Compositions comprising OXA-23 and pal of acinetobacter baumannii, or nucleic acids encoding the same

Abstract

The present invention provides a composition for inducing an immune response against Acinetobacter baumannii in a subject, comprising (i) an OXA-23 protein or (ii) a nucleic acid molecule encoding OXA- 23. The invention also provides a composition for inducing an immune response against Acinetobacter baumannii in a subject, comprising (i) a PAL protein or (ii) a nucleic acid molecule encoding PAL. The invention also provides the use of such compositions for inducing an immune response against A. baumannii and for treating A. baumannii infection.

Description

[0001]COMPOSITIONS FIELD OF THE INVENTIONThe present invention relates to compositions for inducing an immune response against Acinetobacterbaumannii (A. baumannii) in a subject, using OXA-23 and / or PAL as antigens. The invention alsorelates to the use of such compositions for inducing an immune response against A. baumannii andfor treating A. baumannii infection.BACKGROUNDAcinetobacter baumannii (A. baumannii) is a highly antibiotic resistant Gram negative bacteriumresponsible for causing a range of nosocomial infections and hospital outbreaks across the globe. Of most concern is the growing antibiotic resistance seen within A. baumannii. Vaccination is one alternative to antibiotics. An effective vaccine has the potential to reduce the economic burden ofbacterial infection by reducing diagnoses and treatment costs. Reduction of antibiotic use as a resultof vaccination can also lead to reduced development of further antibiotic resistance. Additionally, bacteria are less likely to develop resistances to vaccines in part because antibiotics tend to have a single mechanism of action allowing for easier mutational escape. By comparison, vaccines typically work via multiple mechanisms, inducing different host responses such as specific antibody and / or T cell responses making vaccine resistance more difficult to develop. Due to the ability of A. baumannii to quickly and easily develop new resistances to antibiotics, an effective vaccine provides a viable alternative method of control. One challenge for developing new vaccines against bacterial pathogens is identifying the antigens to target that might induce protective immunity. Traditional bacterial vaccines either target a secreted toxin, surface polysaccharide or contain whole, inactivated bacteria. There are potential limitations to these approaches for the development of new vaccines. Not all bacteria make a toxin or if they do, neutralisation of the toxin may not protect against disease. Surface polysaccharide is poorly immunogenic on its own. Whilst this can be improved through conjugation to protein, polysaccharide is very strain specific and can reduce the breadth of protection offered by the vaccine, or may lead to strain replacement. This has been seen with the pneumococcal conjugate vaccine, the new version of which contains 20 different polysaccharides. Whole cell bacterial vaccines can give a greater breadth and longevity of protection, but are also associated with increased reactogenicity; for example whole cell pertussis was replaced with acellular pertussis in many countries. Therefore, targeting a subset of bacterial proteins may be beneficial. Selecting possible protein target antigens has been done for the acellular pertussis and meningitis B vaccines. For acellular pertussis, selection was based upon the predicted role of the proteins in pathogenicity and relative ease of manufacture. For the MenB vaccine, a reverse vaccinology approach was used where brute force expression of all 350 antigens was used to identify the optimumcombination; this was effective but demanding of resources. A. baumannii is a complex bacterialpathogen, expressing around 3,836 genes. Whilst many of these are intracellular and may not be accessible to antibody mediated vaccine protection, there are still many proteins encoded on thebacterial cell surface that might be potential vaccine targets. One study estimated there were at least300 outer membrane proteins. There therefore exists a need in the art for effective vaccines against A. baumannii. SUMMARY OF THE INVENTIONThe present inventors have surprisingly discovered that the Acinetobacter baumannii antigens OXA-23 (a carbapenem-hydrolysing class D ^-lactamase) and peptidoglycan-associated lipoprotein (PAL)are responsible for inducing an immune response to Acinetobacter baumannii. Here we describe theidentification of these protective A. baumannii vaccine antigens using two different identificationmethods as well as the development of protective recombinant Escherichia coli derived OMVs andmRNA vaccines.Accordingly, in a first aspect the present invention provides a composition for inducing an immuneresponse against Acinetobacter baumannii in a subject, comprising (i) an OXA-23 protein or (ii) anucleic acid molecule encoding OXA-23.In a second aspect the present invention provides a composition for inducing an immune responseagainst Acinetobacter baumannii in a subject, comprising (i) a PAL protein or (ii) a nucleic acidmolecule encoding PAL. DESCRIPTION OF THE SEQUENCESThe OXA-23 and PAL sequences referred to herein are as follows:SEQ ID NO: 1 – OXA-23 amino acid sequenceMNKYFTCYVVASLFLSGCTVQHNLINETPSQIVQGHNQVIHQYF DEKNTSGVLVIQTDKKINLYGNALSRANTEYVPASTFKMLNALIGLENQKTDINEIFK WKGEKRSFTAWEKDMTLGEAMKLSAVPVYQELARRIGLDLMQKEVKRIGFGNAEIGQQ VDNFWLVGPLKVTPIQEVEFVSQLAHTQLPFSEKVQANVKNMLLLEESNGYKIFGKTG WAMDIKPQVGWLTGWVEQPDGKIVAFALNMEMRSEMPASIRNELLMKSLKQLNIISEQ ID NO: 2 – OXA-23 DNA sequence 1 atgaataaat attttacttg ctatgtggtt gcttctcttt ttctttctgg ttgtacggtt 61 cagcataatt taataaatga aaccccgagt cagattgttc aaggacataa tcaggtgatt 121 catcaatact ttgatgaaaa aaacacctca ggtgtgctgg ttattcaaac agataaaaaa 181 attaatctat atggtaatgc tctaagccgc gcaaatacag aatatgtgcc agcctctaca 241 tttaaaatgt tgaatgccct gatcggattg gagaaccaga aaacggatat taatgaaata 301 tttaaatgga agggcgagaa aaggtcattt accgcttggg aaaaagacat gacactagga 361 gaagccatga agctttctgc agtcccagtc tatcaggaac ttgcgcgacg tatcggtctt 421 gatctcatgc aaaaagaagt aaaacgtatt ggtttcggta atgctgaaat tggacagcag 481 gttgataatt tctggttggt aggaccatta aaggttacgc ctattcaaga ggtagagttt 541 gtttcccaat tagcacatac acagcttcca tttagtgaaa aagtgcaggc taatgtaaaa 601 aatatgcttc ttttagaaga gagtaatggc tacaaaattt ttggaaagac tggttgggca 661 atggatataa aaccacaagt gggctggttg accggctggg ttgagcagcc agatggaaaa 721 attgtcgctt ttgcattaaa tatggaaatg cggtcagaaa tgccggcatc tatacgtaat 781 gaattattga tgaaatcatt aaaacagctg aatattattt aaSEQ ID NO: 3 – OXA-23 RNA sequence1 augaauaaau auuuuacuug cuaugugguu gcuucucuuu uucuuucugg uuguacgguu 61 cagcauaauu uaauaaauga aaccccgagu cagauuguuc aaggacauaa ucaggugauu 121 caucaauacu uugaugaaaa aaacaccuca ggugugcugg uuauucaaac agauaaaaaa 181 auuaaucuau augguaaugc ucuaagccgc gcaaauacag aauaugugcc agccucuaca 241 uuuaaaaugu ugaaugcccu gaucggauug gagaaccaga aaacggauau uaaugaaaua 301 uuuaaaugga agggcgagaa aaggucauuu accgcuuggg aaaaagacau gacacuagga 361 gaagccauga agcuuucugc agucccaguc uaucaggaac uugcgcgacg uaucggucuu 421 gaucucaugc aaaaagaagu aaaacguauu gguuucggua augcugaaau uggacagcag 481 guugauaauu ucugguuggu aggaccauua aagguuacgc cuauucaaga gguagaguuu 541 guuucccaau uagcacauac acagcuucca uuuagugaaa aagugcaggc uaauguaaaa 601 aauaugcuuc uuuuagaaga gaguaauggc uacaaaauuu uuggaaagac ugguugggca 661 auggauauaa aaccacaagu gggcugguug accggcuggg uugagcagcc agauggaaaa 721 auugucgcuu uugcauuaaa uauggaaaug cggucagaaa ugccggcauc uauacguaau 781 gaauuauuga ugaaaucauu aaaacagcug aauauuauuu aaSEQ ID NO: 4 – PAL amino acid sequenceMKSIKYLAFPLLSAALVMTGCASRKPATTATTGTTNPSTVNTTG LSEDAALNAQNLAGASSKGVTEANKAALAKRVVHFDYDSSDLSTEDYQTLQAHAQFLM ANANSKVALTGHTDERGTREYNMALGERRAKAVQNYLITSGVNPQQLEAVSYGKEAPV NPGHDESAWKENRRVEINYEAVPPLLKSEQ ID NO: 5 – PAL DNA sequence1 atgaaatcaa ttaaatattt ggcctttcct ctacttagtg cggcacttgt catgacaggt 61 tgtgcaagtc gtaagccagc aacaacggca actacaggta caactaaccc aagcacagta 121 aatacgaccg gcttaagtga agatgctgca ttaaatgctc aaaatctagc aggtgcttct 181 tcaaaaggtg taactgaggc aaacaaggct gccctagcga aacgcgtcgt tcacttcgat 241 tatgacagta gtgatttatc tactgaagat taccaaacac ttcaggctca tgctcagttc 301 ctcatggcaa atgcaaactc aaaagttgca ttaactggtc atacagacga gcgcggtaca 361 cgcgaataca acatggcctt gggtgagcgt cgtgcaaaag cagttcagaa ttatcttatt 421 accagtggtg taaatcctca gcaacttgaa gctgtaagtt atggtaaaga agcgcctgtt 481 aatcctggcc atgatgaatc agcttggaaa gaaaaccgcc gcgttgaaat taactatgaa 541 gcggttcctc ctctattaaa ataaSEQ ID NO: 6 – PAL RNA sequence1 augaaaucaa uuaaauauuu ggccuuuccu cuacuuagug cggcacuugu caugacaggu 61 ugugcaaguc guaagccagc aacaacggca acuacaggua caacuaaccc aagcacagua 121 aauacgaccg gcuuaaguga agaugcugca uuaaaugcuc aaaaucuagc aggugcuucu 181 ucaaaaggug uaacugaggc aaacaaggcu gcccuagcga aacgcgucgu ucacuucgau 241 uaugacagua gugauuuauc uacugaagau uaccaaacac uucaggcuca ugcucaguuc 301 cucauggcaa augcaaacuc aaaaguugca uuaacugguc auacagacga gcgcgguaca 361 cgcgaauaca acauggccuu gggugagcgu cgugcaaaag caguucagaa uuaucuuauu 421 accaguggug uaaauccuca gcaacuugaa gcuguaaguu augguaaaga agcgccuguu 481 aauccuggcc augaugaauc agcuuggaaa gaaaaccgcc gcguugaaau uaacuaugaa 541 gcgguuccuc cucuauuaaa auaaAll sequences are taken from Acinetobacter baumannii strain MS14413 chromosome, completegenome (GenBank: CP054302.1)https: / / www.ncbi.nlm.nih.gov / nuccore / CP054302.1?from=1615478&to=1616299&strand=2 (OXA-23)https: / / www.ncbi.nlm.nih.gov / nuccore / CP054302.1?from=896002&to=896565 (PAL)DETAILED DESCRIPTION OF THE INVENTIONThe invention relates to the use of OXA-23 and PAL as antigens to induce an immune response against Acinetobacter baumannii.The first aspect of the invention relates to a composition for inducing an immune response againstAcinetobacter baumannii in a subject, comprising (i) an OXA-23 