 rpm rotation for 1 hour and 30 minutes. The reaction was quenched with 100 pL of ice-cold PBS added to all wells. The wells were serially diluted and plated to enumerate viable bacteria.

[0120] Determination of Antibody by ELISA. Microtiter plates were coated with UV- inactivated bacteria (106per well) or AMEV2 peptides (1 pM per well) overnight in sodium bicarbonate buffer (pH 9.5). The ELISAs were carried out as previously described (16). Endpoint titers were determined as the highest dilution with an absorbance reading of 0.1 greater than the blank absorbance reading.

[0121] Bone Marrow-Derived Macrophage Generation. C57BL / 6 or B6.129P2- Fcerl^mlRavSA2 mice were sacrificed, and their two hind limbs were collected for bone marrow isolation. 5 x 106bone marrow cells were seeded into 100mm x 15mm Petri dishes in 10 mLs of complete R10 media (RPMI 1640, 10% fetal bovine serum, 1% Pen-Strep, 1% L-glutamine) supplemented with 20 ng / mL of macrophage colony-stimulating factor (M-CSF) (Gibco). On day 3, an additional 10 mLs of R10 supplemented with 20 ng / mL of M-CSF was added to the petri dish. On day 6, 10 mLs of media was removed from the petri dish, and 10 mLs of R10 supplemented with 20 ng / mL of M-CSF was added back. On day 8, the cells were fed one last time, with the removal of 10 mLs and the addition of 10 mLs of supplemented media. On day 9, the media was removed from the Petri dishes, and PBS was added to wash away the nonadherent and semi-adherent cells. The remaining adhered cells were collected with Accutase cell detachment solution (StemCell). The Accutase reaction was quenched and cells were collected, spun down, and counted for plating. The macrophages were seeded into the experimental plates and incubated with 20 ng / mL of M-CSF overnight to allow recovery from the Accutase treatment before being used in downstream in vitro applications.

[0122] Opsonophagocytic Killing Assay and Uptake Assay. A reaction volume of 100 pL containing 50 % diluted A. baumannii, 37.5 % freshly reconstituted baby rabbit serum, and 12.5 % immunized sera was incubated at 37 °C for 30 minutes at 300 rpm for opsonization. The opsonized bacteria were then diluted with R10 without antibiotics to 105CFU / mL. Wells containing 105adhered BMDMs were washed with R10 without antibiotics, and 100 pL of the opsonized bacteria was pipetted into the wells with or without the macrophages. Plates were spun down 300g for 5 minutes and then placed in the 37 °C 5% CO2 incubator for 1 hour. The plate was gently tapped on all sides to mix every 15 minutes. Following the incubation, the plate wasFJ ref. UTSA-P0165WO / Client ref. 2022-032-CIP placed on an ice pack at 4 °C for 20 minutes to cease macrophage function and bacteria growth. The media in the wells was then serially diluted and plated on LB agar to enumerate the remaining viable bacteria. RIO supplemented with 25 pg / mL of Polymyxin B (Sigma- Aldrich) was added to all macrophage wells and incubated at 4 °C for 1 hour to kill the remaining extracellular bacteria. Following the hour, the cells were washed twice with media and 200 pL of 0.2 % Deoxycholate was added to each well to lyse the macrophages. Complete lysis was observed under the tissue culture microscope, and 100 pL was removed from each well, serially diluted, and plated on LB agar to enumerate bacteria taken up by the macrophages.

[0123] Passive Sera Generation. C57BL / 6 mice (n = 5 per group) were vaccinated subcutaneously with either 100 pL of PBS+AddaS03 or AMEV2+AddaS03 (10 pg per dose) on day 0. Mice were boosted with the same dose on days 14 and 28. Vaccinated mice were terminally bled for serum collection two weeks after the second boost.

[0124] Passive Sera Vaccination and Pulmonary Challenge. C57BL / 6 mice (n = 6 per group) were injected intraperitoneally with 100 pL of serum from either mock or AMEV2 vaccinated mice 24 hours before intranasal challenge. The pulmonary challenge was carried out as previously described (16). The mice were monitored daily for weight loss and morbidity for 14 days and total survival for 30 days.

[0125] T-Cell ELISpot Assays. T-cell reactivity to AMEV2 peptides was evaluated using IFNy,IL-4, and IL-5 ELISpot assays as previously described with minor adjustments (16). Splenocytes were evaluated for in vitro recall with AMEV2 peptides (2 pM). Media-only wells served as a negative control for background, pNspec (2 pM) a Coccidioides posadassii peptide served as a specificity control, and a-CD3 (clone: 145-2C11, 1 pg / mL) served as a positive control. PVDF membrane ELISpot plates (Millipore Sigma) were coated overnight with IFNY (clone: AN- 18, 2 pg / mL), IL-4 (clone: 1 IB 11, 4 pg / mL), or IL-5 (clone: TRFK5, 5 pg / mL) capture antibodies. Biotinylated IFNy (clone: R4-6A2, 0.5 pg / mL), IL-4 (clone: BVD6-24G2, 2 pg / mL), or IL-5 (clone: TRFK4, 0.5 pg / mL) were used as detection antibodies.

