COMBINATORIAL PLATFORM FOR THE DISPLAY OF SURFACE ADJUVANTS AND ANTIGENS

Abstract

Engineered bacteria are provided that produce modified lipid A and a polypeptide or polysaccharide antigens. In some aspects, immunogenic compositions are provided comprising a modified a lipid A and a polypeptide or polysaccharide antigen.

Claims

1. An isolated engineered bacteria strain comprising (a) at least one expression vector comprising at least one polynucleotide encoding a lipid A modification enzyme, wherein the gene encoding a lipid A modification enzyme is selected from the group consisting of lpxE, lpxF, lpxO, lpxR, pagL, and pagP; and (b) at least one expression vector comprising at least one polynucleotide encoding a polysaccharide antigen biosynthesis protein, wherein the engineered bacteria strain produces the polysaccharide antigen covalently conjugated to a modified lipid A.

2. The isolated engineered strain of claim 1, further comprising a deletion of the Eck1673 gene.

3. The isolated engineered strain of claim 1, further comprising a deletion of the rfbD gene.

4. The isolated engineered strain of claim 1, further comprising a deletion of the rfbB-wbbL genetic region.

5. The isolated engineered strain of claim 1, comprising at least lpxE, pagL and pagP.

6. The isolated engineered strain of claim 1, comprising at least lpxE, lpxO, pagL and pagP.

7. The isolated engineered strain of claim 1, wherein the polysaccharide antigen is Vibrio cholerae O antigen, Salmonella typhimurium O antigen, or Shigella spcs O antigen.

8. The isolated engineered strain of claim 7, wherein the at least one polynucleotide encoding a polysaccharide antigen biosynthesis protein is selected from the group consisting of a gmhD gene, a manC gene, a manB gene, a gmd gene, a wbeE gene, a wbeG gene, a wzm, a wzt gene, a wbeK gene, a wbeL gene, a wbeM gene, a wbeN gene, a wbeO gene, a wbeP gene, a wbeT gene, a wbeU gene, a wbeV gene, a gale gene, and a wbeW gene, and a wbf region gene.

9. The isolated engineered strain of claim 1, wherein the polysaccharide antigen is Streptococcus pneumonia capsule, Staphylococcus aureus capsule, or Neisseria meningitidis capsule.

10. The isolated engineered strain of claims 1, wherein the bacteria is an E. coli bacteria.

11. The isolated engineered strain of claim 9, wherein the at least one polynucleotide encoding a polysaccharide antigen biosynthesis protein is selected from the group consisting of a wzm gene, a wzt gene, a wzx gene, and a wzy gene.

12. A composition isolated from the engineered bacteria strain of any one of claims 1-11, wherein the composition comprises the polysaccharide antigen covalently conjugated to a modified lipid A.

13. The composition of claim 12, wherein the composition comprises isolated outer membrane vesicles.

14. An isolated engineered bacteria strain comprising (a) at least one expression vector comprising at least one polynucleotide encoding a lipid A modification enzyme, wherein the gene encoding a lipid A modification enzyme is selected from the group consisting of lpxE, lpxF, lpxO, lpxR, pagL, and pagP; and (b) at least one expression vector comprising at least one polynucleotide encoding an antigenic protein fused to a membrane anchor sequence, wherein the engineered bacteria strain produces a modified lipid A and an outer membrane-bound antigenic protein.

15. The isolated engineered strain of claim 14, further comprising a deletion of the Eck1673 gene.

16. The isolated engineered strain of claim 14, further comprising a deletion of the pagP, lpxT, and eptA genes.

17. The isolated engineered strain of claim 14, wherein the antigenic protein comprises the HA2 domain of the hemagglutinin protein.

18. The isolated engineered strain of claim 14, wherein the membrane anchor sequence comprises the membrane anchor sequence from OmpA

19. The isolated engineered strain of claim 18, wherein the antigenic protein is fused to the C-terminus of a Lpp-OmpA fusion protein.

20. The isolated engineered strain of claims 14, wherein the bacteria is an E. coli bacteria.

21. The isolated engineered strain of claim 20, wherein the polynucleotide encoding an antigenic protein fused to a membrane anchor sequence is codon optimized for expression in E. coli.

22. A composition isolated from the engineered bacteria strain of any one of claims 14-22, wherein the composition comprises the antigenic protein fused to a membrane anchor sequence and a modified lipid A.

23. The composition of claim 22, wherein the composition comprises isolated outer membrane vesicles.

24. An immunogenic composition comprising the composition according to any one of claim 12, 13, 22 or 23.

25. An isolated engineered bacteria strain comprising (a) at least one expression vector comprising at least one polynucleotide encoding a glycosylation acceptor protein; (b) at least one expression vector comprising at least one oligosaccharyltransferase; and (b) at least one expression vector comprising at least one polynucleotide encoding a polysaccharide antigen biosynthesis protein, wherein the engineered bacteria strain produces the polysaccharide antigen covalently conjugated to the glycosylation acceptor protein.

26. The isolated engineered strain of claim 25, further comprising a deletion of the rfbD gene.

27. The isolated engineered strain of claim 25, further comprising a deletion of the ribB-wbbL genetic region.

28. The isolated engineered strain of claim 25, further comprising a deletion of the waaL gene.

29. The isolated engineered strain of claim 25, wherein the glycosylation acceptor protein is acrA.

30. The isolated engineered strain of claim 25, wherein the oligosaccharyltransferase is pglB.

31. The isolated engineered strain of claim 25, wherein the polysaccharide antigen is Vibrio cholerae O antigen, Salmonella typhimurium O antigen, or Shigella spcs O antigen.

32. The isolated engineered strain of claim 31, wherein the at least one polynucleotide encoding a polysaccharide antigen biosynthesis protein is selected from the group consisting of a gmhD gene, a manC gene, a manB gene, a gmd gene, a wbeE gene, a wbeG gene, a wzm, a wzt gene, a wbeK gene, a wbeL gene, a wbeM gene, a wbeN gene, a wbeO gene, a wbeP gene, a wbeT gene, a wbeU gene, a wbeV gene, a gale gene, and a wbeW gene, and a wbf region gene.

33. The isolated engineered strain of claims 25, wherein the bacteria is an E. coli bacteria.

34. The isolated engineered strain of claim 25, wherein the polysaccharide antigen is Streptococcus pneumonia capsule, Staphylococcus aureus capsule, or Neisseria meningitidis capsule.

35. The isolated engineered strain of claim 34, wherein the at least one polynucleotide encoding a polysaccharide antigen biosynthesis protein is selected from the group consisting of a wzm gene, a wzt gene, a wzx gene, and a wzy gene.

36. A composition comprising a polysaccharide antigen covalently conjugated to a glycosylation acceptor protein isolated from the isolated engineered bacteria strain of any one of claims 25-35.

37. The composition of claim 36, comprised in a pharmaceutically acceptable carrier.

38. An engineered E. coli strain comprising (a) one or more lipid modification polynucleotides selected from the group consisting of a lpxE, a lpxF, a lpxO, a lpxR, a pagL, and a pagP polynucleotide, and (b) one or more antigen associated polynucleotides, wherein the E. coli strain creates one or more lipid A moieties linked to the one or more antigens.

39. The engineered E. coli strain of claim 38, wherein the one or more lipid modification polynucleotides are the lpxE, pagL and pagP polynucleotides.

40. The engineered E. coli strain of claim 38, wherein the one or more lipid modification polynucleotides are the lpxE, lpxO, pagL and pagP polynucleotides.

