Enterotoxigenic escherichia coli multi-epitope fusion antigen proteins and use

Abstract

Fusion proteins that include a backbone protein and at least one heterologous epitope from enterotoxigenic E. coli are provided. In some aspects, the fusion proteins are multiepitope fusion antigen proteins that include an LT B domain. In some aspects, the LT B domain is directly fused to an LT A domain (for example, the LT B domain does not include a starting methionine or signal sequence). In other aspects, the LT B domain includes a starting methionine and signal sequence. Methods of inducing an immune response in pigs utilizing the fusion proteins, such as an immune response that is protective against porcine post-weaning diarrhea, are also provided.

Description

[0001]7950-112226-02 ENTEROTOXIGENIC ESCHERICHIA COLI MULTI-EPITOPE FUSION ANTIGEN PROTEINS AND USE CROSS REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No.63 / 678,672, filed August 2, 2024, which is incorporated by reference in its entirety. FIELD This disclosure relates to enterotoxigenic Escherichia coli (ETEC) multi-epitope fusion antigen proteins and their use in methods, particularly in inducing a protective immune response against porcine post-weaning diarrhea. ACKNOWLEDGMENT OF GOVERNMENT SUPPORT This invention was made with government support under Grant Nos.2017-67015-26632 and 2017- 67015-31471 awarded by the National Institute of Food and Agriculture. The government has certain rights in the invention. INCORPORATION OF ELECTRONIC SEQUENCE LISTING The Sequence Listing is submitted as an XML file named “Sequence.xml,” created on July 25, 2025, 37,237 bytes, which is incorporated by reference herein. BACKGROUND Post-weaning diarrhea (PWD) is a common disease for newly weaned piglets. While suckling piglets can be protected by passive maternal antibodies from sows immunized with enterotoxigenic E. coli (ETEC) fimbriae antigens or infected naturally with ETEC bacteria, piglets become vulnerable to diarrhea and other infections at the moment of entering weaning. PWD results in slow growth, weight loss, acute death, extra management attention, and prophylaxis and metaphylaxis treatments, leading to significant economic losses to swine producers worldwide. PWD can be caused indirectly by factors including separation from the sows particularly depletion of maternal antibodies as well as stress from co-mingling with piglets from other litters and change of diet. The direct cause, however, is the infection of viral pathogens including rotaviruses, transmissible gastroenteritis viruses (TGE), and porcine epidemic diarrhea viruses (PEDV), parasitic pathogens, or mostly diarrheal bacteria, particularly Escherichia coli. Among the diarrheal E. coli bacteria, enterotoxigenic E. coli (ETEC), a specific group of E. coli that produce enterotoxins is the predominant cause of PWD. A vaccine that induces protective immunity against ETEC fimbriae and enterotoxins has been considered optimal against ETEC diarrhea. Unfortunately, there are no effective vaccines available currently against ETEC-associated PWD. 7950-112226-02 SUMMARY Disclosed herein are fusion proteins including the amino acid sequence of SEQ ID NO: 1 or a backbone protein, wherein the backbone protein includes a consensus sequence with at least 90% identity to SEQ ID NO: 3 and at least one heterologous epitope. In some aspects, the at least one heterologous epitope includes 8-16 amino acids. In some aspects, the backbone protein is an LT toxoid backbone, for example, an LT A protein backbone. In some aspects, the fusion protein includes an LT B subunit domain, for example, fused to the LT A portion of the backbone. In specific examples, the fusion protein includes a consensus sequence with at least 90% sequence identity to SEQ ID NO: 3 and including at least one heterologous epitope (e.g., one or more of SEQ ID NOs: 7, 9, 11, 13, 17, 19, and 21) and an LT B domain (e.g., SEQ ID NO: 23). In other aspects fusion proteins including the amino acid sequence of SEQ ID NO: 25 or a backbone protein, wherein the backbone protein comprises a consensus sequence with at least 90% identity to SEQ ID NO: 29 and at least one heterologous epitope are provided. In some aspects, the at least one heterologous epitope includes 8-16 amino acids. In some aspects, the backbone protein is an LT toxoid backbone, for example, an LT A protein backbone. In some aspects, the fusion protein includes an LT B subunit domain, for example, fused to the LT A portion of the backbone, wherein the LT B subunit includes a starting methionine and signal peptide. In specific non-limiting examples, the fusion protein includes a consensus sequence with at least 90% sequence identity to SEQ ID NO: 29 and including at least one heterologous epitope (e.g., one or more of SEQ ID NOs: 7, 9, 11, 13, 17, 19, and 21) and an LT B domain (e.g., SEQ ID NO: 27). Nucleic acids encoding the fusion proteins disclosed herein are also provided. In some aspects, the nucleic acid includes SEQ ID NO: 2 or includes a consensus sequence at least 90% identical to SEQ ID NO: 4. In some aspects the nucleic acid encoding the at least one heterologous epitope includes one or more of SEQ ID NOs: 8, 10, 12, 14, 18, 20, or 22. In additional aspects, the nucleic acid encoding the fusion protein includes an LT B domain encoded by SEQ ID NO: 24. In other aspects, the nucleic acid includes SEQ ID NO: 26 or includes a consensus sequence at least 90% identical to SEQ ID NO: 30. In some aspects the nucleic acid encoding the at least one heterologous epitope includes one or more of SEQ ID NOs: 8, 10, 12, 14, 18, 20, or 22. In additional aspects, the nucleic acid encoding the fusion protein includes an LT B domain encoded by SEQ ID NO: 28. Vectors including a disclosed nucleic acid are also provided. Further provided are host cells transformed with a disclosed nucleic acid or vector. In some aspects, the host cell is a bacterial cell, such an enterotoxigenic Escherichia coli (ETEC) F4 fimbrial cell and / or ETEC F18 fimbrial cell. Also disclosed are pharmaceutical compositions, which include a pharmaceutically acceptable carrier and any of the fusion proteins, nucleic acids, vectors, or host cells disclosed herein. In some aspects, the pharmaceutical composition can also include an adjuvant. In a non-limiting example, the adjuvant is a deletion mutant heat-labile enterotoxin (dmLT). 7950-112226-02 Methods of inducing an immune response in a pig utilizing the disclosed fusion proteins, nucleic acids, vectors, host cells, or pharmaceutical compositions are also provided. In some aspects, the methods include administering to the pig a fusion protein including the amino acid sequence of SEQ ID NO: 1 or a backbone protein, wherein the backbone protein includes a consensus sequence with at least 90% identity to SEQ ID NO: 3 and at least one heterologous epitope, a nucleic acid encoding the fusion protein, a vector including the nucleic acid or a pharmaceutical composition including the fusion protein, nucleic acid, or vector, followed by administering to the pig a fusion protein including the amino acid sequence of SEQ ID NO: 25 or a backbone protein, wherein the backbone protein comprises a consensus sequence with at least 90% identity to SEQ ID NO: 29 and at least one heterologous epitope, a nucleic acid encoding the fusion protein, a vector including the nucleic acid, a cell expressing the fusion protein, or a pharmaceutical composition including the fusion protein. In some aspects, the immune response is a protective response against porcine post-weaning diarrhea. The foregoing and other features disclosure will become more apparent from the following detailed description, which proceeds with reference to the accompanying figures. BRIEF DESCRIPTION OF THE DRAWINGS FIG.1 is a schematic showing PWD vaccine candidate PWDVax and the vaccine coverage. PWDVax includes two components, an acellular part which is a polyvalent fimbria-toxin multi-epitope fusion antigen (MEFA) protein (top left), and a cellular part which carries an F4-fimbrial E. coli strain and an F18-fimbrial strain to express GM1-binding AB5 holotoxin-structured fimbria-toxin MEFA (bottom left). This fimbria-toxin MEFA consists of a chimeric LT A1 peptide that has backbone epitopes substituted with functional epitopes of fimbriae F4 and F18 and toxins LT, STa, STb, and Stx2e, the LT A2 peptide, and LT B subunit (one copy B subunit in the monomer, and five copies of B subunits in the holotoxin structure). FIG.2 shows ELISA OD650readings to measure anti-F4, -F18, -LT, -STa, -STb, and anti-Stx2e IgG responses from pig serum samples of the control group (filled circles) or the vaccine group (open circles) 14 days after the primary intramuscular immunization with the fimbria-toxin MEFA monomer protein. Bars indicate the mean OD reading in the group. ★ and ★★ show a p-value of <0.05 and <0.001, respectively. FIGS.3A-3B show ELISA OD650 readings to measure anti-F4, -F18, -LT, -STa, -STb, and anti- Stx2e IgA responses from pig fecal samples (FIG.3A) or jejunum washes (FIG.3B) of the control group (filled circles) or the vaccine group (open circles). Fecal samples were collected 14 days after the oral booster with PWDVax cellular component that combining the F4-fimbrial vaccine strain and the F18 vaccine strain, both express GM1-binding AB5 holotoxin-structured fimbria-toxin MEFA protein. Jejunum washes were collected at necropsy 3 days after challenge. Bars indicate the mean OD reading in the group. ★, ★★, and ★★★ show a p-value of <0.05, <0.001, and <0.0001, respectively. FIGS.4A-4B show protection against bacteria colonization of the F18 ETEC challenge strain in pig small intestines and dry fecal matter from the feces of the control or vaccine pigs. (FIG.4A) F18 ETEC bacteria colonization (CFUs per gram of ileum segment) in the control pigs (filled circles) or the immunized 7950-112226-02 pigs (open circles) after oral inoculation with F18 ETEC field isolate 9922 (F18, LT, STa, STb, Stx2e). (FIG.4B) Dry fecal matter (% in weight) from the feces of the control pigs (filled circles) or the immunized pigs (open circles) at necropsy. Bars indicate the mean OD reading in the group. ★ and ★★ show a p-value of <0.05 and <0.001, respectively. SEQUENCES Any nucleic acid and amino acid sequences listed herein are shown using standard letter abbreviations for nucleotide bases and amino acids, as defined in 37 C.F.R. § 1.822. In at least some cases, only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand. SEQ ID NO: 1 is the amino acid sequence of an exemplary PWD MEFA monomer with an E. coli LT backbone, epitopes from LT A, FedF, Sta, Stx2e, FaeG, and STb, and a fusion of LT B domain (without starting methionine or signal sequence): MKNITFIFFILLASPLYANGDKLYRADSRPPDEIKRSGGLMPQPDATGSWYDTQMNINCCELCCSPA CAGCYYDDGYVSTSQSYVSSLNAGQIPSSSGTLTCQAGTATAPNMFNVNDVLGVYSPHPYEQEVS ALGGIPCCELCCSPACAGCYIDERLGRTKEAFATPYRNLNIPMKNAGGTKVGSVKVNQAWREEPWI HHKKDLCENYSRTITGDTCNEETQNLSTIYLRKYQSKVKRQIFSDYQSEVDIYNRIRNELDPRVPSSA PQSITELCSEYRNTQIYTINDKILSYTESMAGKREMVIITFKSGATFQVEVPGSQHIDSQKKAIERMKD TLRITYLTETKIDKLCVWNNKTPNSIAAISMEN SEQ ID NO: 2 is an exemplary nucleic acid sequence encoding a PWD MEFA monomer with an E. coli LT backbone, epitopes from LT A, FedF, Sta, Stx2e, FaeG, and STb, and a fusion of LT B domain (without starting methionine or signal peptide sequence): ATGAAAAACATCACCTTTATCTTCTTTATTCTGCTGGCGAGCCCGCTGTATGCGAATGGCGACA AACTGTACCGTGCGGATAGCCGTCCGCCAGACGAGATCAAGCGTAGCGGTGGCCTGATGCCGC AGCCGGATGCGACCGGCAGCTGGTACGATACCCAAATGAATATTAATTGCTGCGAACTGTGCT GCAGCCCGGCGTGCGCGGGTTGCTACTATGACGATGGCTACGTTAGCACCAGCCAGAGCTATG TGAGCAGCCTGAACGCGGGTCAAATTCCGAGCAGCAGCGGCACCCTGACCTGCCAGGCGGGTA CCGCGACCGCGCCGAACATGTTCAACGTGAACGACGTTCTGGGCGTGTACAGCCCGCACCCGT ATGAGCAAGAAGTTAGCGCGCTGGGTGGAATACCATGCTGCGAGCTGTGTTGTAGCCCGGCGT GCGCGGGCTGCTATATCGATGAGCGTCTGGGTCGTACCAAGGAAGCGTTTGCGACCCCGTACC GTAACCTGAACATTCCGATGAAAAACGCGGGTGGCACCAAAGTGGGTAGCGTGAAAGTTAACC AGGCGTGGCGTGAGGAACCGTGGATTCATCATAAAAAGGACCTGTGCGAGAACTACAGCCGTA CCATCACAGGTGATACTTGTAATGAGGAGACCCAGAATCTGAGCACAATATATCTCAGGAAAT ATCAATCAAAAGTTAAGAGGCAGATATTTTCAGACTATCAGTCAGAGGTTGACATATATAACA GAATTCGGAATGAATTAGATCCCCGGGTACCGAGCTCGGCTCCCCAGTCTATTACAGAACTATG TTCGGAATATCGCAACACACAAATATATACGATAAATGACAAGATACTATCATATACGGAATC GATGGCAGGCAAAAGAGAAATGGTTATCATTACATTTAAGAGCGGCGCAACATTTCAGGTCGA AGTCCCGGGCAGTCAACATATAGACTCCCAAAAAAAAGCCATTGAAAGGATGAAGGACACATT AAGAATCACATATCTGACCGAGACCAAAATTGATAAATTATGTGTATGGAATAATAAAACCCC CAATTCAATTGCGGCAATCAGTATGGAAAAC SEQ ID NO: 3 is an exemplary consensus amino acid sequence for a backbone protein, where ‘X’ indicates a location for insertion or substitution with a heterologous or homologous epitope or where the LT B fusion is located: 7950-112226-02 MKNITFIFFILLASPLYANGDKLYRAXXXXXXXXXXXXXXMPXXXXXXXXXXTQMNINXXXXXX