protein or (ii) a nucleic acid moleculeencoding OXA-23.The composition can further include (i) a PAL protein or (ii) a nucleic acid molecule encoding PAL.The second aspect of the invention relates to a composition for inducing an immune responseagainst Acinetobacter baumannii in a subject, comprising (i) a PAL protein or (ii) a nucleic acidmolecule encoding PAL.The compositions of the invention are capable of inducing an immune response in a subject againstAcinetobacter baumannii and are therefore vaccine compositions.The compositions of the invention typically comprise an OXA-23 protein and / or a PAL protein, or anucleic acid molecule encoding OXA-23 and / or a nucleic acid molecule encoding PAL (rather than amixture of proteins and nucleic acids), as will be described further herein. In some embodiments, compositions of the invention include an OXA-23 protein and / or a PAL protein.The OXA-23 protein typically has the amino acid sequence of SEQ ID NO: 1 or a sequence having atleast 90% identity to SEQ ID NO: 1. For example, the sequence can have at least 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 1.The PAL protein typically has the amino acid sequence of SEQ ID NO: 4 or a sequence having atleast 90% identity to SEQ ID NO: 4. For example, the sequence can have at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 4. “Identity” as known in the art is the relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. In the art, identity also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences. While there exist a number of methods to measure identity between two polypeptide or two polynucleotide sequences, methods commonly employed to determine identity are codified in computer programs. Preferred computer programs to determine identity between two sequences include, but are not limited to, GCG program package (Devereux, et al., Nucleic Acids Research, 12, 387 (1984), BLASTP, BLASTN, and FASTA (Atschul et al., J. Molec. Biol.215, 403 (1990)). One can use a program such as the CLUSTAL program to compare amino acid sequences. This program compares amino acid sequences and finds the optimal alignment by inserting spaces in either sequence as appropriate. It is possible to calculate amino acid identity or similarity (identity plus conservation of amino acid type) for an optimal alignment. A program like BLASTx will align the longest stretch of similar sequences and assign a value to the fit. It is thus possible to obtain a comparison where several regions of similarity are found, each having a different score. Both types of identity analysis are contemplated in the present invention. The percent identity of two amino acid sequences or of two nucleic acid sequences is determined by aligning the sequences for optimal comparison purposes (e.g., gaps can be introduced in the first sequence for best alignment with the sequence) and comparing the amino acid residues or nucleotides at corresponding positions. The “best alignment” is an alignment of two sequences which results in the highest percent identity. The percent identity is determined by the number of identical amino acid residues or nucleotides in the sequences being compared (i.e., % identity = number of identical positions / total number of positions x 100). The determination of percent identity between two sequences can be accomplished using a mathematical algorithm known to those of skill in the art. An example of a mathematical algorithm for comparing two sequences is the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-2268, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877.The NBLAST and XBLAST programs of Altschul, et al. (1990) J. Mol. Biol.215:403-410 haveincorporated such an algorithm. BLAST nucleotide searches can be performed with the NBLAST program, score = 100, wordlength = 12 to obtain nucleotide sequences homologous to nucleic acid molecules. BLAST protein searches can be performed with the XBLAST program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to protein molecules for use in the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilised as described in Altschul et al. (1997) Nucleic Acids Res.25:3389-3402. Alternatively, PSI-Blast can be used to perform an iterated search which detects distant relationships between molecules (Id.). When utilising BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See http: / / www.ncbi.nlm.nih.gov. Another example of a mathematical algorithm utilised for the comparison of sequences is the algorithm of Myers and Miller, CABIOS (1989). The ALIGN program (version 2.0) which is part of the CGC sequence alignment software package has incorporated such an algorithm. Other algorithms for sequence analysis known in the art include ADVANCE and ADAM as described in Torellis and Robotti (1994) Comput. Appl. Biosci., 10 :3-5; and FASTA described in Pearson and Lipman (1988) Proc. Natl. Acad. Sci.85:2444-8. Within FASTA, ktup is a control option that sets the sensitivity and speed of the search. The skilled person is aware that various amino acids have similar properties and thus are “conservative”. One or more such amino acids of a protein, polypeptide or peptide can often be substituted by one or more other such amino acids without eliminating a desired activity of that protein, polypeptide or peptide. Thus the amino acids glycine, alanine, valine, leucine and isoleucine can often be substituted for one another (amino acids having aliphatic side chains). Of these possible substitutions it is preferred that glycine and alanine are used to substitute for one another (since they have relatively short side chains) and that valine, leucine and isoleucine are used to substitute for one another (since they have larger aliphatic side chains which are hydrophobic). Other amino acids which can often be substituted for one another include: phenylalanine, tyrosine and tryptophan (amino acids having aromatic side chains); lysine, arginine and histidine (amino acids having basic side chains); aspartate and glutamate (amino acids having acidic side chains); asparagine and glutamine (amino acids having amide sidechains); and cysteine and methionine (amino acids having sulphur containing side chains). It shouldbe appreciated that amino acid substitutions within the scope of the present invention can be made using naturally occurring or non-naturally occurring amino acids. For example, it is contemplated herein that the methyl group on an alanine may be replaced with an ethyl group, and / or that minor changes may be made to the peptide backbone. Whether or not natural or synthetic amino acids areused, it is preferred that only L- amino acids are present.Substitutions of this nature are often referred to as “conservative” or “semi-conservative” amino acidsubstitutions. The present invention therefore extends to use of a protein or nucleic acid comprisingany of the sequences described above but with one or more conservative substitutions and or one ormore tolerated substitutions in the sequence, such that the amino acid or nucleotide sequence of themolecule, has at least 90% identity, for example at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or 100% identity, to the sequences disclosed herein. The OXA-23 protein and / or PAL protein present in the compositions of the invention are typically produced recombinantly. The OXA-23 protein is typically encoded by the DNA sequence of SEQ ID NO: 2 or a sequence having at least 90% identity to SEQ ID NO: 2. For example, the sequence can have at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 2.The PAL protein is typically encoded by the DNA sequence SEQ ID NO: 5 or a sequence havingat least 90% identity to SEQ ID NO: 5. For example, the sequence can have at least 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 5. For compositions of the invention that include an OXA-23 protein and / or a PAL protein, the OXA-23 and / or PAL protein is typically present in an outer membrane vesicle (OMV).OMVs are immunogenic proteoliposomes that are produced naturally during the growth of manyGram-negative bacteria and bleb off of the outer membrane. They are made up of a phospholipidbilayer with an outer layer made up of lipopolysaccharide, outer membrane proteins and receptors all in their native conformation (Cecil et al Outer Membrane Vesicle-Host Cell Interactions, Microbiol Spectr.2019 January; 7(1)). Native OMVs from the New Zealand outbreak strain of Neisseria meningitidis have been successfully incorporated into the Bexsero 4CMenB vaccine; they may also act as adjuvants in this context. As well as native OMVs, recombinant OMVs can be used as antigen delivery systems. This can be achieved by expressing antigens on the surfaces of bacteria andengineering them to hypervesiculate by deleting components of the Tol-Pal system. For example, onemethod to do this is PCR gene replacement. A DNA fragment encoding for an antibiotic resistancecassette flanked by two short regions of homology to the target gene for deletion is amplified by PCR.The gene targeting fragment is introduced to E. coli expressing ^ red recombinase to generateantibiotic resistant transformants, which can be selected for using agar plates containing the matchingantibiotic (https: / / www.embopress.org / doi / full / 10.1038 / msb4100050#sec-3 ,https: / / www.pnas.org / doi / epdf / 10.1073 / pnas.120163297 ,https: / / www.sciencedirect.com / science / article / pii / S0923250822000481 ). The GSK generalisedmodules for membrane antigens (GMMA) platform utilises OMVs with a modified lipid A structure. Vaccine antigens can then also be conjugated onto the GMMA as a “plug and play” vaccine technology increasing immunogenicity.The OMV is typically a recombinant OMV (rOMV). The recombinant OMV can be produced