41. The engineered E. coli strain of claim 38, wherein the linkage is a co-localization at the E. coli outer membrane.

42. The engineered E. coli strain of claim 41, wherein an expression vector comprises the one or more lipid modification polynucleotides, the one or more antigen associated polynucleotides, an Lpp signal polynucleotide, and an ompA polynucleotide; wherein the E. coli strain has a deletion of a pagP, lpxT, eptA and lpp polynucleotide; and wherein the one or more antigen associated polynucleotides are polypeptide encoding.

43. The engineered E. coli strain of claim 41, wherein the one or more antigen associated polynucleotides are a hemagglutinin polynucleotide and/or a neuraminidase polynucleotide.

44. The engineered E. coli strain of claim 38, wherein the linkage is a covalent linkage.

45. The engineered E. coli strain of claim 44, wherein one expression vector comprises the one or more lipid modification polynucleotides; wherein another expression vector comprises the one or more antigen associated polynucleotides; wherein the E. coli strain has a deletion of an lpp polynucleotide; and wherein the one or more antigen associated polynucleotides are polysaccharide generating.

46. The engineered E. coli strain of claim 45, wherein the E. coli strain has a deletion of an rfbD polynucleotide or a polynucleotide region spanning an rfbB polynucleotide to a wbbL polynucleotide.

47. The engineered E. coli strain of claim 45, wherein the polysaccharide is an O antigen or a non-O antigen capsular polysaccharide.

48. The engineered E. coli strain of claim 47, wherein the O antigen is selected from the group consisting of a V. cholerae, a S. typhimurium, and a Shigella species O antigen.

49. The engineered E. coli strain of claim 48, wherein the one or more V. cholera O antigen associated polynucleotides are selected from the group consisting gmhD gene, a manC gene, a manB gene, a gmd gene, a wbeE gene, a wbeG gene, a wzm gene (ABC transport), a wzt gene (ABC transport), a wbeK gene, a wbeL gene, a wbeM gene, a wbeN gene, a wbeO gene, a wbeP gene, a wbeT gene, a wbeU gene, a wbeV gene, a gale gene, and a wbeW gene, and a wbf region gene.

50. The engineered E. coli strain of claim 47, wherein the non-O antigen capsular polysaccharide is selected from the group consisting of a S. pneumoniae, S. aureus and N, meningitidis capsular antigen.

51. A composition isolated from the engineered E. coli strain of any of claims 38-50, wherein the composition comprises the one or more lipid A moieties linked to the one or more antigens.

52. The composition of claim 51, wherein the composition comprises an outer membrane vesicle.

53. A pharmaceutical composition comprising the composition of claim 51.

54. An engineered E. coli strain comprising an acrA polynucleotide, a pglB polynucleotide, and one or more O antigen associated polynucleotides, wherein the E. coli strain has a deletion of a waal polynucleotide.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0036] 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 specific embodiments presented herein.

[0037] FIG. 1. A schematic representation of the pACYC184 plasmid.

[0038] FIGS. 2A-D. Engineering a protein-based antigen/adjuvant vaccine delivery platform. (A) An illustration of a fusion protein containing an N-terminal outer membrane lipoprotein sorting sequence (Lpp), a transmembrane domain (OmpA) fused to a C-terminal influenza hemagglutinin (HA) containing the conserved HA2 domain is localized to the outer membrane of E. coli with the MPL adjuvant. (B) Western blot analysis of the Lpp-OmpA construct and the tripartite Lpp-OmpA-HA fusion protein from whole cells and isolated OMVs. (C) E. coli cells expressing the tripartite Lpp-OmpA-HA2 protein on the surface of the cells. A FITC-labeled secondary antibody was used to detect the surface localized HA2 protein. (D) MALDI-TOF mass spectrometry of lipid A isolated from E. coli cells.

[0039] FIGS. 3A-C. The gene and protein sequences of HA2 and a timeline of mouse vaccinations studies. (A) The HA2 domain from Influenza A H1N1 PR/8/1934 (SEQ ID NOs: 13-14; Bommakanti et al., 2012) and (B) H3N2 HongKong/68 (SEQ ID NOs: 15-16; Bommakanti et al., 2010) that are used in the influenza vaccines. The genetic coding sequences and the translated amino acid sequence of each are included. (C) Mice are vaccinated with orally with Lpp-OmpA-HA expressing whole bacteria or isolated OMVs. Primary vaccination occurs on day 0 and mice are boost vaccinated on day 28. On day 44 mice are challenged with influenza virus and monitored for illness. On day 7 post infection, mice are sacrificed for serum and lung collection.

[0040] FIGS. 4A-D. Vaccination protects mice from influenza challenge. (A) Oral vaccination of mice with PBS, E. coli expressing MPL and the surface localized Lpp-OmpA protein (MPL), or E. coli expressing MPL and the surface localized tripartite Lpp-OmpA-HA protein (MPL-HA). (B) Intranasal vaccination of mice with PBS, OMVs isolated from E. coli expressing MPL and the surface localized Lpp-OmpA protein (MPL), OMVs isolated from E. coli expressing MPL and the surface localized tripartite Lpp-OmpA-HA protein (MPL-HA) or BPL-inactivated virus. (C) Survival curve of challenged mice after vaccination. (D) ELISA using sera from each group to detect recombinant HA2 protein.

[0041] FIGS. 5A-E. Phenotypic changes after lethal challenge. (A) Mice (n=5) were vaccinated intranasally with OMVs and challenged with a lethal dose of influenza. Changes in weight were monitored and reported as fractional weight for each group. (B) PBS-vaccinated mouse on day seven post-challenge (C) MPL-vaccinated mouse on day seven post infection (D) MPL-HA-vaccinated mice on day seven post-infection (E) Inactivated virus vaccinated mouse on day seven post-infection.

[0042] FIGS. 6A-D. Engineering a glycoconjugate-based antigen/adjuvant vaccine delivery platform. (A) An illustration of a V. cholerae O -antigen linked to E. coli lipooligosaccharide. E. coli MPL is attached to core oligosaccharide that gets covalently linked to the O-antigen of V. cholerae. (B) Pro-Q emerald 300 stain of lipopolysaccharide from V. cholerae, E. coli DH1, BN1 expressing pPM1001, and BN1 with a mutation in the rfbD gene expressing pPM1001. (C) BN1 cells that were exposed to a FITC-labeled secondary antibody specific to the V. cholerae O -antigen. (D) MALDI-TOF mass spectrometry analysis of lipid A isolated from cells expressing the enzymes to make MPL.

[0043] FIG. 7. Outline of the combinatorial platform for the display of surface adjuvants and antigens in E. coli.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0044] Provided herein are engineered Escherichia coli strains that create lipid A moieties linked with antigen and methods for producing these E. coli strains. In some embodiments, the lipid A moiety is covalently linked to the antigen. In other embodiments, the lipid A moiety is linked to the antigen via a co-localization in an outer membrane of the E. coli. The compositions and methods described herein advantageously provide a means for producing a whole vaccine (a linked antigen and an adjuvant) from a bacterium. The compositions and methods further provide a broad range of possible lipid A moiety and antigen combinations, and thereby allows for selection and creation of lipid A and antigen combinations that are specifically tailored to generate a desired immune response. Accordingly, the compositions created by the E. coli strains are highly useful as vaccines or vaccine components.