XXXXXXXXYDDGYVSTSXXXXXXXXAGQXXXXXXXXXXXXXXATAPNMFNVNDVLGVYXXX XXXXXXXXLGGIPXXXXXXXXXXXXXXIDERLXXXXXXXXXXYRNLNIXXXXXXXXXXXXXXX XQAWREEPWIHHXXXXXXXXSRTITGDTCNEETQNLSTIYLRKYQSKVKRQIFSDYQSEVDIYNRIR NELDPRVPSSXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX SEQ ID NO: 4 is an exemplary consensus nucleic acid sequence that encodes a backbone protein, where ‘N’ indicates a location for insertion or substitution with a heterologous or homologous epitope or where the LT B fusion is located: ATGAAAAACATCACCTTTATCTTCTTTATTCTGCTGGCGAGCCCGCTGTATGCGAATGGCGACA AACTGTACCGTGCGNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNATGCC GNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNACCCAAATGAATATTAATNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNTATGACGATGGCTACGTTAGCACCAGCNNNN NNNNNNNNNNNNNNNNNNNNGCGGGTCAANNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNGCGACCGCGCCGAACATGTTCAACGTGAACGACGTTCTGGGCGTGTACNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNGGTGGAATACCANNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNATCGATGAGCGTCTGNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNTACCGTAACCTGAACATTNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNCAGGCGTGGCGTGAGGAACCGTGGATTCATCATNNNNNNNNNNNNN NNNNNNNNNNNAGCCGTACCATCAGCCGTACCATCACAGGTGATACTTGTAATGAGGAGACCC AGAATCTGAGCACAATATATCTCAGGAAATATCAATCAAAAGTTAAGAGGCAGATATTTTCAG ACTATCAGTCAGAGGTTGACATATATAACAGAATTCGGAATGAATTAGATCCCCGGGTACCGA GCTCGNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NN SEQ ID NO: 5 is the amino acid sequence of an exemplary LT A subunit epitope: DSRPPDEIKRSGG SEQ ID NO: 6 is an exemplary nucleic acid sequence encoding an LT A subunit epitope: GATAGCCGTCCGCCAGACGAGATCAAGCGTAGCGGTGGC SEQ ID NO: 7 is the amino acid sequence of an exemplary FedF subunit epitope: QPDATGSWYD SEQ ID NO: 8 is an exemplary nucleic acid sequence encoding an FedF subunit epitope: CAGCCGGATGCGACCGGCAGCTGGTACGAT SEQ ID NO: 9 is the amino acid sequence of an exemplary STa toxoid epitope: CCELCCSPACAGCY SEQ ID NO: 10 is an exemplary nucleic acid sequence encoding an STa toxoid epitope: TGCTGCGAACTGTGCTGCAGCCCGGCGTGCGCGGGTTGCTAC SEQ ID NO: 11 is the amino acid sequence of an exemplary Stx2eA subunit epitope: QSYVSSLN 7950-112226-02 SEQ ID NO: 12 is an exemplary nucleic acid sequence encoding an Stx2eA subunit epitope: CAGAGCTATGTGAGCAGCCTGAAC SEQ ID NO: 13 is the amino acid sequence of an additional exemplary FedF subunit epitope: IPSSSGTLTCQAGT SEQ ID NO: 14 is an exemplary nucleic acid sequence encoding an additional FedF subunit epitope: ATTCCGAGCAGCAGCGGCACCCTGACCTGCCAGGCGGGTACC SEQ ID NO: 15 is the amino acid sequence of an additional exemplary LT A subunit epitope: SPHPYEQEVSA SEQ ID NO: 16 is an exemplary nucleic acid sequence encoding an additional LT A subunit epitope: AGCCCGCACCCGTATGAGCAAGAAGTTAGCGCG SEQ ID NO: 17 is the amino acid sequence of an exemplary FaeG major subunit epitope: GRTKEAFATP SEQ ID NO: 18 is an exemplary nucleic acid encoding an FaeG major subunit epitope: GGTCGTACCAAGGAAGCGTTTGCGACCCCG SEQ ID NO: 19 is the amino acid sequence of an additional exemplary FaeG major subunit epitope: PMKNAGGTKVGSVKVN SEQ ID NO: 20 is an exemplary nucleic acid sequence encoding an additional FaeG major subunit epitope: CCGATGAAAAACGCGGGTGGCACCAAAGTGGGTAGCGTGAAAGTTAAC SEQ ID NO: 21 is the amino acid sequence of an exemplary STb epitope: KKDLCENY SEQ ID NO: 22 is an exemplary nucleic acid sequence encoding an STb epitope: AAAAAGGACCTGTGCGAGAACTAC SEQ ID NO: 23 is the amino acid sequence of an exemplary LT B protein lacking the signal peptide: APQSITELCSEYRNTQIYTINDKILSYTESMAGKREMVIITFKSGATFQVEVPGSQHIDSQKKAIERMK DTLRITYLTETKIDKLCVWNNKTPNSIAAISMEN SEQ ID NO: 24 is an exemplary nucleic acid sequence encoding an LT B protein lacking the signal sequence: GCTCCCCAGTCTATTACAGAACTATGTTCGGAATATCGCAACACACAAATATATACGATAAATG ACAAGATACTATCATATACGGAATCGATGGCAGGCAAAAGAGAAATGGTTATCATTACATTTA AGAGCGGCGCAACATTTCAGGTCGAAGTCCCGGGCAGTCAACATATAGACTCCCAAAAAAAAG 7950-112226-02 CCATTGAAAGGATGAAGGACACATTAAGAATCACATATCTGACCGAGACCAAAATTGATAAAT TATGTGTATGGAATAATAAAACCCCCAATTCAATTGCGGCAATCAGTATGGAAAAC SEQ ID NO: 25 is the amino acid sequence of an exemplary AB5 holotoxin-structured PWD MEFA antigen: MKNITFIFFILLASPLYANGDKLYRADSRPPDEIKRSGGLMPQPDATGSWYDTQNTFYCCELCCSPA CAGCYYDDGYVSTSQSYVSSLNAGQIPSSSGTLTCQAGTATAPNMFNVNDVLGVYSPHPYEQEVS ALNTFYCCELCCSPACAGCYIDERLGRTKEAFATPYRNLNIPMKNAGGTKVGSVKVNQAWREEPW IHHKKDLCENYSRTITGDTCNEETQNLSTIYLRKYQSKVKRQIFSDYQSEVDIYNRIRNELMNKVKC YVLFTALLSSLCAYGAPQSITELCSEYRNTQIYTINDKILSYTESMAGKREMVIITFKSGATFQVEVPG SQHIDSQKKAIERMKDTLRITYLTETKIDKLCVWNNKTPNSIAAISMEN SEQ ID NO: 26 is an exemplary nucleic acid encoding an AB5 holotoxin-structured PWD MEFA antigen (stop of LT A subunit / start of LT B subunit is underlined): ATGAAAAACATCACCTTTATCTTCTTTATTCTGCTGGCGAGCCCGCTGTATGCGAATGGCGACA AACTGTACCGTGCGGATAGCCGTCCGCCAGACGAGATCAAGCGTAGCGGTGGCCTGATGCCGC AGCCGGATGCGACCGGCAGCTGGTACGATACCCAAAACACCTTCTATTGCTGCGAACTGTGCT GCAGCCCGGCGTGCGCGGGTTGCTACTATGACGATGGCTACGTTAGCACCAGCCAGAGCTATG TGAGCAGCCTGAACGCGGGTCAAATTCCGAGCAGCAGCGGCACCCTGACCTGCCAGGCGGGTA CCGCGACCGCGCCGAACATGTTCAACGTGAACGACGTTCTGGGCGTGTACAGCCCGCACCCGT ATGAGCAAGAAGTTAGCGCGCTGAACACCTTTTACTGCTGCGAGCTGTGTTGTAGCCCGGCGTG CGCGGGCTGCTATATCGATGAGCGTCTGGGTCGTACCAAGGAAGCGTTTGCGACCCCGTACCG TAACCTGAACATTCCGATGAAAAACGCGGGTGGCACCAAAGTGGGTAGCGTGAAAGTTAACCA GGCGTGGCGTGAGGAACCGTGGATTCATCATAAAAAGGACCTGTGCGAGAACTACAGCCGTAC CATCACAGGTGATACTTGTAATGAGGAGACCCAGAATCTGAGCACAATATATCTCAGGAAATA TCAATCAAAAGTTAAGAGGCAGATATTTTCAGACTATCAGTCAGAGGTTGACATATATAACAG AATTCGGAATGAATTATGAATAAAGTAAAATGTTATGTTTTATTTACGGCGTTACTATCCTCTCT ATGTGCATACGGAGCTCCCCAGTCTATTACAGAACTATGTTCGGAATATCGCAACACACAAAT ATATACGATAAATGACAAGATACTATCATATACGGAATCGATGGCAGGCAAAAGAGAAATGGT TATCATTACATTTAAGAGCGGCGCAACATTTCAGGTCGAAGTCCCGGGCAGTCAACATATAGA CTCCCAAAAAAAAGCCATTGAAAGGATGAAGGACACATTAAGAATCACATATCTGACCGAGAC CAAAATTGATAAATTATGTGTATGGAATAATAAAACCCCCAATTCAATTGCGGCAATCAGTAT GGAAAAC SEQ ID NO: 27 is the amino acid sequence of an exemplary LT B subunit domain: MNKVKCYVLFTALLSSLCAYGAPQSITELCSEYRNTQIYTINDKILSYTESMAGKREMVIITFKSGAT FQVEVPGSQHIDSQKKAIERMKDTLRITYLTETKIDKLCVWNNKTPNSIAAISMEN SEQ ID NO: 28 is an exemplary nucleic acid sequence encoding an LT B subunit domain: ATGAATAAAGTAAAATGTTATGTTTTATTTACGGCGTTACTATCCTCTCTATGTGCATACGGAG CTCCCCAGTCTATTACAGAACTATGTTCGGAATATCGCAACACACAAATATATACGATAAATGA CAAGATACTATCATATACGGAATCGATGGCAGGCAAAAGAGAAATGGTTATCATTACATTTAA GAGCGGCGCAACATTTCAGGTCGAAGTCCCGGGCAGTCAACATATAGACTCCCAAAAAAAAGC CATTGAAAGGATGAAGGACACATTAAGAATCACATATCTGACCGAGACCAAAATTGATAAATT ATGTGTATGGAATAATAAAACCCCCAATTCAATTGCGGCAATCAGTATGGAAAAC SEQ ID NO: 29 is an exemplary consensus amino acid sequence for a backbone protein, where ‘X’ indicates a location for insertion or substitution with a heterologous or homologous epitope or where LT B domain (with starting methionine and signal peptide sequence) is located: MKNITFIFFILLASPLYANGDKLYRAXXXXXXXXXXXXXLMPXXXXXXXXXXTQNTFYXXXXXX XXXXXXXXYDDGYVSTSXXXXXXXXAGQXXXXXXXXXXXXXXATAPNMFNVNDVLGVYXXX XXXXXXXXLNTFYXXXXXXXXXXXXXXIDERLXXXXXXXXXXYRNLNIXXXXXXXXXXXXXX 7950-112226-02 XXQAWREEPWIHHXXXXXXXXSRTITGDTCNEETQNLSTIYLRKYQSKVKRQIFSDYQSEVDIYNR IRNELXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX XXXXXXXX SEQ ID NO: 30 is an exemplary consensus nucleic acid sequence that encodes a backbone protein, where ‘N’ indicates a location for insertion or substitution with a heterologous or homologous epitope or where LT B domain (with starting methionine and signal peptide sequence) is located: ATGAAAAACATCACCTTTATCTTCTTTATTCTGCTGGCGAGCCCGCTGTATGCGAATGGCGACA AACTGTACCGTGCGNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNCTGATGCC GNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNACCCAAAACACCTTCTATNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNTATGACGATGGCTACGTTAGCACCAGCNNNN NNNNNNNNNNNNNNNNNNNNGCGGGTCAANNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNGCGACCGCGCCGAACATGTTCAACGTGAACGACGTTCTGGGCGTGTACNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNCTGAACACCTTTTACNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNATCGATGAGCGTCTGNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNTACCGTAACCTGAACATTNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNCAGGCGTGGCGTGAGGAACCGTGGATTCATCATNNNNNNNNNNNNNN NNNNNNNNNNAGCCGTACCATCACAGGTGATACTTGTAATGAGGAGACCCAGAATCTGAGCA CAATATATCTCAGGAAATATCAATCAAAAGTTAAGAGGCAGATATTTTCAGACTATCAGTCAG AGGTTGACATATATAACAGAATTCGGAATGAATTNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN SEQ ID NO: 31 is the amino acid sequence of an exemplary deletion mutant heat-labile enterotoxin (dmLT): MKNITFIFFILLASPLYANGDKLYRADSRPPDEIKRSGGLMPRGHNEYFDRGTQMNINLYDHARGTQ TGFVRYDDGYVSTSLSLRSAHLAGQSILSGYSTYYIYVIATAPNMFNVNDVLGVYSPHPYEQEVSAL GGIPYSQIYGWYRVNFGVIDERLHRNREYRDRYYRNLNIAPAEDGYRLAGFPPDHQAWREEPWIH HAPQGCGNSSGTITGDTCNEETQNLSTIYARKYQSKVKRQIFSDYQSEVDIYNRIRNEMNKVKCYV LFTALLSSLCAYGAPQSITELCSEYRNTQIYTINDKILSYTESMAGKREMVIITFKSGATFQVEVPGSQ HIDSQKKAIERMKDTLRITYLTETKIDKLCVWNNKTPNSIAAISEN SEQ ID NO: 32 is an exemplary nucleic acid sequence encoding a deletion mutant heat-labile enterotoxin (dmLT): bold = mature peptide start; bold underlined = 192 and 211 mutations; italics = cistron structure, stop / start ATGAAAAATATAACTTTCATTTTTTTTATTTTATTAGCATCGCCATTATATGCAAATGGCGACAA ATTATACCGTGCTGACTCTAGACCCCCAGATGAAATAAAACGTTCCGGAGGTCTTATGCCCAGA GGGCATAATGAGTACTTCGATAGAGGAACTCAAATGAATATTAATCTTTATGATCACGCGAGA GGAACACAAACCGGCTTTGTCAGATATGATGACGGATATGTTTCCACTTCTCTTAGTTTGAGAA GTGCTCACTTAGCAGGACAGTCTATATTATCAGGATATTCCACTTACTATATATATGTTATAGC GACAGCACCAAATATGTTTAATGTTAATGATGTATTAGGCGTATACAGCCCTCACCCATATGAA CAGGAGGTTTCTGCGTTAGGTGGAATACCATATTCTCAGATATATGGATGGTATCGTGTTAATT TTGGTGTGATTGATGAACGATTACATCGTAACAGGGAATATAGAGACCGGTATTACAGAAATC TGAATATAGCTCCGGCAGAGGATGGTTACAGATTAGCAGGTTTCCCACCGGATCACCAAGCTT GGAGAGAAGAACCCTGGATTCATCATGCACCACAAGGTTGTGGAAATTCATCAGGAACAATTA CAGGTGATACTTGTAATGAGGAGACCCAGAATCTGAGCACAATATATGCCAGGAAATATCAAT CAAAAGTTAAGAGGCAGATATTTTCAGACTATCAGTCAGAGGTTGACATATATAACAGAATTC 7950-112226-02 GGAATGAATTATGAATAAAGTAAAATGTTATGTTTTATTTACGGCGTTACTATCCTCTCTATGTG CATACGGAGCTCCCcAGTCTATTACAGAACTATGTTCGGAATATCGCAACACACAAATATATAC GATAAATGACAAGATACTATCATATACGGAATCGATGGCAGGCAAAAGAGAAATGGTTATCAT TACATTTAAGAGCGGCGCAACATTTCAGGTCGAAGTCCCGGGCAGTCAACATATAGACTCCCA AAAAAAAGCCATTGAAAGGATGAAGGACACATTAAGAATCACATATCTGACCGAGACCAAAA TTGATAAATTATGTGTATGGAATAATAAAACCCCCAATTCAATTGCGGCAATCAGTGAAAACT AA DETAILED DESCRIPTION Piglets commonly wean at the age of around 21 days or even as early as 17 days and can develop diarrhea within 24 to 48 hours after weaned. This leaves a very narrow window for vaccine intervention for protection from PWD. To be protected, piglets need to be vaccinated at an age as early as three days so that they can develop effective immunity against diarrhea by the time they are weaned. This poses a technical challenge for vaccination against PWD. It is believed that oral immunization with a cellular vaccine favors induction of mucosal immunity, and intestinal mucosal immunity plays a more important role in protecting against enteric diseases including PWD. However, oral administration of whole-cell vaccine products is often not very effective at inducing immune responses in very young animals. On the other hand, injectable subunit vaccines are more efficient in inducing responses in young animals, but subunit vaccines elevate mostly systemic immune responses. To resolve this dilemma, this disclosure provides a two-component PWD vaccine candidate including an acellular component and a cellular component applied as a heterologous prime-boost vaccination strategy, with IM injection of an acellular vaccine component as the primary immunization at the age of 3-5 days and then oral booster with a cellular vaccine component before weaning. Results provided herein show that piglets IM immunized with fimbria-toxin monomer protein at day five developed IgG antibody responses to fimbriae (F4, F18) and toxins LT and STb. After the oral booster with the vaccine cellular component, these piglets had a significant increase in IgA responses to the two fimbriae (F4, F18) and four toxins (LT, STa, STb, Stx2e), detected from their fecal samples (two weeks after oral immunization) as well as jejunum washes collected at necropsy (2-3 days after the challenge). This indicated that IM injection of the protein antigen elicited systemic responses in neonatal pigs, and the oral booster with the live cellular product induced vaccine antigen-specific IgA responses in weaned pigs. I. Summary of Terms Unless otherwise noted, technical terms are used according to conventional usage. Definitions of many common terms in molecular biology may be found in Krebs et al. (eds.), Lewin’s genes XII, published by Jones & Bartlett Learning, 2017. As used herein, the singular forms “a,” “an,” and “the,” refer to both the singular as well as plural, unless the context clearly indicates otherwise. For example, the term “a protein” includes singular or plural proteins and can be considered equivalent to the phrase “at least one protein.” As used herein, the term “comprises” means “includes.” It is further to be understood that any and all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for descriptive purposes, unless otherwise indicated. 