by E. col,in particular E. coli that have been engineered to hypervesiculate. For example, E. coli can beengineered to hypervesiculate by deleting the TolR gene. The E. coli may be ΔTolR::AmpR E.coli.The composition can include separate OMVs including the OXA-23 and PAL proteins. Accordingly, thecomposition can include an OMV comprising an OXA-23 protein and an OMV comprising a PALprotein.Alternative compositions of the invention include a nucleic acid molecule encoding OXA-23and / or a nucleic acid molecule encoding PAL.The nucleic acid molecule is typically an RNA, more typically an mRNA.mRNA vaccines have a number of advantages including the speed and ease with which they can be produced and scalability. The SARS-CoV-2 pandemic has shown mRNA vaccines can be highly effective, particularly when they incorporate N1-methylpseudouridine to mask the RNA from the cellintrinsic immune response. The modular properties of mRNA vaccines allow for quick and easyvaccine development.The mRNA encoding OXA-23 typically has the sequence of SEQ ID NO: 3 or a sequence having atleast 90% identity to SEQ ID NO: 3. For example, the sequence can have at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 3.The mRNA encoding PAL typically has the sequence of SEQ ID NO: 6 or a sequence having at least90% identity to SEQ ID NO: 6. For example, the sequence can have at least 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 6.The OXA-23 mRNA typically encodes a protein having the amino acid sequence of SEQ ID NO: 1 or asequence having at least 90% identity to SEQ ID NO: 1. For example, the amino acid sequence can have at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 1.The PAL mRNA typically encodes a protein having the amino acid sequence of SEQ ID NO: 4 or asequence having at least 90% identity to SEQ ID NO: 4. For example, the amino acid sequence canhave at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 4. The OXA-23 mRNA is typically encoded by the DNA sequence of SEQ ID NO: 2 or a sequence having at least 90% identity to SEQ ID NO: 2. For example, the DNA sequence can have at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 2.The PAL mRNA is typically encoded by the DNA sequence SEQ ID NO: 5 or a sequence havingat least 90% identity to SEQ ID NO: 5. For example, the DNA sequence can have at least 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 5.The mRNA may include modified nucleosides, for example pseudouridines such as N1-methylpseudouridine. N1-methylpseudouridine (m1Ψ) can be used to mask the RNA from the cellintrinsic immune response.mRNA vaccines are typically incorporated into a delivery vehicle to aid entry into cells and protect themRNA from degradation by nucleases. Lipid nanoparticles (LNPs) are the most clinically advanced and typically consist of an ionizable lipid, a cholesterol variant, a helper lipid and a PEGylated lipid toencapsulate and protect the mRNA. Alternative approaches may be required for the delivery tomucosal surfaces. Accordingly, in this embodiment of the invention the mRNA is typically encapsulated in a lipid nanoparticle (LNP). However, other carriers may be used including micelles, liposomes or polymers.For example, cationic liposome carriers may be used, such as N4 -cholesteryl-spermine. LNPs formRNA delivery are known in the art and described, for example, in Hou et al., Nature Reviews Materials 6, 1078-1094 (2021). The composition can include separate LNPs including the OXA-23 and PAL mRNAs. Accordingly, thecomposition may include an LNP comprising an OXA-23 mRNA and an LNP comprising a PALmRNA.In the first and second aspects of the invention the composition are pharmaceutical compositions andtherefore typically include one or more adjuvants. diluents, carriers. Since the compositions arevaccine compositions they typically include an adjuvant. Suitable adjuvants are well known in the artand include aluminium, AS01B, AS04, CpG 1018, Matrix-M™ and MF59.In a third aspect, the invention provides a composition of the first or second aspect of the invention foruse in a method of inducing an immune response against Acinetobacter baumannii. In other words,the composition of the first or second aspect of the invention for use in preventing an Acinetobacterbaumannii infection.This aspect of the invention can also be worded as: ^A method of inducing an immune response against Acinetobacter baumannii, the methodcomprising administering to a subject in need thereof a composition of the first or secondaspect of the invention. ^Use of a composition of the first or second aspect of the invention in the manufacture of amedicament for inducing an immune response against Acinetobacter baumannii. ^A method of preventing an Acinetobacter baumannii infection, the method comprisingadministering to a subject in need thereof a composition of the first or second aspect of the invention. ^Use of a composition of the first or second aspect of the invention in the manufacture of amedicament for preventing an Acinetobacter baumannii infection.The compositions of the first and second aspect of the invention can also be used for the treatment ofAcinetobacter baumannii infection.Accordingly, in a fourth aspect, the invention provides a composition of the first or second aspect ofthe invention for use in a method of treating Acinetobacter baumannii infection.This aspect of the invention can also be worded as: ^A method of treating Acinetobacter baumannii infection, the method comprising administeringto a subject in need thereof a composition of the first or second aspect of the invention. ^Use of a composition of the first or second aspect of the invention in the manufacture of amedicament for treating Acinetobacter baumannii infection.In this aspect of the invention, the composition of the first or second aspect of the invention is typically administered to a patient in combination with an antibiotic, for example a carbanepem (for exampleimipenem, meropenem, doripenem or ertapenem), sulbactam or colistin (polymyxin E).Compositions of the invention including OXA-23 as an antigen are particularly useful in combination with treatment with carbanepem antibiotics. OXA-23 is a carbapenemase and therefore an OXA-23 vaccine could help to neutralise OXA-23 carbapenemase and increase sensitivity to carbanepemantibiotics. This can be useful for patients seriously ill with Acinetobacter baumannii infection, forexample being treated in an ICU. The compositions of the first or second aspect of the invention can be administered using any suitable mode of administration, for example subcutaneous, intramuscular, intranasal, intradermal, or byinhalation. The compositions are typically administered intramuscularly or intranasally. Intranasaldelivery is particularly preferred. For example, the composition of the first or second aspect of the invention may include an mRNA encoding OXA-23 and / or an mRNA encoding PAL, encapsulated in one or more LNPs or liposomes and be administered intranasally. As used herein, “treatment” is also intended to cover preventative treatment, i.e., prophylaxis.Dosages of the composition of first and second aspect of the invention can vary between wide limits,depending upon the disease or disorder to be treated, the age and condition of the individual to be treated, whether the treatment is prophylactic or therapeutic, the type, onset, progression, severity, frequency, duration, or probability of the disease to be treated, the clinical endpoint desired, previous or simultaneous treatments, etc. Dosages can be based upon current existing protocols, empiricallydetermined, using animal disease models or optionally in human clinical trials. A physician will ultimatelydetermine appropriate dosages to be used. The dosage may be increased or decreased depending on any adverse side effects, complications or other risk factors of the treatment or therapy and the status of the subject.The composition of the first or second aspect of the invention is typically administered to a subject “inneed thereof”, meaning a subject in need of therapeutic or preventative treatment of Acinetobacterbaumannii infection. The composition of the first or second aspect of the invention is typicallyadministered in a “therapeutically effective amount”. By “therapeutically effective amount” is meant anamount sufficient to show a therapeutic benefit to the subject, i.e., to prevent, reduce or relieve thesymptoms of Acinetobacter baumannii infection. A “subject” is also referred to herein as a “patient”.The subject is typically a human subject. Definitions Below are provided certain definitions of terms, technical means, and embodiments used herein. Aspects and embodiments described herein with the term “comprising” may include other features or steps within the scope. It is also understood that aspects and embodiments described as “comprising” also describes aspect and embodiments wherein the term “comprising” is replaced by the term “consisting essentially of” or “consisting of”. The phrase "selected from the group comprising" may be substituted with the phrase "selected from the group consisting of" and vice versa, wherever they occur herein. It is also understood that the application discloses all combinations of any of the above aspects and embodiments described above with each other, unless the context demands otherwise. Similarly, the application discloses all combinations of the preferred and / or optional features either singly or together with any of the other aspects, unless the context demands otherwise. The invention will now be further described by way of the following Examples, which are meant to serve to assist one of ordinary skill in the art in carrying out the invention and are not intended in any way to limit the