[0126] AMEV2 peptide-specific antibody absorption. Immuno-Blot PVDF membrane (BioRad) was pre-wet with methanol and washed with water. 100 pL (590 pg) of either the pNspec or pTonB peptide was pipetted onto separately activated membranes. The membranes were incubated at 4 °C overnight. The following day, the membranes were washed with PBS and then transferred to tubes containing 1% bovine serum albumin (BSA) for 1 hour at room temperatureFJ ref. UTSA-P0165WO / Client ref. 2022-032-CIP to block. After blocking, the membranes were each washed once more with PBS before being transferred to tubes containing AMEV2-vaccinated serum. The membranes were incubated with the sera with consistent shaking at 1500 rpm for 2 hours at room temperature. After 2 hours, the membrane was removed from each tube, and the serum was aliquoted and frozen for later in vitro assays. Successful absorption and reduction in peptide antibody titer were confirmed by peptide ELISA.

[0127] Statistical Analysis. GraphPad Prism 10.0 was used to determine statistical significance tests. Differences between mock and AMEV2 vaccinated groups, as well as mock and AMEV2 antisera treated bacteria, were assessed using the Student’s t-test, One-way ANOVA, and 2way- ANOVA. Survival rates were analyzed with the Log-rank Mantel-Cox test. Differences were considered statistically significant when p < 0.05.

Claims

FJ ref. UTSA-P0165WO / Client ref. 2022-032-CIPCLAIMS1. A polyclonal antibody composition that specifically binds the pTonB peptide of SEQ ID NO: 11, wherein said antibodies are elicited by immunization with a multi-epitope antigen comprising Acinetobacter baumannii thioredoxin, an EAAAK linker, and the immunogenic kernels of SEQ ID NOs: 6, 7, 11, 12, and 8 connected by KK or GPGPG linkers, and wherein said antibodies enhance opsonophagocytic killing (OPK) of baumannii Ci79 in the presence of bone marrow- derived macrophages (BMDMs) and complement.

2. The polyclonal antibody composition of claim 1, wherein removal of pTonB-specific antibodies by absorption with SEQ ID NO: 11 reduces OPK activity by at least 70 % compared to unabsorbed serum.

3. The polyclonal antibody composition of claim 1, wherein the multi-epitope antigen has the amino acid sequence of SEQ ID NO: 2 (AMEV2).

4. The polyclonal antibody composition of claim 1, wherein said antibodies activate the classical complement pathway and mediate phagocytosis via Fey receptors on BMDMs.

5. The polyclonal antibody composition of claim 1, wherein passive transfer of 100 pL of said composition into naive C57BL / 6 mice 24 hours prior to intranasal challenge with 108CFUs of A. baumannii Ci79 results in at least 60 % survival at 30 days.

6. A pharmaceutical composition comprising the polyclonal antibody composition of any one of claims 1-5 and a pharmaceutically acceptable carrier.

7. The pharmaceutical composition of claim 6, formulated for intravenous, intramuscular, or intraperitoneal administration.

8. A method of treating Acinetobacter baumannii infection in a subject, comprising administering to the subject a therapeutically effective amount of the polyclonal antibody composition of any one of claims 1-5.

9. The method of claim 8, further comprising co-administering one or more antibiotics selected from imipenem, meropenem, and colistin.FJ ref. UTSA-P0165WO / Client ref. 2022-032-CIP10. A method of preventing Acinetobacter baumannii colonization or infection in a high-risk subject, comprising administering to the subject a prophylactically effective amount of the polyclonal antibody composition of any one of claims 1-5.

11. A diagnostic kit for detecting Acinetobacter baumannii exposure, comprising: (a) the pTonB peptide of SEQ ID NO: 11 immobilized on a solid support; and (b) a detection reagent that binds human or mouse IgG.

12. A method of identifying protective anti -4. baumannii antibodies in a serum sample, comprising: (a) contacting the serum sample with the pTonB peptide of SEQ ID NO: 11; (b) measuring binding affinity; and (c) correlating binding above a threshold (OD45O > 0.5 at 1 :5000 dilution) with protective capacity as determined by OPK or passive transfer survival.

13. The polyclonal antibody composition of claim 1, wherein the antibodies are humanized or fully human and retain pTonB-specific binding and OPK activity.

14. A polyclonal antibody composition that binds the pTonB peptide of SEQ ID NO: 11 and enhances OPK of A. baumannii in vitro.

15. The polyclonal antibody composition of claim 14, derived from a mammal immunized with the multi-epitope antigen of SEQ ID NO: 2.

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