7950-112226-02 Although many methods and materials similar or equivalent to those described herein can be used, particular suitable methods and materials are described herein. In case of conflict, the present specification, including explanations of terms, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. To facilitate review of the various aspects, the following explanations of terms are provided: About: Unless otherwise indicated, the term “about” refers to a range of 5% of a reference value, for example, about 100 refers to a range of 95 to 105. Adjuvant: A substance or vehicle that non-specifically enhances the immune response to an antigen (for example, an Escherichia coli antigen). Adjuvants can be used with the compositions disclosed herein, for example, as part of an immunogenic composition provided herein. Adjuvants can include a suspension of minerals (alum, aluminum hydroxide, or phosphate) on which antigen is adsorbed or a water- in-oil emulsion in which antigen solution is emulsified in mineral oil (for example, Freund’s incomplete adjuvant), sometimes with the inclusion of killed mycobacteria (Freund’s complete adjuvant) to further enhance antigenicity. Immunostimulatory oligonucleotides (e.g., those including a CpG motif) can also be used as adjuvants (for example, see U.S. Patent Nos.6,194,388; 6,207,646; 6,214,806; 6,218,371; 6,239,116; 6,339,068; 6,406,705; and 6,429,199). Adjuvants may also include biological molecules, e.g., costimulatory molecules. Exemplary biological adjuvants include IL-2, RANTES, GM-CSF, TNF-α, IFN-γ, G-CSF, LFA- 3, CD72, B7-1, B7-2, OX-40L and 41 BBL. In one example the adjuvant is one or more toll-like receptor (TLR) agonists, for example, an agonist of TLR1 / 2 (which can be a synthetic ligand) (e.g., Pam3Cys), TLR2 (e.g., CFA, Pam2Cys), TLR3 (e.g., polyI:C, poly A:U), TLR4 (e.g., MPLA, Lipid A, and LPS), TLR5 (e.g., flagellin), TLR7 (e.g., gardiquimod, imiquimod, loxoribine, Resiquimod®), TLR7 / 8 (e.g., R0848), TLR8 (e.g., imidazoquionolines, ssPolyU, 3M-012), TLR9 (e.g., ODN 1826 (type B), ODN 2216 (type A), CpG oligonucleotides and / or TLR11 / 12 (e.g., profilin). In one example, the adjuvant is lipid A, for example, lipid A monophosphoryl (MPL) from Salmonella enterica serotype Minnesota Re 595 (for example, Sigma Aldrich Catalog # L6895). In another example the adjuvant is an enterotoxin-based adjuvant, for example, a deletion mutant heat-labile toxin (dmLT). Administer: As used herein, administering a composition (e.g., a composition disclosed herein, for example, a fusion protein, nucleic acid, vector, or pharmaceutical composition disclosed herein) to a subject means to give, apply or bring the composition into contact with the subject. Administration can be accomplished by any of a number of routes, such as, for example, intramuscular, intranasal, pulmonary, topical, oral, subcutaneous, intraperitoneal, intravenous, intrathecal, rectal, vaginal, or intradermal. In specific non-limiting examples, administration of a composition disclosed herein is intramuscular or subcutaneous. In further non-limiting examples, administration of a composition disclosed herein is oral. Antigen or immunogen: A compound, composition, or substance that can stimulate the production of an immune response in a subject, including compositions that are injected or absorbed into a subject. An antigen reacts with the products of specific humoral or cellular immunity, including those induced by heterologous immunogens. 7950-112226-02 Attenuated: In the context of the type of live pathogen, the pathogen (for example, a bacterial pathogen, e.g., E. coli) is attenuated if its ability to produce disease is reduced (or even eliminated) compared with a wild-type pathogen. Fusion protein: A protein containing amino acid sequence from at least two different (heterologous) proteins or peptides. In some examples herein, the fusion protein comprises a backbone protein and one or more heterologous peptides, for example, one or more heterologous epitopes. In some aspects, the backbone and heterologous sequences are contiguous. Fusion proteins can be generated, for example, by expression of a nucleic acid sequence engineered from nucleic acid sequences encoding at least a portion of two different (heterologous) proteins. To create a fusion protein, typically the nucleic acid sequences are in the same reading frame and contain no internal stop codons. Fusion proteins, particularly short fusion proteins, can also be generated by chemical synthesis. Heterologous: A heterologous protein or nucleic acid refers to a protein or nucleic acid derived from a different source (e.g., a peptide or epitope from a different protein) or species. Immune response: A response of a cell of the immune system, for example, a B-cell, T-cell, macrophage, or polymorphonucleocyte, to a stimulus, for example, an antigen / immunogen or vaccine (e.g., a Shigella or E. coli immunogenic composition or vaccine). An immune response can include any cell of the body involved in a host defense response, including for example, an epithelial cell that secretes an interferon or a cytokine. An immune response includes, but is not limited to, an innate immune response. As used herein, a protective immune response refers to an immune response that protects a subject from infection (prevents infection or prevents the development of disease associated with infection). Methods of measuring immune responses include, for example, measuring proliferation and / or activity of lymphocytes (e.g., B or T cells), secretion of cytokines or chemokines, inflammation, antibody production and the like. Isolated: An “isolated” biological component (e.g., a nucleic acid, protein, or cell) has been substantially separated or purified away from other biological components (e.g., cell debris, or other proteins or nucleic acids). Biological components that have been “isolated” include those components purified by standard purification methods. The term also embraces recombinant nucleic acids and proteins and chemically synthesized nucleic acids or peptides. Modification: A change in a nucleic acid or protein sequence. For example, sequence modifications include substitutions, insertions, and deletions, or combinations thereof. For proteins, insertions include amino and / or carboxyl terminal fusions as well as intrasequence insertions of single or multiple amino acid residues. Insertions for nucleic acid sequence include 5' or 3' additions or intrasequence insertions of single or multiple nucleotides. Deletions are characterized by the removal of one or more amino acid residues from a protein sequence or one or more nucleotides from a nucleic acid sequence. Substitutions are those in which at least one amino acid residue or nucleotide has been removed and a different residue or nucleotide inserted in its place. Substitutions, deletions, insertions or any combination thereof may be combined to arrive at a final modified sequence. Protein modifications can be prepared by modification of nucleotides in the DNA encoding the protein, thereby producing DNA encoding the 7950-112226-02 modification. Techniques for making insertion, deletion and substitution mutations at predetermined sites in DNA or RNA having a known sequence are well known in the art. A “modified” protein, nucleic acid or virus is one that has one or more modifications as outlined above. Nucleic acid: A polymeric form of nucleotides, which may include both sense and anti-sense strands of RNA, cDNA, genomic DNA, genomic RNA, and synthetic forms and mixed polymers of the above. A nucleotide refers to a ribonucleotide, deoxynucleotide or a modified form of either type of nucleotide. The term “nucleic acid molecule” as used herein is synonymous with “nucleic acid” and “polynucleotide.” A nucleic acid molecule is usually at least 10 bases in length, unless otherwise specified. The term includes single- and double-stranded forms of DNA. A polynucleotide may include either or both naturally occurring and modified nucleotides linked together by naturally occurring and / or non-naturally occurring nucleotide linkages. “cDNA” is DNA that has been synthesized using messenger RNA (mRNA) as a template, and can be in either single stranded or double stranded form. “Encoding” refers to the inherent property of specific sequences of nucleotides in a polynucleotide, for example, a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (e.g., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. ORF (open reading frame): A series of nucleotide triplets (codons) coding for amino acids without any termination codons. These sequences are usually translatable into a peptide. Operably linked: A first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein-coding regions, in the same reading frame. Pharmaceutically acceptable carriers: Pharmaceutically acceptable carriers are known. Remington’s Pharmaceutical Sciences, by E.W. Martin, Mack Publishing Co., Easton, PA, 22nd Edition, 2013, describes compositions and formulations suitable for pharmaceutical delivery of the disclosed compositions. In general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids, for example, water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. For solid compositions (for example, powder, pill, tablet, or capsule forms), non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral carriers, pharmaceutical compositions (e.g., immunogenic compositions) to be administered can contain minor amounts of non-toxic auxiliary substances, for example, wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate. In some aspects, in compositions suitable for administration to a subject, the carrier may be sterile and / or suspended or 7950-112226-02 otherwise contained in a unit dosage form containing one or more measured doses of the composition suitable to induce the desired immune response. The unit dosage form may be, for example, in a sealed vial that contains sterile contents or a syringe for injection into a subject, or lyophilized for subsequent solubilization and administration or in a solid or controlled release dosage. Peptide: Any chain of amino acids, regardless of length or post-translational modification (for example, glycosylation or phosphorylation). “Peptide” applies to amino acid polymers including naturally occurring amino acid polymers and non-naturally occurring amino acid polymers, including such polymers in which one or more amino acid residues is a non-natural amino acid, for example, an artificial chemical mimetic of a corresponding naturally occurring amino acid. A “residue” refers to an amino acid or amino acid mimetic incorporated in a peptide by an amide bond or amide bond mimetic. A peptide has an amino terminal (N-terminal) end and a carboxy terminal (C-terminal) end. Post-weaning diarrhea (PWD): Porcine PWD occurs 3-10 days after weaning and is characterized by grey-brown watery diarrhea. PWD is typically transient, but may persist, and death due to dehydration or septicemia may occur. PWD is caused predominantly by enterotoxigenic Escherichia coli (ETEC). ETEC bacteria causing PWD produce F4 (K88) or F18 fimbria and various combinations of enterotoxins heat- labile toxin (LT), heat-stable toxin type Ib (STa), heat-stable toxin II (STb), and Shiga toxin type 2e (Stx2e). Preventing, treating or ameliorating a disease: “Preventing” a disease refers to inhibiting the full development of a disease. “Treating” refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition after it has begun to develop. “Ameliorating” refers to the reduction in the number or severity of signs or symptoms of a disease. Recombinant: A recombinant nucleic acid molecule is one that has a sequence that is not naturally occurring, for example, includes one or more nucleic acid substitutions, deletions, or insertions, and / or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence. This artificial combination can be accomplished by chemical synthesis or by the artificial manipulation of isolated segments of nucleic acids, for example, by genetic engineering techniques. A recombinant protein is one that has a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence. In some aspects, a recombinant protein includes a fusion protein, for example, a protein that includes one or more heterologous peptides (e.g., epitopes). Sequence identity: The similarity between amino acid or nucleic acid sequences