scope of the invention, with reference to the following Figures.Figure 1 - Schematic to show phage expression cloning process. A lambda phage library wasgenerated containing random fragments of genomic A. baumannii BAL_276 DNA. This library wasused to screen polyclonal serum from mice immunised intramuscularly with BAL_276 derived OMVs and identify the protein targets of the antibodies within the serum.Figure 2 - Three proteins identified via both phage expression cloning and mass spectrometryusing serum from A. baumannii immunised mice. A. baumannii BAL_276 OMVs separated usingSDS-PAGE to perform a western blot (A) using sera from mice immunised intramuscularly withBAL_276 derived OMVs and for staining with Coomassie blue (B) to extract proteins for massspectrometry analysis. Using an A. baumannii BAL_276 lambda phage library and the anti-sera,phage expression cloning was used to identify putative proteins (C). Underlined proteins representthose identified via both methods. OMVs separated via SDS-PAGE were transferred onto membranesfor western blot using human serum from an infected patient (D).Figure 3 - After infection with A. baumannii antibody responses are raised against OXA-23 andPAL in humans. Different capsule type and GC2 subclade clinical isolate derived OMVs wereseparated via SDS-PAGE and the proteins were transferred onto membranes for western blot usinghuman serum from an infected patient (A) and uninfected control patient (B). The bands where OXA- 23 and PAL were visible were highlighted. Figure 4 | OXA-23 and PAL show high levels of sequence homology between different A.baumannii strains. Amino acid sequences for OXA-23 and PAL were collected from the NCBI proteindatabase and aligned. Using MEGA-11 maximum likelihood phylogenetic trees were generated for OXA-23 (A) and PAL (B). The trees were rooted arbitrarily and the best model was selected for each tree using MEGA-11model selector. Due to the size of the phylogenetic trees large numbers ofidentical sequences were collapsed, this is depicted by triangles with the number of collapsedsequences noted. (C) The percentage of identical amino acid sequences for each of the antigens. (D)The consensus sequence of PAL (SEQ ID NO: 4) and % similarity of each amino acid. (E) Theconsensus sequence of OXA-23 (SEQ ID NO: 1) and % similarity of each amino acid. (F) Alphafold predictions of PAL consensus sequence and (G) OXA-23 consensus sequence. Both protein predictions were coloured from the N to C terminal domains; structured regions indicated by helices, unstructured regions by long chain.Figure 5 - After 24 hours of IPTG treatment rOMVs show varied levels of target antigen.ΔTolR::AmpR E. coli possessing each plasmid to express either OXA-23 or PAL were grown in liquid culture with 400mM IPTG to induce protein expression over 24 hours. The rOMVs were separated by SDS-PAGE for westernblot using serum from mice immunised subcutaneously with BAL_276 derivedOMVs to show expression of OXA-23 (A) and PAL (B) proteins on E. coli derived rOMVs. BALB / cmice were vaccinated subcutaneously with 500 FU (~5μg) E. coli derived rOMVs expressing PAL or OXA-23 individually or in combination in a prime boost approach with 21 day intervals (C).Figure 6 - Subcutaneous vaccination with rOMVs provides protection against intranasalchallenge. Mice were challenged intranasally with A. baumannii BAL_27621 days after the boost andculled 24 hours later. Their whole bacterial cell specific IgM (A) and IgG (B) titres were measured byELISA. Their weight loss (C), inflammation level in the lungs (D) and bacterial loads in their BAL fluid(E), nasal lavage (F), lung homogenate (G) and spleen homogenate (H) were measured 24 hoursafter challenge. One-way ANOVAs with post tests were performed to determine significant differences between groups where ** shows p ≤ 0.01, * shows p ≤ 0.05; n=5 per group.Figure 7 - HEK293T cells produce OXA-23 and PAL proteins from mRNA after 24 hourtransfection. HEK293T cells were transfected with mRNA encoding either OXA-23 (A) or PAL (B) and left for 24 hours. Cell lysates and cell supernatants were separated by SDS-PAGE for western blot with serum from mice immunised subcutaneously with BAL_276 derived OMVs.Figure 8 - Intramuscular vaccination with OXA-23 encoding mRNA provides protection againstintranasal challenge. BALB / c mice were vaccinated intramuscularly with 5μg mRNA encoding PAL orOXA-23 in a prime boost approach with 21 day intervals. Mice were challenged intranasally withA. baumannii BAL_27621 days after the boost and culled 24 hours later. Their whole bacterial cellspecific IgG (A) and IgM (B) titres were measured by ELISA. Their weight loss (C), inflammation levelin the lungs (D) and bacterial loads in their BAL fluid (E), IL-6 nasal lavage (F), lung homogenate (G)and spleen homogenate (H) were measured 24 hours after challenge. Shapiro-Wilk tests for normality were performed using graph pad prism V9.3.1 and either ordinary one-way ANOVAs or Kruskal-Wallistests were performed to determine significant differences between groups where **** shows p ≤0.0001, *** shows p ≤ 0.001, ** shows p ≤ 0.01, * shows p ≤ 0.05.Figure 9 - Incorporation of m1ψ does not improve protection. BALB / c mice were vaccinatedintramuscularly with 5 μg of LNP formulated wild type mRNA or m1ψ mRNA encoding OXA-23 in aprime boost approach with 21 day intervals. Whole bacterial cell specific IgG (F) were measured in serum after prime and boost immunisation. Mice were challenged intranasally with A. baumannii BAL_27621 days after the boost and culled 24 hours later. Their body weight change (B) and bacterial loads in their Nasal lavage (C), BAL fluid (D), lung (E) and spleen homogenate (F) were measured 24 hours after challenge. Cells collected from lung were counted (G) and stained for CD4 Trm (H) and CD8 Trm (I). One-way ANOVAs with post tests were performed to determine significant differences between groups where **** shows p ≤ 0.0001, *** shows p ≤ 0.001, ** shows p ≤ 0.01, * shows p ≤ 0.05; n=5 per group.Figure 10 - Intranasal delivered OXA-23 reduces weight loss and increases bacterial clearance.BALB / c mice were vaccinated intramuscularly (IM) or intranasally (IN) with 1μg of wild type mRNA OXA-23 formulated with GL67 in a prime boost approach with 21 day intervals. Whole bacterial cell specific IgG (A), measured in the serum after prime or boost immunisation. Mice were challenged intranasally with A. baumannii BAL_27621 days after the boost and culled 24 hours later. Their body weight change (B) and bacterial loads in their Nasal Lavage (C), BAL fluid (D), lung (E) and spleen homogenate (F) were measured 24 hours after challenge. Cells collected from lung were counted (G) and stained for CD4 Trm (H) and CD8 Trm (I). One-way ANOVAs with post tests were performed to determine significant differences between groups where **** shows p ≤ 0.0001, *** shows p ≤ 0.001, ** shows p ≤ 0.01, * shows p ≤ 0.05; n=5 per group. EXAMPLES Native OMV productionClinical isolates of A. baumannii strains BAL_276 was isolated from ICU patients in Ho Chi Minh. All A.baumannii strains were grown at 37°C, 200rpm in Lysogeny broth (LB) or statically at 37°C onHiCrome Acinetobacter Agar Base (HIMEDIA M1938: Trafalgar). OMVs were generated as described [Higham]. Briefly, bacteria were grown planktonically in LB overnight. The culture was centrifuged at 7,000g for 15 minutes at 4°C and the supernatant containing the OMVs was vacuum filtered using a 0.45μm filter. The filtered supernatant was then ultracentrifuged at 100,000g for 2 hours at 4°C to pellet the OMVs. OMVs were quantified by incubation with lipophilic dye FM4-64 in a black 96 wellpolypropylene microtiter plate. Fluorescence was then read using an EnVision Plate Reader (PerkinElmer) with excitation 520nm and emission 700nm. OMV Immunisation of mice to generate polyclonal sera All animal studies were performed in accordance with the United Kingdom’s Home Office guidelines under animal study protocol number (P4EE85DED). All work was approved by the Animal Welfare and Ethical Review board at Imperial College London. Studies also followed NC3Rs guidelines, and all work was carried out in biosafety level-two facilities. The studies performed here used specific pathogen free, female, 6–8-week-old BALB / c mice, bought from Charles River Laboratories (Hertford, UK). Mice were maintained in individually ventilated cages,housed in groups of five with water and food provided ad libitum. The cages were kept in a specificpathogen free room at 20 – 24°C with 55±10% humidity.To generate polyclonal sera, mice were intramuscularly with 100 FU of OMVs from A. baumannii (fromstrain BAL_276) in a 50μl volume. Mice were immunised in a prime-boost regime with a 3-week interval between immunisations. Serum was collected 3 weeks after boost. SDS-PAGE, western blotting and mass spectrometry For western blot, 500 fluorescence units of OMV (approximately 5µg) were run down mini-PROTEAN TGX gels and the proteins were transferred onto PVDF membranes using Trans-Blot Turbo Mini PDVF Transfer Packs and a Trans-Blot Turbo. Membranes were blocked with 1% bovineserum albumin in phosphate buffered saline, washed with 0.1% PBS-tween then coated with serumfrom mice intramuscularly immunised with OMVs. Anti-mouse IgG-HRP was used for detection of OMV specific IgG and blots were visualised using western HRP chemiluminescent substrate (Fisher Scientific WBLUR) and imaged using a Biostep, Celvin S Chemiluminescent imager. For identification by mass spectrometry, 500 FU OMV were run on a separate mini-PROTEAN TGX gel in parallel to the Western blot gel. The gel was stained with InstantBlue Coomassie protein stain and imaged. The protein bands corresponding to the western blot were excised