[0045] Provided herein are new bacterial vaccine production platforms where nonpathogenic bacteria produce antigen and adjuvant on the cell surface or where the adjuvant and antigen are purified from whole bacteria using OMVs. Previously, a bacterial system was developed in nonpathogenic E. coli that expressed lipid A modification genes from a plasmid. This work resulted in 61 distinct E. coli strains that each generated a unique lipid A adjuvant molecule on the surface of the cell (Needham et al., 2013). In this work, this new adjuvant technology is built on by adapting both protein and carbohydrate antigens to express coordinately with a bacterial-derived lipid adjuvant on the cell surface. Initial pilot vaccines were produced with a HA2-domain protein that was tested in mice to understand the efficacy of this system. The influenza vaccine successfully induced an antibody response specific to the HA protein, resulted in reduced viral titers after lethal challenge, and protected vaccinated mice from influenza. In addition, this vaccine platform has also been engineered to directly link carbohydrate antigens onto a lipid adjuvant, such as MPL, to produce glycoconjugate vaccines. Carbohydrate antigens and lipid adjuvants are produced on the cell surface and purified as a vaccine to protect from cholera infections.

I. DEFINITIONS

[0046] As used in the specification and claims, the singular form a, an, and the include plural references unless the context clearly dictates otherwise. For example, the term a cell includes a plurality of cells, including mixtures thereof.

[0047] The terms about and approximately are defined as being close to as understood by one of ordinary skill in the art. In one non-limiting embodiment the terms are defined to be within 10%. In another non-limiting embodiment, the terms are defined to be within 5%. In still another non-limiting embodiment, the terms are defined to be within 1%.

[0048] As used herein, the term comprising is intended to mean that the compositions and methods include the recited elements, but not excluding others. Consisting essentially of when used to define compositions and methods, shall mean excluding other elements of any essential significance to the combination. Thus, a composition consisting essentially of the elements as defined herein would not exclude trace contaminants from the isolation and purification method and pharmaceutically acceptable carriers, such as phosphate buffered saline, preservatives, and the like. Consisting of shall mean excluding more than trace elements of other ingredients and substantial method steps for administering the compositions of this invention. Embodiments defined by each of these transition terms are within the scope of this invention.

[0049] The expression control sequences refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. The control sequences that are suitable for prokaryotes, for example, include a promoter, optionally an operator sequence, and a ribosome binding site. DNA for a presequence or secretory leader may be operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, operably linked means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous.

[0050] The term deletion, when referring to a polynucleotide sequence or a gene, is used herein to refer to an effective deletion of the function of the polynucleotide sequence or gene. More specifically, a deletion includes a complete removal, a partial removal, and one or more mutations that render the polynucleotide sequence, the gene, or a polypeptide encoded by the polypeptide or gene, inactive or ineffective for its desired purpose.

[0051] As used herein, expression refers to the process by which polynucleotides are transcribed into mRNA and/or the process by which the transcribed mRNA is subsequently being translated into peptides, polypeptides, or proteins.

[0052] A gene refers to a polynucleotide containing at least one open reading frame that is capable of encoding a particular polypeptide or protein after being transcribed and translated.

[0053] The terms link, linked, and linkage refer to a close proximity and do not require a physical touching. In some embodiments, these terms refer to a covalent bond. In other embodiments, these terms refer to a co-localization such as at an outer membrane.

[0054] A pharmaceutical composition is intended to include the combination of an active agent with a pharmaceutically acceptable carrier, inert or active, making the composition suitable for diagnostic or therapeutic use in vivo or ex vivo.

[0055] The term pharmaceutically acceptable carrier means a carrier or excipient that is useful in preparing a pharmaceutical composition that is generally safe and non-toxic, and includes a carrier that is acceptable for veterinary and/or human pharmaceutical use. As used herein, the term pharmaceutically acceptable carrier encompasses any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water, and emulsions, such as an oil/water or water/oil emulsion, and various types of wetting agents. As used herein, the term carrier encompasses any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, lipid, stabilizer, or other material well known in the art for use in pharmaceutical formulations and as described further below. The pharmaceutical compositions also can include preservatives. A pharmaceutically acceptable carrier as used in the specification and claims includes both one and more than one such carrier.

[0056] The terms polynucleotide and oligonucleotide are used interchangeably, and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three-dimensional structure, and may perform any function, known or unknown. The following are non-limiting examples of polynucleotides: a gene or gene fragment, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component. The term also refers to both double- and single-stranded molecules.

[0057] A polynucleotide is composed of a specific sequence of four nucleotide bases: adenine (A); cytosine (C); guanine (G); thymine (T); and uracil (U) for thymine (T) when the polynucleotide is RNA. Thus, the term polynucleotide sequence is the alphabetical representation of a polynucleotide molecule. This alphabetical representation can be input into databases in a computer having a central processing unit and used for bioinformatics applications such as functional genomics and homology searching.

[0058] As used herein, an antigen associated polynucleotide includes a polynucleotide that encodes the antigen polypeptide and a polynucleotide that encodes a polypeptide, which polypeptide functions to create or modify the antigen. In some embodiments, an antigen associated polynucleotide encodes an enzyme that creates or modifies a polysaccharide antigen such as a capsular antigen. Capsular antigens include O antigens and non-O antigens. These antigen associated polynucleotides are referred to herein as polysaccharide generating.

[0059] The term polypeptide is used in its broadest sense to refer to a compound of two or more subunit amino acids, amino acid analogs, or peptidomimetics. A peptide of three or more amino acids is commonly called an oligopeptide if the peptide chain is short. If the peptide chain is long, the peptide is commonly called a polypeptide or a protein.

[0060] A polynucleotide or polynucleotide region (or a polypeptide or polypeptide region) has a certain percentage (for example, 80%, 85%, 90%, or 95%) of sequence identity to another sequence means that, when aligned, that percentage of bases (or amino acids) are the same in comparing the two sequences. This alignment and the percent homology or sequence identity can be determined using software programs known in the art, for example those described in CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (F. M. Ausubel et al., eds., 1987) Supplement 30, section 7.7.18, Table 7.7.1. Preferably, default parameters are used for alignment. A preferred alignment program is BLAST, using default parameters. In particular, preferred programs are BLASTN and BLASTP, using the following default parameters: Genetic code=standard; filter=none; strand=both; cutoff=60; expect=10; Matrix=BLOSUM62; Descriptions=50 sequences; sort by=HIGH SCORE; Databases=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+SwissProtein+SPupdate+PIR. Details of these programs can be found at the following Internet address: ncbi.nlm.nih.gov/cgi-bin/BLAST.

[0061] Transformation of a cellular organism with DNA means introducing DNA into an organism so that at least a portion of the DNA is replicable, either as an extrachromosomal element or by chromosomal integration.

[0062] The term vector means a DNA construct containing a DNA sequence which is operably linked to a suitable control sequence capable of effecting the expression of the DNA in a suitable host. Such control sequences include a promoter to effect transcription, an optional operator sequence to control such transcription, a sequence encoding suitable mRNA ribosome binding sites, and sequences which control the termination of transcription and translation. The vector may be a plasmid, a phage particle, or simply a potential genomic insert. Once transformed into a suitable host, the vector may replicate and function independently of the host genome, or may in some instances, integrate into the genome itself. A plasmid is the most commonly used form of vector, however, the invention is intended to include such other forms of vectors which serve equivalent function as and which are, or become, known in the art.