is expressed in terms of the similarity between the sequences, otherwise referred to as sequence identity. Sequence identity is frequently measured in terms of percentage identity (or similarity or homology); the higher the percentage, the more similar the two sequences are. Homologs or variants of a given gene or protein will possess a relatively high degree of sequence identity when aligned using standard methods. Methods of alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms are described in: Smith and Waterman, Adv. Appl. Math.2:482, 1981; Needleman and Wunsch, J. Mol. Biol.48:443, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A.85:2444, 1988; Higgins 7950-112226-02 and Sharp, Gene 73:237-244, 1988; Higgins and Sharp, CABIOS 5:151-153, 1989; Corpet et al., Nucleic Acids Research 16:10881-10890, 1988; and Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A.85:2444, 1988. Altschul et al., Nature Genet.6:119-129, 1994. The NCBI Basic Local Alignment Search Tool (BLASTTM) (Altschul et al., J. Mol. Biol.215:403- 410, 1990) is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, MD) and on the Internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx. Subject: Living multi-cellular vertebrate organisms, a category that includes both human and non-human mammals. In some aspects, the subject is a pig (e.g., Sus domesticus). In some examples, a subject is one that is or can be infected with Shigella or E. coli, for example, humans or pigs. Synthetic: Produced by artificial means, for example a synthetic nucleic acid or protein can be chemically synthesized in a laboratory. Therapeutically effective amount (or effective amount): The amount of agent, for example, a disclosed fusion protein, nucleic acid, vector, host cell expressing a disclosed fusion protein, or pharmaceutical composition, that is sufficient to induce a response, for example, an immune response, or is sufficient to treat, reduce, and / or ameliorate symptoms and / or underlying causes of a disorder or disease, for example, to induce an immune response to E. coli or to inhibit and / or treat E. coli infection and / or disease resulting therefrom (e.g., PWD). In some aspects, a therapeutically effective amount is sufficient to reduce or eliminate a symptom of a disease. For instance, this can be the amount necessary to inhibit pathogen (e.g., enterotoxigenic E. coli) replication or to measurably alter outward symptoms of pathogen infection (e.g., diarrhea, for example, porcine post-weaning diarrhea). In one example, a desired response is to inhibit or reduce E. coli infection. E. coli infection does not need to be completely eliminated for the method to be effective. For example, administration of a therapeutically effective amount of the agent can decrease the E. coli infection (for example, as measured by infection of cells, or by number or percentage of subjects infected by E. coli) by a desired amount, for example by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 99% compared to a suitable control. In some aspects, the therapeutically effective amount produces a protective immune response. It is understood that producing a protective immune response against a pathogen can require multiple administrations of one or more immunogenic compositions. For example, a therapeutically effective amount of can be administered in a single dose, or in several doses, during a course of treatment (e.g., a prime-boost vaccination treatment). However, the therapeutically effective amount and timing of administration can depend on the subject being treated, the severity and type of the condition being treated, and the manner of administration. A unit dosage form of the agent can be packaged in a therapeutic amount, or in multiples of the therapeutic amount, for example, in a vial (e.g., with a pierceable lid) or syringe having sterile components. 7950-112226-02 Vaccine: A preparation of immunogenic material capable of stimulating an immune response. The immunogenic material may include attenuated or killed microorganisms (e.g., attenuated viruses) or antigenic proteins, peptides, or nucleic acids encoding an antigen. Vaccines may elicit both prophylactic and therapeutic responses. Methods of administration vary according to the vaccine, but may include inoculation, ingestion, inhalation, or other forms of administration. Inoculations can be delivered by any of a number of routes, including parenteral, for example, intravenous, subcutaneous, or intramuscular. In specific aspects, vaccines can be administered via a subcutaneous or intramuscular route. Vaccines may be administered with an adjuvant to increase the immune response to the vaccine. Vector: A nucleic acid molecule as introduced into a host cell, thereby producing a transformed host cell. A vector may include nucleic acid sequences that permit it to replicate in the host cell, for example, an origin of replication. A vector may also include one or more therapeutic nucleic acids and / or selectable marker genes and other genetic elements known in the art. A vector can transduce, transform or infect a cell, thereby causing the cell to express nucleic acids and / or proteins other than those native to the cell. A vector optionally includes materials to aid in achieving entry of the nucleic acid into the cell, for example, a viral particle, liposome, protein coating or the like. A vector can be a viral vector. Virulence factors: Virulence factors enable a host to replicate and disseminate bacteria within a host in part by subverting or eluding host defenses. In example aspects, virulence factors include E.coli virulence factors, for example, heat-labile enterotoxin (LT A or LT B subunits), heat-stable enterotoxin-a (STa), heat-stable enterotoxin-b (STb), and Shiga toxin (e.g., Stx2e). II. Fusion Proteins and Compositions Fusion proteins that include a backbone protein and at least one heterologous epitope from enterotoxigenic E. coli are provided. In some aspects, the fusion proteins are multiepitope fusion antigen proteins that include an LT B domain. In some aspects, the LT B domain is directly fused to an LT A domain (for example, the LT B domain does not include a starting methionine or signal sequence). In other aspects, the LT B is not fused to the LT A domain (for example, the LT B domain includes a starting methionine and signal sequence). Proteins, peptides, and nucleic acids that are similar to those disclosed herein can be used as well as fragments thereof that retain biological activity. These proteins, peptides, and nucleic acids may contain variations, substitutions, deletions, or additions. In some aspects, the differences can be in regions not significantly conserved among different species. Such regions can be identified by aligning the amino acid sequences of related proteins, peptides, and nucleic acids from various species. Generally, the biological effects of a molecule are retained, for example, immunogenicity or ability to elicit an immune response in a subject. For example, a protein, peptide, and / or nucleic acid at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to one of these molecules can be utilized. Proteins or peptides (or nucleic acids encoding such proteins or peptides) may include at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative amino acid substitutions. Generally, modified proteins or peptides (or nucleic acids encoding such proteins or 7950-112226-02 peptides) retain at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of the biological function of the native (or parent) molecule or have increased biological function as compared to the native (or parent) molecule. Also included are derivatives or modifications of the backbone proteins, epitopes (e.g., heterologous or homologous epitopes), or fusion proteins disclosed herein, which are differentially modified during or after synthesis, for example, by benzylation, glycosylation, acetylation, phosphorylation, amidation, pegylation, derivatization by known protecting / blocking groups. In some aspects, peptides can include at least one amino acid or every amino acid that is a D stereoisomer. Other peptides can include at least one amino acid that is reversed. The amino acid that is reversed may be a D stereoisomer. Every amino acid of a peptide may be reversed and / or every amino acid can be a D stereoisomer. A. PWD MEFA Monomer In some aspects, the fusion protein includes a backbone protein and at least one heterologous epitope. In some aspects, the backbone protein is derived from E. coli heat-labile toxin (LT) A. In some aspects, the backbone protein includes a consensus sequence with at least 90% identity to SEQ ID NO: 3 and at least one heterologous epitope. In some aspects, the fusion protein also includes one or more homologous epitopes (e.g., one or more LT A epitopes). Epitope insertion sites within the backbone protein of SEQ ID NO: 3 are designated by a string of “X” amino acids (Xn). The number of Xs included at each insertion site is not intended to limit the size of an inserted epitope, and rather the number of amino acids at each epitope site can vary, depending on the length of the inserted epitope. The backbone protein also includes a fusion to an LT B subunit domain, wherein the LT B subunit lacks the signal peptide and is fused to the C-terminal of the LT A portion of the backbone. In specific, non-limiting examples, the backbone protein includes a consensus sequence with at least 90% identity (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identity) to SEQ ID NO: 3. The consensus sequence (or the part of the sequence denoted with a specific amino acid) denotes the portion that is considered the backbone, whereas ‘X’ can vary and may include an epitope sequence. In specific, non-limiting examples, the backbone protein is encoded by a nucleic acid including a consensus sequence with at least 90% identity (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identity) to SEQ ID NO: 4. The consensus sequence (or the part of the sequence denoted with a specific nucleotide) denotes the portion that is considered the backbone, whereas ‘N’ can vary and may include an epitope sequence (e.g., a heterologous or homologous sequence). In some aspects, the disclosed fusion proteins include at least one heterologous epitope. In some aspects, one or more homologous epitopes are also included. Included epitopes can range from 5-30 amino acids in length, including ranges of about 5-10, 8-16, 8-20, 10-14, 10-16, 10-20, and 10-30 amino acids, for example, at least about 5, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 20, 25, or 30 amino acids in length, or about 8, 9, 10, 11, 12, 13, 14, 15, or 16 amino acids long. The epitopes may be contiguous or non-contiguous with one another in the backbone protein. In some aspects, all of the epitopes are non-contiguous. In other 7950-112226-02 aspects, one or more of the epitopes are contiguous, while one or more other epitopes are non-contiguous with the other epitopes. In some aspects, the epitope is a peptide derived from one or more ETEC virulence factors (e.g., LT, STa, STb, or Stx2e) or one or more fimbriae subunit (e.g., F4 and / or F18 fimbriae). In some aspects, the backbone protein includes one or more peptides (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more peptides) from at least one ETEC virulence factor and / or fimbriae subunit. In a specific non-limiting example, the backbone protein is derived from LT A, one or more homologous epitopes from LT A are included, and one or more heterologous epitopes from STa, STb, Stx2e, F4 (K88) major subunit FaeG, and / or F18 adhesin subunit FedF are included in the fusion protein. Exemplary epitope sequences include or consist of the following: DSRPPDEIKRSGG (SEQ ID NO: 5, an exemplary LT A epitope amino acid sequence, e.g., a homologous epitope where the backbone protein is derived from LT A); QPDATGSWYD (SEQ ID NO: 7, an exemplary FedF epitope amino acid sequence, e.g., a heterologous epitope where the backbone protein is derived from LT A); CCELCCSPACAGCY (SEQ ID NO: 9, an exemplary STa toxoid epitope amino acid sequence, e.g., a heterologous epitope where the backbone protein is derived from LT A); QSYVSSLN (SEQ ID NO: 11, an exemplary Stx2e A epitope amino acid sequence, e.g., a heterologous epitope where the backbone protein is derived from LT A); IPSSSGTLTCQAGT (SEQ ID NO: 13, an exemplary FedF epitope amino acid sequence, e.g., a heterologous epitope where the backbone protein is derived from LT A); SPHPYEQEVSA (SEQ ID NO: 15, an exemplary LT A epitope amino acid sequence, e.g., a homologous epitope where the backbone protein is derived from LT A); GRTKEAFATP (SEQ ID NO: 17, an exemplary FaeG epitope amino acid sequence, e.g., a heterologous epitope where the backbone protein