and an in gel tryptic digestion kit (Thermo Scientific 89871) was used to extract the protein ready for analysis by mass spectrometry. The digested samples were analysed by online nano-LC-ESI-MS and MS / MS using a Synapt G2-S (Waters), quadrupole time-of-flight mass spectrometer. Online analysis was performed using a nano- Acquity Ultra Performance LC (Waters). A BEH C18 column (75 μm inner diameter x 15 cm length) was used for all analyses. Samples were eluted from the column using 0.1% formic acid in water and a 10%-45% acetonitrile gradient over 45 minutes. For data-independent acquisition, LC-MSE was performed, with fragmentation achieved using CAD with argon as the collision gas.Protein identification – Raw LC-MS and MS / MS data were processed with ProteinLynx GlobalSERVER version 3.01 (Waters) to identify proteins, searching against UniProt specific databases (UniProt Consortium, 2021). Phage expression cloningPhage expression cloning was performed using the methods presented by Michael J. Lodes et. Al andusing the Agilent Lambda ZAP II Undigested Vector Kit (236201-12). In brief (Fig.1), the lambdaphage library was generated by extracting genomic DNA from A. baumannii BAL_276 and sheeringvia sonication. The ends were blunted with DNA ligase and dNTPs before adding on EcoRI adaptors for cloning. Finally, fragments were selected using DNA size selection columns and ligated into the lambda ZAP II expression vector pBluescript SK. The phage library was titrated to estimate plaque forming units per ml and the quality of the library was checked by colony PCR and amplified DNA fragments were analysed via gel electrophoresis to confirm a variation in the size of gDNA fragments. After confirming the phage library was of good quality, the library was amplified and aliquots were stored at -80°C for future use. Negative phage containing empty vector was also produced to be used as negative pick phage. This allowed for sera from mice, immunised intramuscularly with BAL_276 derived OMVs, to be pre- adsorped with Escherichia coli proteins and remove any antibodies from the sera binding to E. coliprior to screening against the A. baumannii phage library.To perform the primary screen, E. coli XL1-Blue MRF cells were grown overnight in LB broth at 30°C, shaking at 200rpm. The following morning the culture was centrifuged, and the bacterial pellet was resuspended in 10mM MgSO4 and diluted to an OD600 of 0.5 in 10mM MgSO4. In a microcentrifuge tube, the E. coli XL1-Blue MRF cells were mixed with the positive phage and incubated at 37°C for 15 minutes, to allow the phage to attach to and begin to infect the E. coli XL1-Blue MRF cells, prior to mixing the cells and phage in NZY top agar and pouring the mixture on top of NZY agar plates. After the top agar had set, the plates were incubated at 42°C until plaques began to form. Nitrocellulose membranes that had been pre-soaked in 10mM IPTG were placed on top of the plates and the plates were incubated at 37°C overnight. The following morning, the plates were cooled at 4°C for 15 minutes before removing the membranes, which were then washed in 0.1% PBS-Tween20 and then blocked using 1% bovine serum albumin in 1% PBST with rocking. The membranes were washed again before adding the pre-adsorped mouse serum to each membrane and incubating at room temperature with rocking. The membranes were washed again prior to the addition of AP conjugated anti-mouse IgG to membranes and incubation at room temperature with rocking. Finally, membranes were washed more before adding NBT / BCIP until purple spots developed. Positive plaques corresponding to the purple spots on the membrane were removed (plugged phage) and placed into SM buffer containing chloroform and stored at 4°C. To reduce potential contamination, a secondary screen was performed. At the initial step of mixing the E. coli XL1-Blue cells and the phage the plugged phage from the primary screen was used instead of the phage from the phage library. To excise the phagemids, plugged phage from the secondary screen were vortexed and incubated at room temperature before storing at 4°C. E. coli XL1-Blue MRF and SOLR cells were grown overnight in LB broth with supplements, centrifuged and resuspended to an OD600 of 1.0 in 10mM MgSO4. The E. coli XL1-Blue MRF cells, plugged positive phage and ExAssist helper phage were mixed and incubated at 37°C. LB broth with supplements was then added to the tubes and the tubes were incubated at 37°C with shaking for 2-3 hours. The lambda phage particles were then lysed by incubating the tubes for 20 minutes at 65-70°C and the cell debris was pelleted at 1,000 G for 15 minutes. The supernatant was added to a microcentrifuge tube containing SOLR cells and incubated for 15 minutes at 37°C before plating the mixture onto LB ampicillin plates and incubating overnight at 37°C. Individual colonies were picked into LB broth containing ampicillin to grow overnight and extractplasmids to be sequenced. The sequencing data was then blasted against the A. baumannii genometo identify the proteins. Human sera Serum samples from the ‘Microbial Products and effects on the host’ project obtained at the point of admission were linked to anonymised patient data provided by an NHS clinician including the date the sample was taken in relation to the point of admission. (West London REC reference 06 / Q0406 / 20). Phylogenetic tree analysis To produce phylogenetic trees, amino acid sequences were collected from the NCBI protein database for blaOXA-23 and pal for A. baumannii. Any duplicate sequences were removed and following the methods published by B.G. Hall27, the sequences were aligned using MEGA11 and muscle alignment with standard settings. For each protein, the model selector was used to determine the best model to use when generating the maximum likelihood tree. Each collection of aligned amino acid sequences was then used to generate phylogenetic tree files in MEGA11 using the JTT model for Oxa-23 and JTT + G for PAL. Finally, the trees were formatted in FigTree to collapse long branches containing identical sequences. rOMV production To produce recombinant OMVs the pDSG254 vector was digested with NotI and the blaOXA-23 andpal genes were amplified from A. baumannii BAL_276 gDNA using the following primers:blaOXA-23-Forward: GCTAGGCCCAGCCGGCCATGAATAAATATTTTACTTGCTATGTGGTTGC (SEQ ID NO: 7) blaOXA-23-Reverse:GTCAGCGGCCGCTTAAATAATATTCAGCTGTTTTAATGATTTCATCAATAATTC (SEQ ID NO: 8)pal-Forward: GCTAGGCCCAGCCGGCCATGAAATCAATTAAATATTTGGCCTTTCCTC (SEQ ID NO:9)pal-Reverse: GTCAGCGGCCGCTTATTTTAATAGAGGAGGAACCGCTTC (SEQ ID NO: 10)The pDSG254 vector was digested using NotI and the amplified genes were ligated into the vector. Hypervesiculating ΔTolR::AmpR E. coli were made electrocompetent and transformed via electroporation with the pDSG254 vectors containing the blaOXA-23 and pal genes. The transformedE. coli were grown in LB broth with ampicillin and chloramphenicol at 37°C with shaking until theyreached OD6000.5 – 0.6 and IPTG was added to a concentration of 400μM to induce proteinexpression and the tubes were incubated overnight at 37°C overnight. To separate the rOMVs, the bacterial culture was centrifuged at 7,200 xg and the supernatant was removed and filtered using a 0.45μm stericup. The filtered supernatant was then ultracentrifuged at 100,000 xg to pellet the rOMVs and the rOMVs were resuspended in PBS. To quantify the rOMVs, they were incubated at room temperature with FM4-64 lipophilic dye. The fluorescence was read using a FLUOstar Omega with excitation 520 nm, emission 700 nm. The data was expressed as fluorescence units (FU) relative to the volume of sample added to the well. mRNA production and formulationTo produce mRNA blaOXA-23 and pal were amplified out of A. baumannii BAL_276 gDNA using thefollowing primers:blaOXA-23-Forward: GCTAGCTCTTCCATGAATAAATATTTTACTTGCTATGTGGTTGC (SEQ ID NO:11) blaOXA-23-Reverse:GCTAGCTCTTCGTTAAATAATATTCAGCTGTTTTAATGATTTCATCAATAATTC (SEQ ID NO: 12)pal-Forward: GCTAGCTCTTCCATGAAATCAATTAAATATTTGGCCTTTCCTC (SEQ ID NO: 13)pal-Reverse: GCTAGCTCTTCGTTATTTTAATAGAGGAGGAACCGCTTC (SEQ ID NO: 14)The genes were cloned into the LG005 vector, an mRNA vaccine plasmid with a T7 promoter (Φ6.5), clean cap AG initiation site, alpha globin 5’UTR, Kozak sequence, beta globin 3’UTR and a segmented poly-A tail consisting of 60A’s-G-60A’s. Amplified genes were digested with SapI and the genes were ligated into the vector. The vector was linearised with BamHI and NsiI before blunting with T4 DNA polymerase and dNTPs (NEB) following manufactures instructions to remove the 3’ overhang and reduce dsRNA formation. The mRNA was in vitro transcribed using the HiScribe T7 High Yield RNA Synthesis Kit (NEB E2040S) with clean cap AG (Trilink) followed by LiCl purification. Using an N / P ratio of 6 the mRNA was formulated in lipid nanoparticles using the lipid components C12-200, DSPC, cholesterol and DMPE-PEG2000, in a PBS 10% sucrose solution. Using the RiboGreen RNA quantitation assay kit (Life Technologies, UK) the amount of mRNA present in the LNP formulations was quantified both with and without 2% Triton X-100 to determine the concentration of mRNA in the formulation and % of encapsulation compared to an RNA standard. Additionally, the zeta potential and polydispersity index were determined using the ZetaSizer Ultra (Malvern Instruments), folded capillary cuvettes were used to make the measurements. The mean diameters and polydispersity were determined using dynamic light scattering. In vivo vaccination and challenge Mice were vaccinated in a prime boost approach with 21-day intervals. When vaccinated with mRNA mice were administered with 5μg total mRNA formulated in LNPs intramuscularly. Where mice were immunised with a