II. ASPECTS OF THE PRESENT EMBODIMENTS

[0063] Influenza virus is a highly transmissible respiratory pathogen that results in about 40,000 deaths annually and kills millions during pandemic years (Fan et al., 2004). Influenza viruses are classified into two epidemiologically interesting types including A and B. While only one serologically distinct influenza B virus exists, influenza A viruses are highly variable and strains are subtyped based on two antigenic surface glycoproteins called hemagglutinin (HA) and neuraminidase (Lamb and Krug, 2001). While influenza A viruses include 16 different HA proteins (H1-H16) and nine neuraminidase (N1-N9), Human-infectious influenza viruses are primarily A-type H1N1, H2N2, H3N2, and B-type viruses, but cross-species infections from avian-associated H5N1 and H7N7, and H9N2 and swine-associated strains have recently emerged. These avian influenza outbreaks are extremely worrisome because of close human interaction with birds present the possibility of a new influenza pandemic (Horimoto and Kawaoka, 2005).

[0064] Currently, vaccination is considered the most effective way to prevent influenza transmission. The most common influenza vaccine consists of heat-inactivated H1N1, H3N2, and B-type viruses that are grown in chicken eggs, but recently, a cold-adapted live virus has been developed that actively replicates in the nasal passages to generate immunity. While each of these vaccination methods produce protective antibodies to protect from influenza, each has drawbacks. Due to antigenic drift and shift of the A-type viruses, influenza vaccines require regular modifications based on emergent viral strains. However, production of each virus strain takes at least six months. Therefore, quick production of vaccines in epidemics/pandemics is not possible with current technology. Furthermore, a recombinant vaccine that can be produced in a timeframe of days or weeks that offers long-term protection from influenza would be ideal for controlling viral outbreaks.

[0065] Influenza HA is a highly immunogenic protein that coats the surface of the virus and it has been the target of many vaccines (Cox, 2005). HA is required for influenza infection by promoting fusion with host cells. Cellular proteases cleave the HA protein into HA1 and HA2 domains, which comprise a necessary step for viral infection (Skehel and Wiley, 2000). Viral entry is mediated by the HA1 domain binding to salic acid receptors on the surface of the host cell. Through endocytosis the virion enters the cell where it is transported to the endosome where the acidic pH promotes structural changes in the HA protein. Conformational shifts expose the HA2 domain, and promote fusion of the viral and endosomal membranes. The crystal structures of HA have revealed that cleavage of the HA1 and HA2 domains and the low pH of the endosome are required for structural alterations that promote viral infection (Chen et al., 1998; Bullough et al., 1994; Sauter et al., 1992).

[0066] While the HA2 domain of HA is considerably more conserved than the HA1 domain, neutralizing antibodies directed at both protein domains offer protection from influenza challenge (Skehel and Wiley, 2000; Gocnik et al., 2008; Smirnov et al., 2000; Okuno et al., 1994). In fact, several neutralizing antibodies has been isolated that target the conserved HA2 domain. These antibodies target conserved epitopes in the HA2 protein of several influenza A subtypes to offer broad protection (Ekiert et al., 2009; Sui et al., 2009; Throsby et al., 2008; Okuno et al., 1993; Sancheck-Fauquier et al., 1987). Mechanistically, the antibodies presumably inhibit the conformational changes that are necessary for viral and host membrane fusion at low pH. Blocking viral entry by targeting the conserved HA domain could offer widespread protection from influenza viruses in human, chickens, and swine, which are the major reservoirs for these viruses.

[0067] A bacterial-based vaccine was engineered that targets the conserved domain of HA2 to offer widespread protection against influenza A viruses. A mouse model was used to demonstrate the efficacy of the influenza protein-based vaccine. The benefits of this new technology are that it is a quick, low cost, high yield production of influenza vaccines that does not require growth of viruses in chicken eggs. Incorporating a conserved antigenic HA2 epitope into a bacterial based vaccine system could be valuable against the threat of epidemics/pandemics.

[0068] In addition to this protein-based vaccine, this system has also been adapted to produce glycoconjugate vaccines that target carbohydrate epitopes of pathogenic bacteria. The efficacy of this system has been demonstrated by generating a vaccine against the conserved O-antigen of Vibrio cholerae. Cholera disease is a potentially lethal diarrheal disease that affects millions of people every year (Harris et al., 2012; Kaper et al., 1995). The well-conserved Vibrio cholerae O-antigen has been shown to provide a protective immune response against infection (Seed et al., 2012). Therefore, a bacterium was engineered to produce the V. cholerae O -antigen as the antigen in the bacterial-based vaccine system.

III. NUCLEIC ACID-BASED EXPRESSION SYSTEMS

[0069] A wide range of nucleic acid-based expression systems may be used for the expression of polypeptide antigens or genes controlling synthesis of polysaccharide antigens of the embodiments. For example, one embodiment of the invention involves transformation of bacteria with the coding sequences of fusion polypeptides comprising a polypeptide antigen linked to a membrane anchor sequence (and section signal). Numerous expression systems exist that comprise some or all of the sequence components discussed below.

[0070] The term expression vector refers to a vector containing a nucleic acid sequence coding for at least part of a gene product capable of being transcribed and then translated into a polypeptide. Expression vectors can contain a variety of control sequences, which refer to nucleic acid sequences necessary for the transcription and possibly translation of an operably linked coding sequence in a particular host organism (e.g., gram positive or gram negative bacteria). In addition to control sequences that govern transcription and translation, vectors and expression vectors may contain nucleic acid sequences that serve other functions as well and are described infra.

[0071] 1. Promoters and Enhancers

[0072] A promoter is a control sequence that is a region of a nucleic acid sequence at which initiation and rate of transcription are controlled. It may contain genetic elements at which regulatory proteins and molecules may bind such as RNA polymerase and other transcription factors. The phrases operatively positioned, operatively linked, under control, and under transcriptional control mean that a promoter is in a correct functional location and/or orientation in relation to a nucleic acid sequence to control transcriptional initiation and/or expression of that sequence. A promoter may or may not be used in conjunction with an enhancer, which refers to a cis-acting regulatory sequence involved in the transcriptional activation of a nucleic acid sequence.

[0073] Preferably a promoter a promoter for use according to the embodiments is a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with a nucleic acid sequence in its natural environment. A recombinant or heterologous enhancer refers also to an enhancer not normally associated with a nucleic acid sequence in its natural environment. Such promoters or enhancers may include promoters or enhancers of other genes, and promoters or enhancers isolated from any other prokaryotic, viral, or eukaryotic cell, and promoters or enhancers not naturally occurring, i.e., containing different elements of different transcriptional regulatory regions, and/or mutations that alter expression. Furthermore, it is contemplated that the control sequences that direct transcription and/or expression of sequences within non-nuclear organelles such as mitochondria, chloroplasts, and the like, can be employed as well.

[0074] In preferred aspects, a promoter (or promoter enhancer system) for use according to the embodiments is an inducible promoter that provides expression of a sequence based on an external stimulus. For example, the inducible promoter may be a promoter that provides expression only in the presence of a particular compound (e.g., IPTG), at a particular pH, or in specific environmental (e.g., lighting) conditions.

[0075] 2. Initiation Signals and Internal Ribosome Binding Sites

[0076] A specific initiation signal also may be required for efficient translation of coding sequences. These signals include the ATG initiation codon or adjacent sequences. Exogenous translational control signals, including the ATG initiation codon, may need to be provided. One of ordinary skill in the art would readily be capable of determining this and providing the necessary signals. It is well known that the initiation codon must be in-frame with the reading frame of the desired coding sequence to ensure translation of the entire insert. The exogenous translational control signals and initiation codons can be either natural or synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements.