is derived from LT A); PMKNAGGTKVGSVKVN (SEQ ID NO: 19, an exemplary FaeG epitope amino acid sequence, e.g., a heterologous epitope where the backbone protein is derived from LT A); and KKDLCENY (SEQ ID NO: 21, an exemplary STb epitope amino acid sequence, e.g., a heterologous epitope where the backbone protein is derived from LT A). In some aspects, the epitope sequences are encoded by nucleic acids including or consisting of SEQ ID NO: 6 (encoding an exemplary LT A epitope), SEQ ID NO: 8 (encoding an exemplary FedF subunit epitope), SEQ ID NO: 10 (encoding an exemplary STa toxoid epitope), SEQ ID NO: 12 (encoding an Stx2e A epitope), SEQ ID NO: 14 (encoding an exemplary FedF subunit epitope), SEQ ID NO: 16 (encoding an exemplary LT A epitope), SEQ ID NO: 18 (encoding an exemplary FaeG major subunit epitope), SEQ ID NO: 20 (encoding an exemplary FaeG major subunit epitope), or SEQ ID NO: 22 (encoding an exemplary STb epitope). The fusion protein also includes a native LT B subunit (eltB) (without the signal sequence) segment fused to the C-terminal of the LT A backbone. In some aspects, the LT B subunit includes an amino acid 7950-112226-02 sequence with at least 90% identity (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identity) to SEQ ID NO: 23. In some aspects, the LT B subunit includes or consists of the amino acid sequence of SEQ ID NO: 23. In some aspects, the LT B subunit is encoded by a nucleic acid with at least 90% identity (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identity) to SEQ ID NO: 24. In some aspects, the LT B subunit is encoded by a nucleic acid including or consisting of the nucleic acid sequence of SEQ ID NO: 24. In specific non-limiting examples, the fusion protein includes a backbone protein derived from LT A that includes one or more epitopes (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more epitopes) from ETEC virulence factors STa, STb, Stx2e and F4 fimbriae subunit FaeG and F18 fimbriae subunit FedF. The fusion protein also includes a C-terminal fusion of an LT B subunit lacking the signal peptide. In some aspects, a fusion protein disclosed herein includes at least 90% identity (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identity) to the amino acid sequence of SEQ ID NO: 1. In some aspects, a fusion protein disclosed herein includes or consists of the amino acid sequence of SEQ ID NO: 1. In some aspects, a fusion protein disclosed herein is encoded by a nucleic acid sequence at least 90% identical (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identity) to SEQ ID NO: 2. In some aspects, a fusion protein disclosed herein is encoded by a nucleic acid sequence including or consisting of SEQ ID NO: 2. B. PWD AB5 Holotoxin-Structured MEFA Protein In some aspects, the fusion protein includes a backbone protein and at least one heterologous epitope. In some aspects, the backbone protein is derived from E. coli heat-labile toxin (LT) A. In some aspects, the backbone protein includes a consensus sequence with at least 90% identity to SEQ ID NO: 29 and at least one heterologous epitope. In some aspects, the fusion protein also includes one or more homologous epitopes (e.g., one or more LT A epitopes). Epitope insertion sites within the backbone protein of SEQ ID NO: 3 are designated by a string of “X” amino acids (Xn). The number of Xs included at each insertion site is not intended to limit the size of an inserted epitope, and rather the number of amino acids at each epitope site can vary, depending on the length of the inserted epitope. The backbone protein also includes a fusion to an LT B subunit, wherein the LT B subunit includes a starting methionine and signal peptide and is fused to the C-terminal of the LT A portion of the backbone. In specific, non-limiting examples, the backbone protein includes a consensus sequence with at least 90% identity (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identity) to SEQ ID NO: 29. The consensus sequence (or the part of the sequence denoted with a specific amino acid) denotes the portion that is considered the backbone, whereas ‘X’ can vary and may include an epitope sequence. In specific, non-limiting examples, the backbone protein is encoded by a nucleic acid including a consensus sequence with at least 90% identity (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 7950-112226-02 98%, at least 99%, or at least 100% identity) to SEQ ID NO: 30. The consensus sequence (or the part of the sequence denoted with a specific nucleotide) denotes the portion that is considered the backbone, whereas ‘N’ can vary and may include an epitope sequence (e.g., a heterologous or homologous sequence). In some aspects, the one or more epitopes include an amino acid sequence including or consisting of SEQ ID NO: 5 (an exemplary LT A epitope), SEQ ID NO: 7 (an exemplary FedF subunit epitope), SEQ ID NO: 9 (an exemplary STa toxoid epitope), SEQ ID NO: 11 (an Stx2e A epitope), SEQ ID NO: 13 (an exemplary FedF subunit epitope), SEQ ID NO: 15 (an exemplary LT A epitope), SEQ ID NO: 17 (an exemplary FaeG major subunit epitope), SEQ ID NO: 19 (an exemplary FaeG major subunit epitope), and / or SEQ ID NO: 21 (an exemplary STb epitope). In other aspects, the one or more epitopes are encoded by nucleic acids including or consisting of SEQ ID NO: 6 (encoding an exemplary LT A epitope), SEQ ID NO: 8 (encoding an exemplary FedF subunit epitope), SEQ ID NO: 10 (encoding an exemplary STa toxoid epitope), SEQ ID NO: 12 (encoding an Stx2e A epitope), SEQ ID NO: 14 (encoding an exemplary FedF subunit epitope), SEQ ID NO: 16 (encoding an exemplary LT A epitope), SEQ ID NO: 18 (encoding an exemplary FaeG major subunit epitope), SEQ ID NO: 20 (encoding an exemplary FaeG major subunit epitope), and / or SEQ ID NO: 22 (encoding an exemplary STb epitope). The fusion protein also includes a native LT B subunit (eltB) segment fused to the C-terminal of the LT A backbone. The LT B subunit is separated from the LT A sequence by a stop codon, and includes a starting methionine and the native signal peptide sequence. In some aspects, the LT B subunit includes an amino acid sequence with at least 90% identity (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identity) to SEQ ID NO: 27. In some aspects, the LT B subunit includes or consists of the amino acid sequence of SEQ ID NO: 27. In some aspects, the LT B subunit is encoded by a nucleic acid with at least 90% identity (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identity) to SEQ ID NO: 28. In some aspects, the LT B subunit is encoded by a nucleic acid including or consisting of the nucleic acid sequence of SEQ ID NO: 28. In specific non-limiting examples, the fusion protein includes a backbone protein derived from LT A that includes one or more epitopes (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more epitopes) from ETEC virulence factors STa, STb, Stx2e and F4 fimbriae subunit FaeG and F18 fimbriae subunit FedF. The fusion protein also includes a C-terminal fusion of an LT B subunit. In some aspects, a fusion protein disclosed herein includes at least 90% identity (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identity) to the amino acid sequence of SEQ ID NO: 25. In some aspects, a fusion protein disclosed herein includes or consists of the amino acid sequence of SEQ ID NO: 25. In some aspects, the fusion protein is encoded by a nucleic acid sequence at least 90% identical (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identity) to SEQ ID NO: 26. In some aspects, the fusion protein is encoded by a nucleic acid sequence including or consisting of SEQ ID NO: 26. 7950-112226-02 C. Vectors and Host Cells Nucleic acids encoding the fusion proteins disclosed herein are also provided. These nucleic acids include DNA, cDNA and RNA sequences which encode the fusion protein, for example, including the nucleic acid sequences disclosed herein. The coding sequence includes variants that result from the degeneracy (e.g., redundancy) of the genetic code, whereby more than one codon can encode the same amino acid residue. Thus, for example, leucine can be encoded by CTT, CTC, CTA, CTG, TTA, or TTG; serine can be encoded by TCT, TCC, TCA, TCG, AGT, or AGC; asparagine can be encoded by AAT or AAC; aspartic acid can be encoded by GAT or GAC; cysteine can be encoded by TGT or TGC; alanine can be encoded by GCT, GCC, GCA, or GCG; glutamine can be encoded by CAA or CAG; tyrosine can be encoded by TAT or TAC; and isoleucine can be encoded by ATT, ATC, or ATA. A nucleic acid encoding the fusion protein can be cloned or amplified by in vitro methods, for example, by the polymerase chain reaction (PCR), the ligase chain reaction (LCR), the transcription-based amplification system (TAS), the self-sustained sequence replication system (3SR) and the Qβ replicase amplification system (QB). For example, a polynucleotide encoding the protein can be isolated by polymerase chain reaction of cDNA using primers based on the DNA sequence of the molecule. A wide variety of cloning and in vitro amplification methodologies are known and have been described. Polynucleotides also can be isolated by screening genomic or cDNA libraries with probes selected from the sequences of the desired polynucleotide under stringent hybridization conditions. A nucleic acid sequence encoding the fusion protein can be operatively linked to expression control sequences. An expression control sequence operatively linked to a coding sequence is linked such that expression of the coding sequence is achieved under conditions compatible with the expression control sequences. The expression control sequences include, but are not limited to, appropriate promoters, enhancers, a ribosome binding site, transcription terminators, transcriptional regulators (e.g., AraC and LacI), a start codon (e.g., ATG) in front of a protein-encoding gene, splicing signal for introns, maintenance of the correct reading frame of that gene to permit proper translation of mRNA, and / or stop codons. The nucleic acid molecules encoding the fusion protein may include a recombinant DNA, which is incorporated into a vector for example, an autonomously replicating plasmid or virus or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., a cDNA) independent of other sequences. The nucleotides can be ribonucleotides, deoxyribonucleotides, or modified forms of either nucleotide. The term includes single and double forms of DNA. In some aspects, vectors are used for fusion protein expression. A person of skill in the art will understand that there are many options for protein expression systems, including systems that express protein in yeast, insect, and bacterial cells. In specific, non-limiting examples, E. coli cells can be used. In some aspects, protein expression systems can include vectors with one or more tags for purification of the fusion protein, for example, histidine (His), chitin-binding protein (CBP), maltose-binding protein (MBP), or glutathione-S-transferase (GST), or a streptavidin tag. In specific, non-limiting examples, a vector with a His tag, for example, a pET28a vector. 