combination of two mRNA vaccines, 2.5μg of each antigen were mixed prior to vaccine administration. For rOMVs, mice were given 500 FU subcutaneously. Where mice were immunised with a combination of the two antigens, 250 FU each antigen were administered subcutaneously.Mice were challenged intranasally 21-days after being boosted with A. baumannii BAL_276 and culled24 hours after challenge. For intranasal challenge mice were anaesthetised via inhalation of isoflurane before being intranasally challenged with 5x107 CFU per mouse in 100μl sterile PBS. Mice were weighed prior to challenge and then again 24 hours later before being culled. To determine bacterial loads in the bronchoalveolar lavage and nasal lavage fluid the samples were serially diluted 1:10 and plated onto HiChrome Acinetobacter Agar Base plates, which were incubated overnight at 37°C to determine the colony forming units per ml. For the lungs and spleens, both lobes of the lungs and the whole spleens were mashed using the gentleMACS tissue dissociator. The lung and spleen homogenates were then serially diluted 1:10 and plated onto HiChrome Acinetobacter Agar Base plates and incubated overnight at 37°C. Bacterial specific ELISAELISAs were performed on A. baumannii BAL_276 whole bacterial cells. Bacteria were cultured untilthey reached OD6001.0 and pelleted via centrifugation to remove the LB broth and resuspend in carbonate coating buffer to OD6000.5. Bacterial suspension was added to sample wells and anti-κ anti-λ antibodies were used to coat the standard curve wells. After washing with 0.05% PBST and blocking with 1% BSA in PBS, plates were coated with mouse serum on the sample wells or isotype control IgG / IgM on the standard wells. Anti-mouse IgG HRP or anti mouse IgM HRP were used to detect total IgG and IgM and TMB substrate was used to develop the ELISA, using 2N H2SO4 to stop the reaction before reading the plates at 450nm using a FLUOstar omega microplate reader. Cytokine ELISA To measure IL-6 levels in the BAL fluid and lungs, the R&D systems mouse IL-6 DuoSet ELISA kit was used (DY406). Statistical Analysis Calculations as described in figure legends were performed using GraphPad Prism 9 (GraphPad Software Inc., La Jolla, CA, USA). Results Use of polyclonal mouse and human sera to identify potential antigens. To identify potential vaccine antigens, serum from mice vaccinated intramuscularly with A. baumannii BAL_276 derived OMVs was used in two different antigen identification assays: mass spectrometryand phage expression cloning. A. baumannii OMVs were run on an SDS-PAGE gel and polyclonalmouse sera used to identify proteins recognised following immunisation. The corresponding bands of a Coomassie stained SDS-PAGE gel were extracted and analysed by mass spectrometry (Fig.2A). This method identified 5 proteins: putative pilus assembly protein FilF (FilF), outer membrane proteinA (OmpA), carbapenem hydrolysing class D ^ lactamase OXA-23 (OXA-23), peptidoglycan-associatedlipoprotein (PAL) and domain of unknown function 333 (DUF333). In parallel, phage expression cloning was performed using a lambda phage library containing randomfragments of the A. baumannii BAL_276 genome. This library was screened using the same polyclonalmouse sera, leading to multiple positive hits. Sequencing of the positive phage identified many proteins (Fig.2B), 3 of which overlapped with those identified via mass spectrometry. The three antigens recognised by antisera and identified via both methods were FilF, OXA-23 and PAL. Having identified the antigens using mouse sera, we confirmed that they were recognised by humanantibodies. We performed a Western blot against A. baumannii BAL_276 OMVs using convalescentserum from patients hospitalised with A. baumannii infection. Compared to sera from control bloodthere were much stronger bands for both OXA-23 and PAL (Fig.3A) which were more intense than an uninfected control human sera (Fig.3B). This suggests that upon natural infection an immune response against OXA-23 and PAL is raised by the immune system. We therefore focussed on these 2 antigens. To determine the level of similarity of the identified antigens between different strains of A. baumannii, amino acid sequences were collected from the NCBI database. The sequences were aligned and the best model was chosen to produce maximum likelihood phylogenetic trees using MEGA11 for OXA-23 (Fig.4A) and PAL (Fig.4B). The longest branches containing identical sequences were collapsed, with the number of identical sequences in each collapsed branch noted next to it. PAL was the most highly conserved, with 86.5% of sequences were identical and 83.6% of the OXA-23 sequences were identical (Fig.4C). Having identified 2 potential antigens, we tested them for immunogenicity and protection using two different vaccine platforms rOMV and mRNA. rOMVs expressing OXA-23 and PAL were immunogenic and protected against infection Hypervesiculating ΔTolR::AmpR E. coli can be used to produce rOMVs expressing antigens of interest on their surface. We generated E. coli expressing either OXA-23 or PAL on its surface. The presence of OXA-23 and PAL on the surface of the E. coli derived rOMVs was confirmed via western blot usingpolyclonal serum from mice intramuscularly immunised with A. baumannii BAL_276 derived OMVs.Bands were visible for both OXA-23 (Fig.5A) at around 30 kDa and PAL (Fig.5B) at around 19 kDa after 24 hour induction with IPTG during rOMV production. To determine whether E. coli derived rOMVs could provide any protection against intranasal challengewith A. baumannii BAL_276, mice were immunised subcutaneously in a prime boost regime (21 daysapart) with OXA-23 rOMVs and PAL rOMVs individually or in combination. There was no difference inthe level of whole A. baumannii bacterial cell specific IgM at day 21 between any of the mice (Fig.6A).By day 43 the immunised mice had developed a whole bacterial cell specific IgM response that was significantly increased in the PAL immunised mice compared to the PBS and combined groups. Micedid not generate much of an anti A. baumannii whole bacterial cell specific IgG response 21 days afterprime (Fig.6B). However, at day 43 an IgG response began to develop in the immunised groups that was significantly increased in the OXA-23 immunised mice compared to the PBS (p≤0.05).Mice were intranasally challenged with A. baumannii BAL_276 on day 42. All mice lost weight 24hours after challenge (Fig.6C). All immunised groups had significantly reduced inflammation in their lungs, measured by IL-6 in the lung supernatant (p≤0.05, Fig.6D). Significantly less bacteria were recovered from BAL fluid of the mice immunised with OXA-23 and PAL both individually and in combination compared to the naïve mice (Fig.6E, p≤0.05). The amount of bacteria recovered from the nose was significantly reduced in the mice immunised with OXA-23 rOMVs compared to the naïve mice (Fig.6F, p≤0.05). However, there was no significant difference between the mice immunised with PAL or the two antigens in combination compared to the naïve mice. In the lungs, only the mice vaccinated with OXA-23 and PAL in combination had significantly reduced bacterial recovery compared to the naïve mice (Fig.6G, p≤0.01). There was very little dissemination of bacteria from the lungs to spleen across all of the groups, including the naïve mice (Fig.6H). As a whole, the results show that the rOMVs are able to induce a systemic IgG and IgM response in the mice, and a significant reduction of bacterial load in the airways. This suggests both antigens in combination and individually have some level of protective efficacy in an rOMV format. OXA-23 and PAL mRNA vaccines induce systemic IgG responses and reduce bacterial burden in the airways We tested whether a second vaccine platform, mRNA, was able to induce a protective immune response to the same antigens. The genes OXA-23 and PAL were cloned into an mRNA plasmid vaccine backbone for in vitro transcription. Since bacterial proteins have different post-translational modification to eukaryotes, we wanted to confirm that antigens expressed by eukaryotic cells could berecognised by antibodies from mice exposed to native A. baumannii antigens. To determine whethermammalian cells could produce recognisable vaccine antigens from the mRNA vaccine constructs, HEK293T cells were transfected with mRNA encoding the OXA-23 and PAL proteins. The proteins within the cell lysates and supernatants were evaluated by Western blot using serum from miceimmunised intramuscularly with A. baumannii BAL_276 nOMVs. The antisera bound strongly to OXA-23 expressed by HEK293T cells (Fig.7A), which was located within the cell lysate. The antisera also bound strongly to the PAL expressed by HEK293T cells (Fig.7B), but PAL was found in the cell supernatant. Therefore, mammalian cells are capable of producing bacterial antigens OXA-23 and PAL from mRNA vaccine constructs that are recognised by sera mice immunised with A. baumannii, at least when denatured. To determine in vivo efficacy of the OXA-23 and PAL mRNA vaccines the mRNA was formulated into LNPs and mice were immunised in a prime boost regime (21 days apart) with LNP formulated mRNAvaccines individually. There wasn’t a strong IgM response to either of the vaccinations (Fig.8A). Therewasn’t much difference in the whole cell specific IgG detected in the serum at day 21 (Fig.8B), however both the OXA-23 and PAL IgG titres increased by day 43. The amount of whole cell specific IgG detectable in the mice immunised with OXA-23 was significantly increased at day 43 compared to naïve and PAL immunised mice (p≤0.0001).Mice were then challenged on day 42 intranasally with A. baumannii BAL_276 and culled 24 hourslater. All mice lost weight (Fig.8C) and