[0077] 3. Multiple Cloning Sites

[0078] Vectors can include a multiple cloning site (MCS), which is a nucleic acid region that contains multiple restriction enzyme sites, any of which can be used in conjunction with standard recombinant technology to digest the vector (see Carbonelli et al., 1999, Levenson et al., 1998, and Cocea, 1997, incorporated herein by reference.) Restriction enzyme digestion refers to catalytic cleavage of a nucleic acid molecule with an enzyme that functions only at specific locations in a nucleic acid molecule. Many of these restriction enzymes are commercially available. Use of such enzymes is understood by those of skill in the art. Frequently, a vector is linearized or fragmented using a restriction enzyme that cuts within the MCS to enable exogenous sequences to be ligated to the vector. Ligation refers to the process of forming phosphodiester bonds between two nucleic acid fragments, which may or may not be contiguous with each other. Techniques involving restriction enzymes and ligation reactions are well known to those of skill in the art of recombinant technology.

[0079] 4. Termination Signals

[0080] The vectors or constructs prepared in accordance with the present invention will generally comprise at least one termination signal. A termination signal or terminator is comprised of the DNA sequences involved in specific termination of an RNA transcript by an RNA polymerase. Thus, in certain embodiments, a termination signal that ends the production of an RNA transcript is contemplated. A terminator may be necessary in vivo to achieve desirable message levels.

[0081] Terminators contemplated for use in the invention include any known terminator of transcription described herein or known to one of ordinary skill in the art, including but not limited to, for example, rhp dependent or rho independent terminators. In certain embodiments, the termination signal may be a lack of transcribable or translatable sequence, such as due to a sequence truncation.

[0082] 5. Origins of Replication

[0083] In order to propagate a vector in a host cell, it may contain one or more origins of replication sites (often termed ori), which is a specific nucleic acid sequence at which replication is initiated. Alternatively an autonomously replicating sequence (ARS) can be employed if the host cell is yeast.

[0084] 6. Selectable and Screenable Markers

[0085] In certain embodiments of the invention, cells containing a nucleic acid construct of the present invention may be identified in vitro or in vivo by including a marker in the expression vector. Such markers would confer an identifiable change to the cell permitting easy identification of cells containing the expression vector. Generally, a selectable marker is one that confers a property that allows for selection. A positive selectable marker is one in which the presence of the marker allows for its selection, while a negative selectable marker is one in which its presence prevents its selection. An example of a positive selectable marker is a drug resistance marker, such an antibiotic resistance marker.

[0086] 7. Fusion Polypeptides

[0087] As described above, in some aspects a vector of the embodiments comprises a sequence for expression, which comprises a fusion of a membrane anchor sequence and an antigen polypeptide. Furthermore, in some aspects, the fusion polypeptide comprises a secretion signal that directs the fusion protein to the bacterial (outer) membrane. Optionally, the fusion polypeptide further comprises a linker positions between the antigen polypeptide sequence and the membrane anchor sequence.

[0088] a. Signal Sequences

[0089] In some aspects, a fusion polypeptide of the embodiments comprises a signal sequence that targets the fusion polypeptide to the membrane (and may be cleaved away from the fusion). In certain aspects, the signal sequence can be from a gram negative bacteria (e.g., E. coli). For example, the signal sequence can be from Lpp. In further aspects, the signal sequence can be a signal sequence from an autotransporter polypeptide of a gram negative bacteria. For example, the signal sequence can be from AIDA-I, EstA, MisL, Hbp, Ag43, BrkA, OmpA, OmpC, OmpX, LamB, FhuA, PfaI, EspP, IgAP, Pet or Yfal (see, e.g., Nicolay et al., 2015 and van Bloois et al., 2011, each incorporated herein by reference).

[0090] b. Membrane Anchor Sequence

[0091] Certain aspect of the embodiments concern fusion polypeptides that comprise a bacterial membrane anchor sequence. For example, the membrane anchor sequence can be composed of all or part of an integral membrane protein from a gram negative bacteria. In further aspects, the membrane anchor sequence can be a non-integral membrane polypeptide, such as a lipoprotein or a component of a bacterial surface appendage. In particular aspects, the bacterial membrane anchor sequence can be an outer membrane anchor sequence. In some aspects, the sequence can be a beta-barrel domain from an autotransporter polypeptide of a gram negative bacteria. For example, the membrane anchor sequence can comprise a membrane anchor domain from AIDA-I, EstA, MisL, Hbp, Ag43, BrkA, OmpA, OmpC, OmpX, LamB, FhuA, PfaI, EspP, IgAP, Pet, Yfal or MraY (see, e.g., Nicolay et al., 2015 and van Bloois et al., 2011, each incorporated herein by reference). In further aspects, the bacterial membrane anchor sequence comprises the membrane anchor sequence from OmpA.

[0092] c. Linker Sequence

[0093] It will be understood that in certain cases, a fusion polypeptide may comprise additional amino acids positioned between the antigen polypeptide sequence and the membrane anchor sequence. In general these sequences are interchangeably termed linker sequences or linker regions. One of skill in the art will recognize that linker regions may be one or more amino acids in length and often comprise one or more glycine residue(s) which confer flexibility to the linker. A variety of linkers can be used as part of fusion polypeptide of the embodiments. In preferred aspects, the optional linker sequence is positioned between the membrane anchor sequence and the antigen polypeptide sequence. In certain aspects the linker sequence comprises at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids. In still further aspects the linker comprises between about 10 and 200, 10 and 100, 20 and 100, 40 and 100 or 50 and 90 amino acids. In certain aspects, the linker sequence may comprise two, three, four or more Gly positions or a poly Gly sequence having two or more consecutive Gly positions.

IV. EXAMPLES

[0094] The following examples 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 which follow represent techniques discovered by the inventor 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, in light of the present disclosure, 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.

Materials and Methods

[0095] Bacterial strains and growth. For all experiments, E. coli strains were initially grown from freezer stocks on LB agar overnight at 37 C. After initial growth, isolated colonies were inoculated into 10 ml cultures and incubated overnight at 37 C. for use in experiments. Ampicillin was used at 100 ng/ml, while chloramphenicol was used at 15 ng/ml for all experiments. IPTG was used at a concentration of 100 M.

[0096] Immunoblot analysis. To detect 6-his tagged proteins, E. coli preparations were grown and diluted to OD600 of 1.0 for whole cell lysates. 5 ng of total protein from whole bacterial cells or isolated OMVs was separated by SDS-PAGE and transferred onto nitrocellulose Immunoblotting was performed with mouse-anti-5-his antibody (Qiagen) at a dilution of 1:2000 or anti-Vibrio antibody at a dilution of 1:40,000 (KPL-seracare). Anti-mouse or anti-rabbit conjugated-HRP secondary antibody (GE Healthcare) or was used at a concentration of 1:10,000, respectively. Detection was performed using Pierce ECL Western Blotting Substrate (Thermo Scientific).

[0097] Fluorescent analysis. E. coli cells were examined as previously described with some modifications (Lam et al., 1994). In brief, cells were grown to mid log phase OD600 of 0.5 and 1 ml was fixed in 4% paraformaldehyde for 20 minutes at 25 C. Samples were washed twice with 2 ml of PBS and then incubated in 10% BSA resuspended in PBS for 20 minutes. Cells were resuspended in 10% BSA-PBS with a 1:100 dilution of mouse-anti-5-his antibody (Qiagen) or anti-Vibrio antibody (KPL-seracare) and incubated at 25 C. for 1 h. Samples were washed three times in PBS and a fluorescein-conjugated anti-mouse or an anti-rabbit secondary antibody was diluted 1:200 in 10% BSA-PBS and incubated at 25 C. for 1 h. The cells were washed four times with PBS. 10 l of cells were mounted on glass slides under a coverslip and examined on a Nikon Eclipse 80i microscope.