7950-112226-02 In other aspects, vectors are used for expression in yeast, for example, Saccharomyces cerevisiae or Kluyveromyces lactis. Several promoters are known to be of use in yeast expression systems for example, the constitutive promoters plasma membrane H+-ATPase (PMA1), glyceraldehyde-3-phosphate dehydrogenase (GPD), phosphoglycerate kinase-1 (PGK1), alcohol dehydrogenase-1 (ADH1), and pleiotropic drug-resistant pump (PDR5). In addition, many inducible promoters are of use, e.g., GAL1–10 (induced by galactose), PHO5 (induced by low extracellular inorganic phosphate), and tandem heat shock HSE elements (induced by temperature elevation to 37°C). Promoters that direct variable expression in response to a titratable inducer include the methionine-responsive MET3 and MET25 promoters and copper- dependent CUP1 promoters. Any of these promoters may be cloned into multicopy (2µ) or single copy (CEN) plasmids to give an additional level of control in expression level. The plasmids can include nutritional markers (e.g., URA3, ADE3, HIS1, and others) for selection in yeast and antibiotic resistance (e.g., AMP) for propagation in bacteria. Plasmids for expression on K. lactis are known, e.g., pKLAC1. Thus, in one example, after amplification in bacteria, plasmids can be introduced into the corresponding yeast auxotrophs by methods similar to bacterial transformation. The polynucleotides can also be designed to be expressed in insect cells. The fusion protein can be expressed in a variety of yeast strains. For example, seven pleiotropic drug-resistant transporters, YOR1, SNQ2, PDR5, YCF1, PDR10, PDR11, and PDR15, together with their activating transcription factors, PDR1 and PDR3, have been simultaneously deleted in yeast host cells, rendering the resultant strain sensitive to drugs. Yeast strains with altered lipid composition of the plasma membrane, e.g., the erg6 mutant defective in ergosterol biosynthesis, can also be utilized. Proteins that are highly sensitive to proteolysis can be expressed in a yeast lacking the master vacuolar endopeptidase Pep4, which controls the activation of other vacuolar hydrolases. Heterologous expression in strains carrying temperature-sensitive (ts) alleles of genes can be employed if the corresponding null mutant is inviable. Viral vectors can also be prepared encoding the fusion protein disclosed herein. Thus, in one aspect, the polynucleotide encoding a fusion protein is included in a viral vector. Suitable vectors include retrovirus vectors, orthopox vectors, avipox vectors, fowlpox vectors, capripox vectors, suipox vectors, adenoviral vectors, herpes virus vectors, alpha virus vectors, baculovirus vectors, Sindbis virus vectors, vaccinia virus vectors, and poliovirus vectors. DNA sequences encoding the fusion protein can be expressed in vitro by DNA transfer into a suitable host cell. In some aspects, a host cell is transformed with a nucleic acid disclosed herein. The cell may be prokaryotic or eukaryotic. The term also includes any progeny of the subject host cell. It is understood that all progeny may not be identical to the parental cell since there may be mutations that occur during replication. Methods of stable transfer, meaning that the foreign DNA is continuously maintained in the host, are known in the art. Hosts cells also include microbial, insect, and mammalian host cells. Methods of expressing DNA sequences having eukaryotic or viral sequences in prokaryotes are well known in the art. Non-limiting examples of suitable host cells include bacteria, archea, insect, fungi (for example, yeast), plant, and animal 7950-112226-02 cells (for example, mammalian cells, e.g., human). Exemplary cells of use include, but are not limited to, Escherichia coli, Bacillus subtilis, Saccharomyces cerevisiae, Salmonella typhimurium, SF9 cells, C129 cells, 293 cells, Neurospora, and immortalized mammalian myeloid and lymphoid cell lines. In some aspects, the host cell is an E. coli cell, for example, an ETEC cell. In some aspects, the cell is a non-pathogenic or attenuated E. coli cell. In some aspects, the cell is an E. coli cell that expresses F4 or F18 fimbria but does not express enterotoxins. In one specific example, the host cell is a live F4ac+ E.coli strain, e.g., strain 1836-2 (Zhang et al., Infect. Immun.74:3107-3114, 2006). In other aspects the host cell is an F18-fimbrial E. coli strain, e.g., isolate 8532 (Zhang et al., Vet. Microbiol.123:145-152, 2007). In certain non-limiting examples, the host cell includes a nucleic acid encoding a disclosed fusion protein (e.g., AB5 holotoxin-structure PWD MEFA fusion protein), for example, the host cell includes a vector (e.g., a plasmid vector) including a nucleic acid encoding the AB5 holotoxin-structure PWD MEFA fusion protein. Techniques for the propagation of mammalian cells in culture are well-known (see, Jakoby and Pastan (eds), Methods in Enzymology: Cell Culture, volume 58, Academic Press, Inc., Harcourt Brace Jovanovich, N.Y., 1979). Examples of commonly used mammalian host cell lines include VERO, HeLa, CHO, WI38, BHK, and COS cell lines, although other cell lines may be used, for example, cells designed to provide higher expression desirable glycosylation patterns, or other features. Transformation of a host cell with recombinant DNA can be carried out by conventional techniques as are well known to those skilled in the art. Where the host is prokaryotic, for example, but not limited to, E. coli, competent cells which are capable of DNA uptake can be prepared from cells harvested after exponential growth phase and subsequently treated by the CaCl2 method using procedures well known in the art. Alternatively, MgCl2 or RbCl can be used. Transformation can also be performed after forming a protoplast of the host cell if desired, or by electroporation. When the host is a eukaryote, methods of transfection of DNA, for example, calcium phosphate coprecipitates, conventional mechanical procedures for example, microinjection, electroporation, insertion of a plasmid encased in liposomes, or virus vectors can be used. Eukaryotic cells can also be co-transformed with polynucleotide sequences encoding a C-terminal endostatin polypeptide, and a second foreign DNA molecule encoding a selectable phenotype, for example, the herpes simplex thymidine kinase gene. Another method is to use a eukaryotic viral vector, for example, simian virus 40 (SV40) or bovine papilloma virus, to transiently infect or transform eukaryotic cells and express the protein (see, for example, Eukaryotic Viral Vectors, Cold Spring Harbor Laboratory, Gluzman ed., 1982). D. Pharmaceutical Compositions Further disclosed are pharmaceutical compositions including a fusion protein, nucleic acid, vector, or host cell disclosed herein and a pharmaceutically acceptable carrier. In some aspects, the pharmaceutical composition includes a host cell expressing a disclosed fusion protein and / or including a nucleic acid encoding a disclosed fusion protein, and a pharmaceutically acceptable carrier. Actual methods for preparing administrable compositions will be known or apparent to those skilled in the art and are described 7950-112226-02 in more detail in such publications as Remington’s Pharmaceutical Sciences, by E.W. Martin, Mack Publishing Co., Easton, PA, 22nd Edition, 2013. Fusion proteins, nucleic acids, vectors, or host cells described herein can be formulated with pharmaceutically acceptable carriers. Potential carriers include, but are not limited to, physiologically balanced culture medium, phosphate buffered saline solution, water, emulsions (for example, oil / water or water / oil emulsions), various types of wetting agents, cryoprotective additives or stabilizers for example, proteins, peptides or hydrolysates (for example, albumin, gelatin), sugars (for example, sucrose, lactose, sorbitol), amino acids (for example, sodium glutamate), or other protective agents. Resulting aqueous solutions may be packaged for use as is or lyophilized. Lyophilized preparations are combined with a sterile solution prior to administration for either single or multiple dosing. Formulated compositions, especially liquid formulations, may contain a bacteriostat to prevent or minimize degradation during storage, including but not limited to effective concentrations (usually ≤1% w / v) of benzyl alcohol, phenol, m-cresol, chlorobutanol, methylparaben, and / or propylparaben. A bacteriostat may be contraindicated for some patients; therefore, a lyophilized formulation may be reconstituted in a solution either containing or not containing such a component. The compositions of the disclosure can contain as pharmaceutically acceptable carriers substances as required to approximate physiological conditions, for example, pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, and triethanolamine oleate. The disclosed compositions may optionally include an adjuvant to enhance an immune response of the subject. Adjuvants, for example, aluminum hydroxide (ALHYDROGEL®, available from Brenntag Biosector, Copenhagen, Denmark and Amphogel®, Wyeth Laboratories, Madison, NJ), Freund’s adjuvant, MONTANIDE™ or SEPIVAC SWE™ (Seppic Inc., Fairfield, NJ), MPL^ (3-O-deacylated monophosphoryl lipid A; Corixa, Hamilton, IN), IL-12 (Genetics Institute, Cambridge, MA), TLR agonists (e.g., TLR-9 agonists), among many other suitable adjuvants, can be included in the compositions. Suitable adjuvants include, for example, toll-like receptor agonists, alum, AlPO4, alhydrogel, Lipid-A and derivatives or variants thereof, dmLT, oil-emulsions, saponins, neutral liposomes, liposomes containing the vaccine and cytokines, non-ionic block copolymers, and chemokines. Non-ionic block polymers containing polyoxyethylene (POE) and polyxylpropylene (POP), such as POE-POP-POE block copolymers, MPL^ (3- O-deacylated monophosphoryl lipid A; Corixa, Hamilton, IN) and IL-12 (Genetics Institute, Cambridge, MA), among many other suitable known adjuvants. In some instances, the adjuvant formulation includes a mineral salt, for example, a calcium or aluminum (alum) salt, for example calcium phosphate, aluminum phosphate or aluminum hydroxide. In some aspects, the adjuvant includes an oil and water emulsion, for example, an oil-in-water emulsion (e.g., MF59 (Novartis) or AS03 (GlaxoSmithKline). One example of an oil-in-water emulsion comprises a metabolizable oil (e.g., squalene), a tocol (e.g., a tocopherol, for example, alpha-tocopherol), and a 7950-112226-02 surfactant (e.g., sorbitan trioleate (Span™ 85) or polyoxyethylene sorbitan monooleate (Tween® 80)), in an aqueous carrier. In other aspects, the adjuvant may be a composition (e.g., a protein or antigen) that increases IgA antibody responses. In some aspects, the adjuvant is a deletion mutant heat-labile enterotoxin (dmLT). dmLT includes R192G and L211A amino acid substitutions compared to the wild type LT amino acid sequence (see, e.g., Clements et al., mSphere 3:e00215-18, 2018). In some non-limiting examples, the dmLT includes or consists of the amino acid sequence of SEQ ID NO: 31 or is encoded by a nucleic acid sequence including or consisting of SEQ ID NO: 32. Thus, in some aspects a disclosed composition further includes a dmLT protein adjuvant. In some aspects, the pharmaceutical composition is provided as a sterile composition. The pharmaceutical composition typically contains an effective amount of a disclosed fusion protein, nucleic acid encoding the fusion protein, vector, or host cell expressing the fusion protein. Typically, the amount of fusion protein, nucleic acid, vector, or host cell in each dose of the composition is selected as an amount which induces an immune response without significant, adverse side effects. In some aspects, the composition is provided in unit dosage form for use to induce an immune response in a subject, for example, to prevent or inhibit PWD in the subject. A unit dosage form contains a suitable single preselected dosage for administration to a subject, suitable marked or measured multiples of two or more preselected unit dosages, and / or a metering mechanism for administering the unit dose or multiples thereof. III. Methods of Inducing an Immune Response Provided herein are methods of inducing an immune response in a pig (an animal from the Sus genus, including, for example, Sus domesticus). In some aspects, the immune response is to an agent that causes porcine post-weaning diarrhea (PWD), for example, enterotoxigenic Escherichia coli (ETEC). In some aspects, the immune response is protective against ETEC infection and / or development of symptoms of PWD. The methods include administering to a subject (e.g., a pig (e.g., Sus domesticus)) (i) an acellular composition including an effective amount of a fusion protein, nucleic acid, or vector disclosed herein, and (ii) a cellular composition including an effective amount of a host cell (e.g., a bacterial cell) expressing a fusion protein disclosed herein. In some aspects, the fusion protein of step (i) includes a backbone consensus sequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 3 and includes at least one heterologous epitope. In some aspects, the fusion protein of step (i) includes or consists of the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical thereto. In some aspects, the fusion protein of step (i) is a fusion protein described herein in section A “PWD MEFA Monomer.” In some aspects, the fusion protein of step (ii) includes a backbone consensus sequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 29 and includes at least one heterologous epitope. In 7950-112226-02 some aspects, the fusion protein of step (ii) includes or consists of the amino acid sequence of SEQ ID NO: 25, or an amino acid sequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical thereto. In some aspects, the fusion protein of step (ii) is a fusion protein described herein in section B “PWD AB5 Holotoxin-Structured MEFA Protein.” In some aspects, the acellular or cellular composition further comprises a pharmaceutically acceptable carrier, for example, a pharmaceutically acceptable carrier described herein. The acellular or cellular composition can further comprise an adjuvant, for example, an adjuvant described herein (e.g., dmLT). In some aspects, the acellular composition comprises or consists of an effective amount of a fusion protein, nucleic acid, or vector disclosed herein, a pharmaceutically acceptable carrier, and an adjuvant. In some