all mice had high levels of inflammation in their lungs, measured by IL-6 levels in the lung supernatant (Fig.8D) 24 hours after challenge. Although mice immunised with OXA-23 and PAL both had reduced bacterial recovery from their BAL fluid of ~2.5 logs(Fig.8E), only OXA-23 had significantly reduced bacterial burden compared to naïve mice (p≤0.05).Similar results were seen in the bacteria recovered from the nose (Fig.8F) where mice immunised with OXA-23 and PAL both had a reduction in bacterial burden. However, only OXA-23 was significantly decreased compared to naïve mice (p≤0.01). Despite a reduction in bacterial burden in the lungs of both immunised groups, only the OXA-23 immunised mice had significantly reduced bacterial loads compared to the naïve mice (Fig.8G, p≤0.05). Immunising mice with PAL had no effect on bacterial dissemination to the spleen (Fig.8H). However, OXA-23 immunisation significantly reduced dissemination of bacteria to the spleen (p≤0.05) compared to both the naïve group and PAL immunised mice. Only one mouse from the OXA-23 immunised group had bacteria recoverable from the spleen compared to all 5 mice in both the PAL and naïve groups. This data suggests in an mRNA format that OXA-23 is able to significantly reduce bacterial burden and induce a significant systemic IgG response. For viral derived antigens, the incorporation of N1-methylpseudouridine (m1Ψ) instead of uridine can improve the level of protection for virally derived antigens, we wished to evaluate the impact of m1Ψ incorporation on responses to OXA-23. To investigate whether the substitution of uridine (unmodified) with m1ψ uridine (modified) would improve the protective efficacy of the vaccine, three groups of 5 mice were treated with PBS or 5µg of either wildtype UTP (WT mRNA) or m1ψ UTP (m1ψ mRNA) mRNA vaccine. Both vaccines led to statistically significant increases in whole bacteria specific serumIgG compared to the PBS group on both pre-boost and pre-challenge timepoints of 21- and 41-dayspost immunisation (Fig.9A). There was no significant difference in the antibody concentration between the vaccines at any timepoint. Across the naïve and vaccinated groups of mice, there were no statistically significant protection against weight loss from infection (Fig.9B). Both mRNA vaccines significantly reduced bacterial load recovered from the nasal lavage (Fig.9C), but only the group immunised with WT mRNA had significantly reduced bacterial load compared to the PBS control (Fig.9D). Both groups had significantly reduced bacterial recovery from the lung compared to the PBSgroup (Fig.9E). Neither group reduced the bacterial load in the spleen (Fig.9F). We also assessed the impact of RNA type on T cell responses in the lung after infection. From flow cytometry analysis of T cells harvested from the lungs, similar lung cell counts were observed across modified and unmodified mRNA vaccine (Fig.9G). The WT mRNA immunised group had significantly more CD4 Trm (defined as CD69+ / CD103+) in the lungs (Fig.9H) compared to PBS or m1Ψ groups; likewise they had significantly more CD8 Trm (Fig.9I). Having seen no advantage in m1Ψ incorporation, we investigated whether other route of immunisation could improve protection or bacterial clearance. With an OMV based vaccine, we observed improved clearance with intranasal vaccination and hypothesised that intranasal vaccination might also improvemRNA vaccine efficacy. We formulated WT mRNA in cationic lipid Genzyme lipid (GL) 67 (N4 -cholesteryl-spermine), which has previously been used to deliver DNA for gene therapy (Emerson,Michael et al. “Transfection efficiency and toxicity following delivery of naked plasmid DNA and cationic lipid-DNA complexes to ovine lung segments.” Molecular therapy : the journal of the American Society of Gene Therapy vol.8,4 (2003): 646-53. doi:10.1016 / s1525-0016(03)00233-8). Mice were immunised with 1μg mRNA formulated in GL67 either intramuscularly or intranasally. Both vaccines led to statistically significant increases in whole bacteria specific serum IgG compared to the PBSgroup on both pre-boost and pre-challenge timepoints of 21- and 41-days post immunisation (Fig.10A). There was no significant difference in the antibody concentration between the vaccines at any timepoint. Mice were then intranasally challenged with A. baumannii; the intranasal immunised group lost significantly less weight than the PBS group (Fig.10B). Intranasal immunisation protected against bacterial infection, with extremely low levels of bacterial recovery from the upper (Fig.10C) or lower airways (Fig.10D). There was also a significant reduction in bacterial numbers recovered from the lungs (Fig.10E) or spleen (Fig.10F) of the IN immunised group. IN immunisation led to a significant greater clearance of bacteria from the nose than IM immunisation. We also assessed the impact of RNA type on T cell responses in the lung after infection. IN immunisation was associated with fewer total cells in the lung following infection (Fig.10G), but an increase in both CD4 (Fig.10H) and CD8 (Fig.10I) Trm. We therefore conclude it is possible to deliver mRNA vaccines IN and they offer improved protection. Discussion Potential vaccine antigens were identified via phage expression and mass spectrometry, two antigens were carried forward to develop novel rOMV and mRNA vaccines against A. baumannii. Two key antigens were identified through phage expression cloning and mass spectrometry, OXA-23 and PAL. High levels of identical sequences across the majority of sequences collected from the NCBI databasesuggests these antigens should be broadly protective. The inventors showed that rOMV and LNPformulated mRNA vaccines are immunogenic in BALB / c mice and that both antigens can induce systemic antibody responses and reduce bacterial burden in mice intranasally challenged with A. baumannii.Here we showed that mRNA vaccines encoding A. baumannii antigens were able to induce whole cellspecific IgG responses in mice and reduce bacterial burden in the lungs, respiratory tract and reduce dissemination to the spleen. The difference in vaccine efficacy between OXA-23 and PAL could be due to PAL being secreted from mammalian cells, subjecting PAL to post-translational modifications inthe secretory pathway, which have been reported previously to dampen immune responses to nucleicacid vaccines. During mammalian protein expression the addition of N-linked sugars onto bacterial proteins can alter their epitopes and affect antigen presentation to MHC II molecules. However, by modifying nucleosides and codon optimising the sequence it’s possible to dampen the innate immune response to sequence and secondary structures formed by mRNA thereby improving protein translation efficiency and antibody recognition. Despite previous suggestion that RNA based bacterial vaccines resulted in inferior protection against bacterial infection, our work suggests that the level of protection given by rOMV vaccines are comparable to mRNA based vaccines; this is supported by previous work. In addition to comparable levels of protection mRNA-LNP vaccines are self- adjuvanting, induce strong humoral and cellular immune responses and are easy to produce and modify allowing for quick response to new isolates of concern or new antigens. Suggesting that mRNA-LNP vaccines provide an effective platform for the development of novel bacterial vaccines and potential for use in combatting antimicrobial resistance.Here we identified a novel vaccine antigen for A. baumannii not previously described, OXA-23, andshow its efficacy in two vaccine types, E. coli derived rOMV and mRNA-LNP based vaccines. Inaddition to this, this is the first time an mRNA-LNP vaccine has been used for A. baumannii and thefirst intranasal delivery of a bacterial antigen, reducing bacterial burden during in vivo infection as wellas protecting against weight loss. We have shown OXA-23 to provide protection against intranasal A.baumannii challenge in BALB / c mice being capable of reducing inflammation and bacterial burdenduring in vivo infection. In addition to helping to prevent infection, it could also lead to improved treatment outcomes and work synergistically with antibiotic therapies to better treat A. baumannii infection. This work was supported by BBSRC (grant number BB / M011178 / 1), Imperial College LondonConfidence in Concept funding, and the Bacterial Vaccines (BactiVac) Network funded by the GCRFNetworks in Vaccines Research and Development which was co-funded by the MRC and BBSRC. Additional support was provided by The Department of Health and Social Care as part of the Global AMR Innovation Fund (GAMRIF), a UK aid programme that supports early-stage innovative research in underfunded areas of antimicrobial resistance (AMR) research and development for the benefit ofthose in low- and middle-income countries (LMICs), who bear the greatest burden of AMR. The viewsexpressed in this publication are those of the author(s) and not necessarily those of the UK Department of Health and Social Care. This work was also supported by MR / X502959 / 1. EQUIVALENTS AND SCOPE Those skilled in the art will appreciate that the present invention is defined by the appended claims and not by the Examples or other description of certain embodiments included herein. Similarly, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. Unless defined otherwise above, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention. Generally, nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, genetics and protein and nucleic acid chemistry described herein are those well-known and commonly used in the art, or according to manufacturer’s specifications. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited.