[0098] Mass spectrometry. For mass spectrometry, lipid A was analyzed using a MALDI-TOF/TOF (ABI 4700 Proteomics Analyzer) mass spectrometer in the negative mode as previously described (Hankins et al., 2011).

[0099] Immunization and challenge studies. Female Balb/C mice were immunized orally with 110.sup.8 bacterial cfu or intranasal administration with OMVs containing 20 g of total protein. All bacterial strains were expression enzymes from the pELOP plasmid (Needham et al., 2013) to generate MPL. All animals were boosted four weeks later with equivalent amounts. At week six, the mice were challenged with 35LD.sub.50 of A/PR/8/1934 (H1N1). At day seven, five mice from each group were sacrificed for lung viral titers, while the rest were monitored for seventeen days. Serum was collected two weeks after the last vaccination for antibody analysis.

[0100] Lung viral titers. A confluent layer of MDCK cells was washed with PBS and lung homogenate was added at 1:10, 1:100, 1:1000 etc. dilutions. Cells were incubated at 33 C. for 1 h with shaking every 15 minutes to ensure an equal distribution of virus throughout the plate. After 1 h the media was removed and the cells were overlayed with 25 ml of 2% DMEM containing 2% penicillin/streptomycin, 2.5 g/ml of NAT, and 2% agarose. Solidified plates were inverted and incubated at 33 C. for three days. Plaques were reported as viral forming units/lung. All titers were performed in triplicate and averaged.

[0101] ELISAs. For antibody analysis, 3 g of recombinant soluble HA2 was purified and immobilized on a 96-well plate. Wells were probed with serial dilutions of mouse serum diluted in PBS. After washing, anti-mouse conjugated-HRP secondary antibody (GE Healthcare) was used at a concentration of 1:10,000. Detection was performed using Pierce ECL Western Blotting Substrate (Thermo Scientific). The absorbance was read at 405 nm.

Example 1

Plasmids and E. coli Strains for the Preparation of Lipid A/Polysaccharide Antigen Vaccines

[0102] Six genes, lpxE, lpxF, lpxO, lpxR, pagL, and pagP, were cloned individually into pQLinkN and expressed in wild-type E. coli for co-expression as described in U.S. Patent Application Publication No. 2013/0230555, which is hereby incorporated by reference in its entirety. The six genes originated from the following species: pagP from E. coli; pagL, lpxR, and lpxO from Salmonella enterica serovar Typhimurium; and lpxE and lpxF from Francisella tularensis. Specifically, the primers listed below in Table 1 were used for said cloning.

TABLE-US-00001 TABLE1 Primersequences LpxEBamHIfor 5-GCGGATCCATGCTC SEQIDNO:1 AAACAGACATTA-3 LpxEBamHIrev 5-GCGCGGCCGCCTAA SEQIDNO:2 ATAATCTCTCTATT-3 LpxFBamHIfor 5-GCGGATCCTTGGCA SEQIDNO:3 AGATTTCATATC-3 LpxFBamHIrev 5-GCGCGGCCGCTCAA SEQIDNO:4 TATTCTTTTTTACG-3 PagLBamHIfor 5-GCGGATCCATGTAT SEQIDNO:5 ATGAAGAGAATA-3 PagLBamHIrev 5-GCGCGGCCGCTCAG SEQIDNO:6 AAATTATAACTAAT-3 LpxOEcoRIfor 5-GCGAATTCATGTTC SEQIDNO:7 GCAGCAATCATT-3 LpxOBamHIrev 5-GCGGATCCTCAGAG SEQIDNO:8 GAGGCTGAAAAG-3 PagPBamHIfor 5-GCGGATCCATGAAC SEQIDNO:9 GTGAGTAAATAT-3 PagPNotIrev 5-GCGCGGCCGCTCAA SEQIDNO:10 AACTGAAAGCGCAT-3 LpxRBamHIfor 5-GCGGATCCATGAAC SEQIDNO:11 AAATACAGCTAT-3 LpxRNotIrev 5-GCGCGGCCGCTCAG SEQIDNO:12 AAGAAGAAGGTGAT-3

[0103] Transformation of wild-type E. coli with pQlinkN-derived plasmids that contained various combinations of the lpxE, lpxF, lpxO, lpxR, pagL, and pagP genes yielded E. coli strains that produce diverse lipid A species. The lipid A species are described in U.S. Patent Application Publication No. 2013/0230555. However, for E. coli expression of linked lipid A/polysaccharide antigen compositions, the lipid A modification genes (lpxE, lpxF, lpxO, lpxR, pagL, and pagP) and their IPTG inducible promoters were transferred to a pACYC184 plasmid and two plasmids were employed. First, the lipid A modification genes (lpxE, lpxF, lpxO, lpxR, pagL, and pagP) and their promoters were amplified from the pQlinkN-derived plasmid with engineered Sall sites and the digested fragments were cloned into SaII-digested pACYC184, which contains a p15A replicon. A schematic of the pACYC184 plasmid is provided in FIG. 1.

[0104] Next, a plasmid, pPM1001, was obtained from Monash University that contains the genes required to synthesize the O1 O-antigen from Vibrio cholerae (Manning et al., 1986). More specifically, the plasmid contained one or more of: gmhD gene, a manC gene, a manB gene, a gmd gene, a wbeE gene, a wbeG gene, a wzm gene (ABC transport), a wzt gene (ABC transport), a wbeK gene, a wbeL gene, a wbeM gene, a wbeN gene, a wbeO gene, a wbeP gene, a wbeT gene, a wbeU gene, a wbeV gene, a gale gene, and a wbeW gene, and a wbf region gene. For optimal expression of O antigen genes in E. coli, either the rfbD gene (Eck2034; see, for example, GenBank Accession No. NC_007779) or the entire E. coli O antigen biosynthetic genetic region from ribB to wbbL (Eck2025-Eck2035) were chromosomally deleted by P1 vir phage transduction using Keio mutants or lambda red recombination, respectively (Baba et al., 2006; Touze et al., 2008). E. coli manifesting one of these deletions were then transformed with the plasmid pPM1001 and a pACYC184 plasmid selected from the group of plasmids containing the various combinations of the lpxE, lpxF, lpxO, lpxR, pagL, and pagP genes.

[0105] All Escherichia coli W3110 strains were grown from freezer stock on Luria-Bertani Broth (LB) or 2 Nutrient broth with 5 g/L of NaCl at 25 C., 30 C., or 37 C. and supplemented with 100 mg/mL ampicillin, 15 mg/mL chloramphenicol, or 30 mg/mL kanamycin when appropriate. When required, 50 M-1mM isopropyl -D-1-thiogalactopyranoside (IPTG) was added as determined by TLC analysis of lipid A enzyme activity. To increase production of outer membrane vesicles (OMVs), the gene Eck1673, which encodes the major outer membrane lipoprotein Lpp, was deleted from the E. coli genome. The Eck1673 gene is also known as lpp. One exemplary lpp polynucleotide is provided in GenBank Accession No. NC_007779. Deletion of the Lpp lipoprotein resulted in a 100- to 1000-fold increased production of OMVs in E. coli strains.

[0106] It is believed that upon expression of the various lipid A species and the O antigen in the E. coli, the E. coli O antigen ligase WaaL covalently conjugated the synthesized V. cholerae O antigen to the lipid A core region of the modified lipid A adjuvant. In a manner similar to the V. cholerae O antigen, the conserved O antigen genetic coding region of Salmonella typhimurium and Shigella spcs. are cloned and expressed in E. coli for covalent attachment to lipid A. In addition, capsule biosynthesis genetic coding regions from Streptococcus pneumoniae and Staphylococcus aureus including, but not limited to, a wzm gene (ABC transport), a wzt gene (ABC transport), a wzx gene (flippase) and/or a wzy gene (polymerase), are cloned in a similar manner and ligated to engineered E. coli lipid A molecules.

[0107] The above-described glycolipids were then isolated in outer membrane vesicles, or OMVs. Outer membrane vesicles from E. coli were purified as previously described (Hug and Feldman, 2011; Mashburn and Whiteley, 2005; Schertzer and Whiteley, 2012). In brief, E. coli strains were grown to stationary phase and pelleted. The cell supernatants were filtered using a 0.45 nm syringe filter and centrifuged at 346249g for 1 h at 4 C. The pellet was resuspended in MV buffer (50 mM Tris pH=7.2, 5 mM NaCl, and 1 mM MgSO.sub.4) and centrifuged at 346249g for 1 h at 15 C. The vesicles were then washed in MV buffer and protein concentration was quantified using Coomassie Plus (Bradford) assay (Thermo).

Example 2

Engineering a Carbohydrate Antigen Based Vaccine Platform

[0108] While most infectious agents express surface proteins that can be targeted in vaccines, most pathogens also express carbohydrates on their surface. Glycoconjugate vaccines, such as those to Streptococcus pneumoniae capsule can prevent bacterial infections (De Roux et al., 2008). Using the bacterial adjuvant system described in patent US20130230555 A1, a system was developed to directly conjugate carbohydrates to the core region of E. coli lipopolysaccharide (FIG. 6A). The O-antigen region of V. cholerae lipopolysaccharide can be targeted by antibodies to prevent infection (Alam et al., 2014). The V. cholerae O-antigen coding sequences have been cloned into plasmid pPM1001 and O-antigen has been detected on the lipopolysaccharide of DH1 E. coli (FIG. 4B) (Manning et al., 1986). To engineer an E. coli strain that produced MPL directly conjugated to the V. cholerae O-antigen, the rfbD gene of E. coli was deleted from the genome, presumably because it disrupts V. cholerae O-antigen biosynthesis in E. coli. Co-expression of pPM1001 and pELOP in the ArfbD background allowed synthesis of the V. cholera O-antigen on E. coli lipopolysaccharide; however, the wild-type background did not produce the O-antigen. Surface exposure of the O-antigen was confirmed by using a V. cholerae O-antigen specific FITC-conjugated antibody (FIG. 6C). Last, lipid A was isolated and MALDI-TOF mass spectrometry indicated that a mixture of lipid was present that included the FDA-approved monophosphoryl lipid A with an m/z of 1729.1 (FIG. 6D). Also present was a dephosphorylated (lpxE) and deacylated (pagL) species corresponding to 1489.8 and a dephosphorylated (lpxE) and acylated (pagP) species corresponding to m/z 1954.4 (FIG. 6D).

Example 3

Plasmids and E. coli Strains for the Preparation of Lipid A/Influenza Polypeptide Vaccines

[0109] The pQLinkN plasmids containing the various combinations of the lipid A modification genes lpxE, lpxF, lpxO, lpxR, pagL, and pagP as described in U.S. Patent Application Publication No. 2013/0230555 and Example 1 above were provided. Genes encoding the influenza hemagglutinin (HA) protein from Influenza strain A/PR/8/34 H1N1 (ACCESSION NP 040980.1 and NP 040981.1) and A/HK/03V6205/2003 H3N2 (ACCESSION EU502208 and EU516332.1) were introduced into these Lipid A modification enzyme encoding pQLinkN plasmids along with genes encoding Lpp signal sequence and OmpA polypeptides. See U.S. Pat. No. 5,348,867, which is hereby incorporated by reference in its entirety, for descriptions and sequences of the Lpp signal sequence and OmpA polypeptides.

[0110] More specifically, the genes encoding the above-described HA proteins were codon optimized by Genescript for optimal expression in E. coli. In order to target the conserved regions of the HA proteins, the genes included polypeptides encoding the HA2 domains for the H1N1 and H3N2 influenza viruses that were codon optimized by Genescript as previously described (Bommakanti et al., 2010; Bommakanti et al., 2012). The HA2 subunit of the hemagglutinin protein is well known to those of ordinary skill in the art as being created upon cleavage of hemagglutinin by cellular proteases. In some embodiments, a codon optimized polypeptide encoding amino acid residues 1-172 or 1-160 of HA2 is introduced into the pQLinkN plasmid. All genes were engineered with a 5 EcoRI site, a 3 BamHI site, and a C-terminal 6-histidine tag.

[0111] After amplification of the coding sequences, DNA fragments were digested with EcoRI and BamHI and cloned into EcoRI- and BamHI-digested pTX101. Plasmid pTX101 is disclosed in U.S. Pat. No. 5,348,867, which is hereby incorporated by reference in its entirety. pTX101 fuses the target influenza protein to the C-terminus of an Lpp-OmpA protein fusion (Francisco et al., 1992). The tripartite fusion protein localizes to the E. coli outer membrane and surface displays the C-terminal influenza protein to the extracellular environment. The tripartite Lpp-OmpA-influenza protein coding sequence was amplified from pTX101 with primers that engineer 5 BamHI and 3 NotI restriction sites. Following the BamHI/NotI digestion, genes encoding the complete tripartite influenza fusion were cloned into BamHI/NotI-digested pQlinkN plasmid to allow expression from an IPTG-inducible promoter, resulting in co-expression of the Lipid A modification enzymes and the tripartite Lpp-OmpA-influenza protein.

[0112] These pQLinkN plasmids, which encode both the lipid A modification enzymes and the tripartite influenza fusion, were transformed into a W3110 E. coli strain mutated as follows. Deletions of pagP, lpxT and eptA resulted in a strain producing >95% of the prototypical, hexa-acylated bis-phosphorylated lipid A species (Needham et al., 2013). To increase production of outer membrane vesicles (OMVs), the gene Eck1763, which encodes the major outer membrane lipoprotein Lpp was also deleted from the E. coli genome. Deletion of the Lpp lipoprotein resulted in a 100- to 1000-fold increased production of OMVs in E. coli strains. E. coli W3110 gene deletions and antibiotic cassette removals were performed by P1 vir phage transduction using Keio mutants, as previously described (Baba et al., 2006; Touze et al., 2008). All Escherichia coli W3110 strains (mutated or wild-type) were grown from freezer stock on Luria-Bertani Broth (LB) or 2 Nutrient broth with 5 g/L of NaCl at 25 C., 30 C., or 37 C. and supplemented with 100 g/mL ampicillin, 15 g/mL chloramphenicol, or 30 g/mL kanamycin when appropriate. When required, 50 M-1 mM isopropyl -D-1-thiogalactopyranoside (IPTG) was added as determined by TLC analysis of lipid A enzyme activity (Needham et al., 2013).

[0113] The above-described E. coli strain produced surface-exposed influenza proteins that were packaged into OMVs with the modified lipid A species. Outer membrane vesicles from E. coli were purified as previously described (Mashburn and Whiteley, 2005; Schertzer and Whiteley, 2012). In brief, E. coli strains were grown to stationary phase and pelleted. The cell supernatants were filtered using a 0.45 m syringe filter and centrifuged at 346249g for 1 h at 4 C. The pellet was resuspended in MV buffer (50 mM Tris pH=7.2, 5 mM NaCl, and 1 mM MgSO.sub.4) and centrifuged at 346249g for 1 hour at 15 C. The vesicles were then washed in MV buffer and protein concentration was quantified using Coomassie Plus (Bradford) assay (Thermo).