Claims

CLAIMS1. A composition for inducing an immune response against Acinetobacter baumannii in a subject,comprising (i) an OXA-23 protein or (ii) a nucleic acid molecule encoding OXA-23.

2. The composition according to claim 1, wherein the composition further includes (i) a PALprotein or (ii) a nucleic acid molecule encoding PAL.

3. A composition for inducing an immune response against Acinetobacter baumannii in a subject,comprising (i) a PAL protein or (ii) a nucleic acid molecule encoding PAL.

4. The composition according to any preceding claim, wherein the composition comprises anOXA-23 protein and / or a PAL protein.

5. The composition according to claim 4, wherein the OXA-23 protein has the amino acidsequence of SEQ ID NO: 1 or a sequence having at least 90% identity to SEQ ID NO: 1.

6. The composition according to claim 4, wherein the PAL protein has the amino acid sequenceSEQ ID NO: 4 or a sequence having at least 90% identity to SEQ ID NO: 4.

7. The composition according to any one of claims 4-6, wherein the OXA-23 and / or PAL proteinis present in an outer membrane vesicle (OMV).

8. The composition according to claim 7, wherein the OMV is a recombinant OMV (rOMV).

9. The composition according to claim 8, wherein the recombinant OMV is produced by E. coli.

10. The composition according to claim 9, wherein the E.coli are hypervesiculating E.coli.

11. The composition according to claim 9 or 10, wherein the E. coli possess a deletion of TolR.

12. The composition according to any one of claims 9-11, wherein the E. coli are ΔTolR::AmpRE.coli.

13. The composition according to any one of claims 7-12, wherein the composition includes anOMV comprising an OXA-23 protein and an OMV comprising a PAL protein.

14. The composition according to any one of claims 1-3, wherein the composition comprises anucleic acid molecule encoding OXA-23 and / or a nucleic acid molecule encoding PAL,optionally wherein the nucleic acid molecule is an mRNA.

15. The composition according to claim 14, wherein the mRNA encoding OXA-23 has thesequence of SEQ ID NO: 3 or a sequence having at least 90% identity to SEQ ID NO: 3.

16. The composition according to claim 14 or 15, wherein the mRNA encoding PAL has thesequence of SEQ ID NO: 6 or a sequence having at least 90% identity to SEQ ID NO: 6.

17. The composition according to any one of claims 14-16, wherein the mRNA is encapsulated ina lipid nanoparticle (LNP) or liposome.

18. The composition according to any one of the preceding claims, wherein the compositionfurther includes an adjuvant.

19. The composition according to any one of the preceding claims for use in a method of inducingan immune response against Acinetobacter baumannii.

20. A method of inducing an immune response against Acinetobacter baumannii, the methodcomprising administering to a subject in need thereof the composition according to any one of claims 1-18.

21. The composition according to any one of claims 1-18 for use in a method of treatingAcinetobacter baumannii infection.

22. A method of treating Acinetobacter baumannii infection, the method comprising administeringto a subject in need thereof the composition according to any one of claims 1-18.

23. The composition for use or the method according to claim 21 or 22, wherein the composition isadministered in combination with an antibiotic.

24. The composition for use or the method according to claim 23, wherein the antibiotic is acarbapenem, sulbactam or colistin (polymyxin E).

25. The composition for use or the method according to any one of claims 19-24, wherein thecomposition is administered intramuscularly, intranasally or subcutaneously.

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