Example 4

Engineering a Protein Antigen Based Vaccine Platform

[0114] Nonpathogenic E. coli have been engineered to co-produce lipid A adjuvants and either protein or carbohydrate antigens on the surface of the bacterial cell. These vaccine components can be purified from the bacteria as OMVs and used as vaccines. Influenza was used herein to demonstrate the efficacy of this system. As a model for the vaccine, the HA2 domain of influenza hemagglutinin protein was co-localized to the surface of the bacterial strain by fusing it with an N-terminal Lpp sorting sequence and an OmpA domain (FIG. 2A) (Francisco et al., 1992; Georgiou et al., 1996). The Lpp sorting sequence and OmpA domains are membrane bound while the 6his-tagged HA2 influenza protein is fused to the surface exposed loop at Asn159 through a Gly-Ile-Pro-Gly linker (FIG. 3A). This construct firmly anchors the HA2 protein onto the surface of the cell. For this study, the tripartite Lpp-OmpA-HA2 protein was linked into the pQlink plasmid system with the plasmid pELOP from U.S. Pat. Publn. No. US20130230555 for co-expression in E. coli to engineer expression of the HA2 antigen with the Federal Drug Administration approved monophosphoryl lipid A adjuvant on the surface of the cell (FIG. 2A) (Needham et al., 2013). Whole cells expressing this construct were isolated and separated using SDS-PAGE. Proteins were transferred to a nitrocellulose membrane and probed with an anti-5-his antibody (Qiagen). The antibody detected the tripartite HA2 fusion protein at the correct molecular weight of 45.54 kD (FIG. 2B). Furthermore, a mutation was introduced into the background BN1 strain that genetically deleted the native Lpp sequence from the genome. This mutation has been shown to result in excess production of OMVs. Purified OMVs from the strain co-expressing the tripartite HA2 fusion and pELOP also contained high levels of the tripartite fusion protein as detected by Western blot (FIG. 2B). To confirm surface accessibility of the HA2 protein on the surface of the cell, bacteria were grown and labeled with a secondary antibody conjugated to a FITC molecule. These bacteria fluoresced indicating the localization of the HA2 protein onto the surface of the bacterial cell (FIG. 2C). Last, lipid A was isolated from this strain and MALDI-TOF Mass spectrometry indicated that a mixture of lipid was present that included the FDA-approved monophosphoryl lipid A with an m/z of 1729.1 (FIG. 2D). Also present was a dephosphorylated (via LpxE) and deacylated (via PagL) species corresponding to 1489.8 and a dephosphorylated and acylated (via LpxE and PagP) species corresponding to m/z 1954.4 (FIG. 2D). The tripartite HA2 constructs that we used are indicated in FIGS. 3A and 3B.

Example 5

Efficacy of the Protein Antigen Vaccine Platform in a Mouse Vaccination Model

[0115] Either whole bacteria or purified OMVs were used to vaccinate eight week old mice on day 0 and boost on day 28. A subset of mice were used to collect serum before challenge and all other mice were challenged with a lethal dose of mouse-adapted H1N1 influenza virus on day 44. Animals were sacrificed seven days post infection and homogenized lung tissue was used to determine viral infectivity (FIG. 3C).

[0116] First, mice (n=5) were orally vaccinated with either PBS and whole E. coli that were producing the MPL adjuvant only or MPL and the HA2 expression construct. Upon sacrifice, titers indicted almost a 100-fold decrease in infectious viral load of the lungs suggesting that oral vaccination with whole bacteria could protect from influenza infection (FIG. 4A). Furthermore, OMVs were purified from bacteria containing the MPL or MPL-HA2 constructs. Mice vaccinated with the MPL-HA2 constructs had 10,000-fold less infectious influenza particles in their lungs after seven days when compared to MPL-vaccinated or PBS vaccinated mice. In addition, the MPL-HA2 construct protected the mouse lungs from influenza infection better than when an inactivated-influenza virus was used as the vaccine (FIG. 4B). The lung viral loads strongly correlate with survival of the mice, where those vaccinated with MPL-HA and inactive virus survived, but the PBS and MPL-vaccinated mice all died (FIG. 4C). Last, ELISAs were performed using serum isolated from each group. Mice vaccinated with MPL-HA2 and inactivated virus had antibodies that recognized the purified HA2 protein, whereas the MPL- and PBS-vaccinated mice did not (FIG. 4D).

[0117] The weight of all mice groups was monitored for up to seventeen days post-challenge. The PBS and MLP-vaccinated groups rapidly lost weight and infection manifested as a ruffled coat, lethargy, and dehydration post challenge (FIGS. 5A-C). In contrast the MPL-HA2 and inactivated virus-vaccinated groups recovered fully from the challenge and maintained a healthy coat (FIGS. 5A, 5D, and 5E).

Example 6

Plasmids and E. coli Strains for the Preparation of Carrier Protein/Polysaccharide Antigen

[0118] To engineer glycoprotein vaccines, acrA (cjj0390) and pglB (cjj1143) genetic coding sequences are amplified from Campylobacter jejuni 81-176 with BamHI and NotI sites and cloned into BamHI and NotI-digested pQlinkN for expression from an IPTG-inducible promoter (Scheich et al., 2007). A C-terminal 6-histidine tag is engineered onto the acrA coding sequence. AcrA is an acceptor protein that is glycosylated by the ligase Pg1B in C. jejuni (Feldman et al., 2005). The two genes, acrA and pglB are linked into the same vector for co-expression as previously described (Scheich et al., 2007; Ihssen et al., 2010). The acrA and pglB genes and their IPTG-inducible promoters are then amplified from the pQlinkN-derived vector with SaII sites and cloned into SaII-digested pACYC184.

[0119] The V. cholerae O-antigen plasmid pPM1001 described in Example 1 above is then provided and both the pPM1001 plasmid and the pACYC184 plasmid containing acrA and pglB genes are transformed into an E. coli strain having the following characteristics: All E. coli W3110 strains are grown from freezer stock on Luria-Bertani Broth (LB) or 2 Nutrient broth with 5 g/L of NaCl at 25 C., 30 C., or 37 C. and supplemented with 100 g/mL ampicillin, 15 g/mL chloramphenicol, or 30 g/mL kanamycin when appropriate. When required 50 M-1 mM isopropyl -D-1-thiogalactopyranoside (IPTG) is added as determined by TLC analysis of lipid A enzyme activity (Needham et al., 2013). For optimal expression of O-antigen/capsule genes in E. coli, either the rfbD gene (Eck2034) or the entire E. coli O-antigen biosynthetic genetic region from rfbB to wbbL (Eck2025-Eck2035) are chromosomally deleted by P1 vir phage transduction using Keio mutants or lambda red recombination, respectively (Baba et al., 2006; Touze et al., 2008). For production of all glycoprotein vaccines, efficient ligation of polysaccharides (O-antigen or capsule) onto an acceptor protein, called AcrA (Cjj0390) by the ligase Pg1B (Cjj1143) requires deletion of the E. coli gene encoding WaaL (Eck3612).

[0120] Once transformed into the E. coli as described above, the V. cholerae O-antigen plasmid pPM1001 is co-expressed with the pACYC-derived plasmid with IPTG to ligate the V. cholerae O-antigen onto the AcrA protein. AcrA glycoproteins are purified using Ni-affinity chromatography and size exclusion chromatography for use as glycoconjugate vaccines. A similar method is used to generate other O-antigen (S. typhimurium, Shigella) and capsule (S. pneumoniae, S. aureus) glycoprotein vaccines using their respective plasmids, which plasmids are described in the Examples above.

[0121] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

REFERENCES

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