aspects, the cellular composition comprises or consists of an effective amount of a bacterial cell expressing a fusion protein disclosed herein, and a pharmaceutically acceptable carrier. In some aspects, the methods include administering to a pig an effective amount of (1) an acellular composition including a disclosed PWD MEFA monomer fusion protein, a nucleic acid encoding the PWD MEFA monomer fusion protein, or a vector including a nucleic acid encoding the PWD MEFA monomer fusion protein and (2) a cellular composition including a bacterial cell expressing a disclosed AB5 holotoxin- structured PWD MEFA antigen fusion protein or including a nucleic acid encoding the AB5 holotoxin- structured PWD MEFA antigen fusion protein. In some aspects, the acellular composition is administered to the pig prior to the cellular composition (for example, as a prime-boost vaccination treatment). In some aspects, the acellular composition is administered to the pig about 12-18 days (e.g., about 12, 13, 14, 15, 16, 17, or 18 days) prior to the cellular composition. In some aspects, the acellular composition is administered to the pig at about 3- 5 days post-natal (e.g., about 3, 4, or 5 days after birth). In further aspects, the cellular composition is administered to the pig at about 17-21 days post-natal (e.g., about 17, 18, 19, 20, or 21 days after birth) or later. In some aspects, the cellular composition is administered to the pig at weaning. In some aspects, the cellular composition is administered one or more times (for example, 1, 2, 3, 4, or more times) after weaning. The disclosed compositions can be administered to subjects by a variety of administration modes known to the person of ordinary skill in the art, for example, oral, intramuscular, subcutaneous, intravenous, intra-arterial, intra-articular, intraperitoneal, intradermal, or parenteral routes. In specific non-limiting examples, the compositions are administered via oral or intramuscular routes. In some aspects, the acellular composition (e.g., PWD MEFA monomer fusion protein composition) is administered intramuscularly, subcutaneously, or intradermally. In a non-limiting example, the acellular composition is administered intramuscularly. In some aspects, the cellular composition (e.g., bacterial cell expressing a disclosed AB5 holotoxin-structured PWD MEFA antigen fusion protein) is administered orally. In some aspects, oral administration is performed by adding the cellular composition to drinking water or feed of the subject. In some aspects, the acellular composition includes a fusion protein disclosed herein (e.g., PWD MEFA monomer fusion protein) and about 25 µg to about 250 µg of the fusion protein is administered to the 7950-112226-02 subject, for example 25 µg to 75 µg, 50 µg to 100 µg, 75 µg to 125 µg, 100 µg to 150 µg, 125 µg to 175 µg, 150 µg to 200 µg, 175 µg to 225 µg, or 200 µg to 250 µg fusion protein is administered to the subject. In some aspects, about 25 µg, 50 µg, 75 µg, 100 µg, 125 µg, 150 µg, 175 µg, 200 µg, 225 µg, or 250 µg of the fusion protein is administered to the subject. In a specific, non-limiting example, the acellular composition includes about 200 µg of the fusion protein. In another non-limiting example, the acellular composition includes about 100 µg of the fusion protein. In some aspects, the acellular composition further includes an adjuvant, for example, an adjuvant described herein. In some aspects, the acellularcomposition includes dmLT protein as the adjuvant. In certain non-limiting examples, the acellular composition includes about 0.5-2% dmLT adjuvant relative to the fusion protein. In one example, the acellular composition includes about 1% dmLT adjuvant relative to the fusion protein, for example, if the composition includes 200 µg of fusion protein, the dmLT is included at 2 µg. Similarly, if the composition includes 100 µg fusion protein, dmLT is included at 1 µg. However, one of ordinary skill in the art can select other appropriate amounts of dmLT for inclusion in the composition. In some aspects, the cellular composition (e.g., bacterial cells expressing the AB5 holotoxin- structured PWD MEFA antigen fusion protein) includes about 1 x 108to about 1 x 1011colony forming units (CFU) of the bacterial cell, for example, 1 x 108to 1 x 109, 5 x 108to 5 x 109, 1 x 109to 1 x 1010, 5 x 109to 5 x 1010, or 1 x 1010to 1 x 1011CFUs. In some aspects, the cellular composition includes about 1 x 108, about 5 x 108, about 1 x 109, about 5 x 109, about 1 x 1010, about 5 x 1010, or about 1 x 1011CFUs. In a specific, non-limiting example, the composition includes about 5 x 109CFU of bacterial cells expressing a fusion protein disclosed herein (e.g., a AB5 holotoxin-structured PWD MEFA antigen fusion protein). In some aspects, the cellular composition includes about 1 x 108to about 1 x 1011colony forming units (CFU) (e.g., for example, 1 x 108to 1 x 109, 5 x 108to 5 x 109, 1 x 109to 1 x 1010, 5 x 109to 5 x 1010, or 1 x 1010to 1 x 1011CFUs ) of the bacterial cell per mL of the cellular composition. In some aspects, 0.1 mL to 10 mL (e.g., 0.1 mL to 5 mL, 0.5 mL to 5 mL, 1 mL to 5 mL, 0.1 mL to 3 mL, 0.5 mL to 3 mL, 1 mL to 3 mL, 1 mL to 10 mL, 1 mL to 8 mL, etc.) of the cellular composition is administered to the subject. In some aspects, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 mL of the cellular composition is administered to the subject. In some aspects, about 1 mL of the cellular composition is administered to the subject. In some aspects, the cellular composition includes two or more strains of bacterial cells expressing a fusion protein disclosed herein. In some aspects, the cellular composition includes two different strains of bacterial cells mixed at a 1:1 ratio to achieve the final dose (total CFUs) administered to the subject. In a specific, non-limiting example, the composition administered to the subject includes about 2.5 x 109CFU of an F4-fimbrial strain expressing a fusion protein disclosed herein (e.g., a AB5 holotoxin-structured PWD MEFA antigen fusion protein) and about 2.5 x 109CFU of an F18-fimbrial strain expressing a fusion protein disclosed herein (e.g., a AB5holotoxin-structured PWD MEFA antigen fusion protein). 7950-112226-02 Administration of the disclosed compositions elicits an immune response sufficient to reduce or prevent E. coli (e.g., ETEC) infection. In some aspects, the methods disclosed herein decrease E. coli (e.g., ETEC) infection, for example, as measured by the bacterial load in a biological sample, or by the number or percentage of subjects in a vaccinated population that show symptoms of E. coli infection. In some aspects, E. coli infection is decreased by a desired amount, for example, by at least 10%, at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 99%, as compared to a suitable control. In some aspects, the methods disclosed herein induce a protective immune response that reduces or eliminates one or more symptoms of porcine PWD, for example, reduces or eliminates one or more of: diarrhea (including mild diarrhea, watery diarrhea, or severe diarrhea), vomiting, dehydration (for example, skin turgor, sunken eye orbit, or prominent backbone), lethargy, and death. In some aspects, the methods disclosed herein decrease PWD symptoms by a desired amount, for example, by at least 10%, at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 99%, as compared to a suitable control. In some aspects, the control is an unvaccinated individual or population. Clauses Clause 1. A fusion protein comprising: (i) the amino acid sequence of SEQ ID NO: 1; or (ii) a backbone protein, wherein the backbone protein comprises a consensus sequence with at least 90% identity to SEQ ID NO: 3 and at least one heterologous epitope. Clause 2. The fusion protein of clause 1, wherein the at least one heterologous epitope comprises 8- 16 amino acids. Clause 3. The fusion protein of clause 1 or clause 2, wherein the backbone protein comprises an LT toxoid backbone. Clause 4. The fusion protein of any one of the prior clauses, wherein the fusion protein comprises an LT B subunit domain. Clause 5. The fusion protein of any one of the prior clauses, wherein the LT B domain comprises SEQ ID NO: 23. Clause 6. The fusion protein of any one of the prior clauses, wherein the at least one heterologous epitope comprises one of more of SEQ ID NOs: 7, 9, 11, 13, 17, 19, or 21. Clause 7. The fusion protein of any one of the prior clauses, comprising SEQ ID NO: 1. Clause 8. A nucleic acid encoding the fusion protein of any one of the prior clauses. Clause 9. The nucleic acid of clause 8, wherein the nucleic acid comprises SEQ ID NO: 2. Clause 10. The nucleic acid of clause 8 or clause 9, wherein the nucleic acid encoding the backbone protein comprises a consensus sequence at least 90% identical to SEQ ID NO: 4. Clause 11. The nucleic acid of any one of clauses 8 to 10, wherein the nucleic acid encoding the backbone protein comprises SEQ ID NO: 4. 7950-112226-02 Clause 12. The nucleic acid of any one of clauses 8 to 11, wherein the nucleic acid encoding the at least one heterologous epitope comprises one or more of SEQ ID NOs: 8, 10, 12, 14, 18, 20, or 22. Clause 13. A vector comprising the nucleic acid of any one of clauses 8 to 12. Clause 14. An isolated host cell transformed with the vector of clause 13. Clause 15. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and: the fusion protein of any one of clauses 1 to 7; the nucleic acid of any one of clauses 8 to 12; or the vector of clause 13. Clause 16. A fusion protein comprising: (i) a fusion protein comprising the amino acid sequence of SEQ ID NO: 25; or (ii) a backbone protein, wherein the backbone protein comprises a consensus sequence with at least 90% identity to SEQ ID NO: 29 and at least one heterologous epitope. Clause 17. The fusion protein of clause 16, wherein the at least one heterologous epitope comprises 8-16 amino acids. Clause 18. The fusion protein of clause 16 or clause 17, wherein the backbone protein comprises an LT toxoid backbone. Clause 19. The fusion protein of any one of clauses 16 to 18, wherein the fusion protein comprises an LT B domain. Clause 20. The fusion protein of any one of clauses 16 to 19, wherein the LT B domain comprises a starting methionine and a signal peptide. Clause 21. The fusion protein of any one of clauses 16 to 20, wherein the LT B domain comprises SEQ ID NO: 27. Clause 22. The fusion protein of any one of clauses 16 to 21, wherein the at least one heterologous epitope comprises one of more of SEQ ID NOs: 7, 9, 11, 13, 17, 19, and 21. Clause 23. The fusion protein of any one of clauses 16 to 22, comprising SEQ ID NO: 25. Clause 24. A nucleic acid encoding the fusion protein of any one of clauses 16 to 23. Clause 25. The nucleic acid of clause 24, wherein the nucleic acid comprises SEQ ID NO: 26. Clause 26. The nucleic acid of clause 24, wherein the nucleic acid encoding the backbone protein comprises a consensus sequence at least 90% identical to SEQ ID NO: 30. Clause 27. The nucleic acid of clause 26, wherein the nucleic acid encoding the backbone protein comprises SEQ ID NO: 30. Clause 28. The nucleic acid of clause 26 or clause 27, wherein the nucleic acid encoding the at least one heterologous epitope comprises one or more of SEQ ID NOs: 8, 10, 12, 14, 18, 20, and 22. Clause 29. A vector comprising the nucleic acid of any one of clauses 24 to 28. Clause 30. An isolated host cell transformed with the vector of clause 29. Clause 31. The isolated host cell of clause 30, wherein the host cell is a bacterial cell. Clause 32. The isolated host cell of clause 31, wherein the bacterial cell is an enterotoxigenic Escherichia coli (ETEC) F4 fimbrial cell and / or ETEC F18 fimbrial cell. 7950-112226-02 Clause 33. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and: the fusion protein of any one of clauses 16 to 23; the nucleic acid of any one of clauses 24 to 28; the vector of clause 29; or the isolated host cell of any one of clauses 30 to 32. Clause 34. A method of inducing an immune response in a pig, comprising administering to the pig an effective amount of: a) the fusion protein of any one of clauses 1 to 7; the nucleic acid of any one of clauses 8 to 12; the vector of clause 13; or the pharmaceutical composition of clause 15; and b) the fusion protein of any one of clauses 16 to 23; the nucleic acid of any one of clauses 24 to 28; the vector of clause 29; the isolated host cell of any one of clauses 30 to 32; or the pharmaceutical composition of clause 33, wherein a) is administered to the pig before b). Clause 35. The method of clause 34, wherein a) is administered by parenteral administration. Clause 36. The method of clause 35, wherein the parenteral administration is intramuscular, intradermal, or subcutaneous administration. Clause 37. The method of any one of clauses 34 to 36, wherein b) is administered orally. Clause 38. The method of any one of clauses 34 to 37, wherein a) further comprises an adjuvant. Clause 39. The method of clause 38, wherein the adjuvant comprises a deletion mutant heat-labile enterotoxin (dmLT). Clause 40. The method of clause 39, wherein the dmLT comprises the amino acid sequence of SEQ ID NO: 31. Clause 41. The method of any one of clauses 34 to 40, wherein the isolated host cell of b) is a mixture of ETEC F4 fimbrial subtype cell and ETEC F18 fimbrial subtype cell. Clause 42. The method of any one of clauses 34 to 41, wherein a) is administered about 12-18 days before b). Clause 43. The method of any one of clauses 34 to 42, wherein a) is administered at 3-5 days post- natal. Clause 44. The method of any one of clauses 34 to 43, wherein b) is administered at 17-21 days post-natal. Clause 45. The method of any one of clauses 34 to 44, wherein the immune response is a protective response against porcine post-weaning diarrhea. EXAMPLES The following examples are provided to illustrate particular features of certain aspects of the disclosure, but the scope of the claims should not be limited to those features exemplified. Example 1 Materials and Methods This examples provides the materials and methods used for the experiments described in Examples 2 and 3. 7950-112226-02 Bacteria strains: The bacteria strains and plasmids used for this study are listed in Table 1. E. coli BL21 (DE3) CodonPlus strain (Agilent Technologies, Santa Clara, CA) was used to express the fimbria- toxin MEFA monomer protein. An F4-fimbrial field isolate (1836-2) (Zhang et al., Infect. Immun.74:3107- 3114, 2006) and an F18-fimbrial isolate (8532) (Zhang et al., Vet. Microbiol.123:145-152, 2007) were used as two vaccine host strains to produce the GM1-binding AB5holotoxin-structured fimbria-toxin MEFA. An F18 ETEC field strain isolated from a recent PWD outbreak, 9922 (F18, LT, STa, STb, Stx2e) which was acquired from Iowa State University by Dr. Joseph Connor (Carthage Veterinary Service, Ltd), was used as the challenge study to assess protection of PWD vaccine candidate PWDVax against infection from F18 ETEC strain 9922. Table 1. Escherichia coli strains and plasmids Strain Relevant properties Source DH fh A2 Δ F l Z 1 h A l V44 P 7 7950-112226-02 Strain Relevant properties Source pBR322 Gene clone vector Promega on: To construct a chimeric PWD fimbria-toxin multiepitope fusion antigen gene, the LT A subunit gene (eltA; without signal nucleotides) of porcine ETEC strain 3030-2 (Zhang et al., Infect. Immun.74:3107-3114, 2006) was used as a template. While retaining the nucleotide segments coding two best functional epitopes on the A1 domain (Huang et al., Appl. Env. Microbiol.84:e00849-18, 2018), the nucleotide segments coding the surface-exposed but less immunodominant epitopes on the A1 domain were replaced with the nucleotide segments coding two functional epitopes of F4 major subunit FaeG (Lu et al., Appl. Environ. Microbiol. 85:e00329-19, 2019) two epitopes of F18 adhesin subunit FedF (Lu et al., Vet. Microbiol.230:171-177, 2019), toxin domain of STa toxoid STaN11S, STb, and Stx2e epitope (Rausch et al., Vet. Microbiol.202:79- 89, 2017) for a chimeric LT A gene, as described previously (Lu et al., Appl. Environ. Microbiol. 86:e00274-20, 2020). This chimeric mutant eltA gene was then genetically fused to the LT B subunit gene (eltB; without signal nucleotides) to code a single peptide named PWD fimbria-toxin MEFA monomer. This PWD fimbria-toxin MEFA monomer gene was cloned into expression vector pET28a (Novogen, Madison, WI) and expressed by E. coli BL21 (DE3) CodonPlus strain (Agilent). Recombinant PWD fimbria-toxin MEFA monomer protein expression and purification were carried out. Briefly, a single colony of the recombinant E. coli strain was cultured in 200 mL 2xYT medium supplemented with 30 μg / mL Kanamycin (Sigma, St. Louis, MO) at 37ºC in a shaker incubator until OD600reached 0.5 to 0.7. After four more hours of incubation, bacteria were harvested by centrifugation, lysed with B-PER (bacteria protein extraction reagent, in phosphate buffer, Thermo Fisher, Waltham, MA), 10 mg / mL lysozyme, sonication, and centrifugation to extract inclusion body proteins. Inclusion body proteins were solubilized with solubilization buffer [500 mM CAPS (pH11.0), 30% N-lauroylsarcosine, 1 mM DTT], and then refolded and dialyzed with dialysis buffer (50 mM Tris buffer with or without 0.1 mM DTT, pH 8.5). Solubilized and refolded protein was examined by SDS-PAGE with Coomassie blue staining and characterized by Western blot with anti-CT rabbit polyclonal antibodies (Sigma), as well as anti-mouse sera specific F18 FedF subunit protein. PWD GM1-binding AB5 holotoxin-structured fimbria-toxin MEFA construction and PWDVax cellular component preparation: To construct GM1-binding holotoxin-structured (eltAB genes-like) PWD fimbria-toxin MEFA genes, mutant eltAB genes (mutated at the 192 residue of the A1 domain) were used as the template and the counterpart on the A1 segment with the PWD fimbria-toxin monomer gene was substituted for eltAB-like chimeric genes. The resultant chimeric genes had the cistron segment between the 7950-112226-02 chimeric A subunit gene (eltA) and the native B subunit gene (eltB), as well as the native signal peptide nucleotides of the eltA gene and eltB gene for an LT-like GM1-binding holotoxin-structured PWD fimbria- toxin protein (FIG.1); the chimeric genes were cloned into vector pBR322 and hosted by E. coli DH5α. F4-fimbrial E. coli isolate 1836-2 and F18-fimbrial E. coli isolate 8532 were used to host the plasmid carrying the eltAB-like PWD fimbria-toxin MEFA genes as two vaccine strains. Isolates 1836-2 and 8532 were tested in PCR and confirmed for the presence of the F4 faeG gene or F18 fedF gene, as well as in bacteria adherence assays and verified their attachment to pig intestinal cell line IPEC-J2. Additionally, 1836-2 was demonstrated previously to be capable of hosting an LT plasmid (eltAB genes cloned in pBR322) to express and secrete LT (Zhang et al., Infect. Immun.74:3107-3114, 2006). Expression and outer-membrane secretion of the PWD fimbria-toxin MEFA protein and protein binding to GM1 were examined in GM1 ELISA. In brief, overnight culture filtrates from each strain were incubated with ELISA wells coated with GM1(Sigma), with CT as a positive control and PBS as a negative control. Anti-CT rabbit polyclonal antibody (Sigma) and HRP-conjugated goat anti-rabbit IgG were used as the primary and second antibodies respectively. Additionally, sera from mice immunized with K88 or F18 and HRP-conjugated goat anti-mouse IgG were used as the primary and secondary antibodies. Wells were washed and then incubated with peroxidase substrates; OD600 values were recorded. After the expression and secretion of the GM1-binding holotoxin-structured PWD fimbria-toxin MEFA from the F4 fimbrial vaccine strain (9938) and the F18 fimbrial vaccine strain (9957), these two vaccine strains were examined for growth curve and further compatibility of co-culture in an equal volume and CFUs. After confirming growth compatibility, these two strains were combined as the cellular component of PWD vaccine candidate, PWDVax (with recombinant PWD fimbria-toxin MEFA monomer protein as PWDVax acellular component). Heterologous prime-boost immunization of piglets with PWDVax: Sows and boars from the University Swine Research Center were tested with DNA markers for susceptibility to F18 ETEC by using PCR and RFLP as described by Meijerink et al. (Immunogenetics 52:129-136, 2000). Three susceptible sows were selected for breeding and two were used for this study. Ten days before farrowing, sows were surface cleaned and transported to the university BSL-II research facility. A total of 30 piglets were born by two sows on the same day, 15 piglets each. On day one, after wiping with iodine, half of the piglets were randomly swapped between the two litters, by coin flipping, with male and female equally distributed. One litter was randomly designated as the control group and the other as the vaccination group. Two piglets in the control group were crushed by the mother after the primary vaccination, leaving 13 piglets in the control group. The heterologous prime-boost vaccine strategy consists of an intramuscular immunization with vaccine PWDVax acellular component, recombinant PWD fimbria-toxin MEFA monomer protein as the primary, followed with an oral inoculation with PWDVax cellular component, an equal combination of two vaccine strains as the booster. For primary immunization, each piglet in the vaccination group was intramuscularly injected with 200 μg (in 200 μL) PWD fimbria-toxin MEFA monomer protein, and piglets 7950-112226-02 in the control group intramuscularly injected with 200 μL PBS, at the age of five days. For booster immunization on day 19, each piglet in the vaccination group was orally inoculated with a total of 5 x 109CFUs of PWDVax cellular component (in 1 mL PBS), 2.5 x 109CFUs of each vaccine strain. For the control piglets, each was orally inoculated with 1 mL PBS. Two μg adjuvant double mutant LT (dmLT, in 2 μL; ATCC BEI Resources) was included in the injection or oral inoculation to all piglets. Piglets were weaned on day 21. Serum samples were collected from each piglet before the primary (day 5), before the booster (day 19), and at necropsy (day 35). Fecal samples were collected before the oral booster and before the challenge (day 32), and jejunum washes were collected at necropsy. Sera were stored at -80°C until use. Pig feces were suspended in fecal reconstitution buffer (10 mM Tris, 100 mM NaCl, 0.05% Tween-20, 5 mM sodium azide, pH7.4) supplemented with 0.5 mM phenylmethylsulfonyl fluoride, at a ratio of 1 gram feces in 5 mL buffer. After centrifugation, fecal suspension supernatants were collected and stored at -80°C until use. Intestinal washing samples were collected after adding 10 mL fecal reconstitution buffer into a 10 cm-long jejunum and rinsing a few times. Pig challenge with an F18 ETEC strain and vaccine efficacy assessment against PWD: Piglets in each litter were separated from the sow on day 21. All pigs were orally inoculated with F18 ETEC field strain 9922, 2.5 x 109CFUs (in 2 mL LB broth) for each pig, on day 32. Pigs were observed twice daily on the first day post-inoculation, and then every 2-4 hours on the following two days. Clinical signs including vomiting, diarrhea, lethargy, dehydration, and death were included for examination and recording. Watery diarrhea was recorded for pigs with a wet rear end, unformed watery stool; severe diarrhea was defined with watery diarrhea and signs of dehydration (turgor skin, sunken eye orbit, prominent backbone); mild diarrhea was recorded for pigs with a stained rear end and semi-formed stool. Diarrhea was recorded for a pig if it developed diarrhea in any of the three days post-challenge. Piglets were also weighed before the challenge and at necropsy. Pigs were euthanized three days after the challenge. At necropsy, intestinal content and ileum segments were corrected from each pig. Fecal content was weighed, dried, and weighed again to calculate dry fecal matter percentage. Ileum tissues were rinsed clean with sterile PBS, weighed, ground in PBS (1 gram tissue in 9 mL PBS), serially diluted, plated on LB agar plates. CFUs were counted after overnight growth at 37°C and used to quantify colonization of the challenge bacteria in small intestines. Fifty colonies randomly selected were tested in PCR with primers specific to LT gene eltAB to confirm the ETEC challenge strain. Vaccine efficacy against PWD was assessed based on the number of diarrheal pigs in the control group versus the vaccine group, using the formula (percentage of diarrhea in the control pigs - percentage of diarrhea in the vaccine group) / (percentage of diarrhea in the control pigs). Vaccine efficacy against watery diarrhea and any diarrhea (watery diarrhea and mild diarrhea) was assessed separately. Statistical analyses: Data of pig IgG and IgA antibody responses (in OD values), bacterial intestinal colonization (CFUs per gram ileum tissue), and fecal dry matter (%) were analyzed with the 7950-112226-02 GraphPad Prism® software (GraphPad Software, San Diego, CA) and presented in means and standard deviations. One-way ANOVA was used to examine the significance of differences between the control group and the vaccine group, and Bonferroni’s comparison test was to indicate significance of differences based on a p-value of less than 0.05, 0.001, or 0.0001. Example 2 A multivalent two-component vaccine candidate for PWD Two chimeric fimbria-toxin MEFA genes coding a monomeric fimbria-toxin MEFA protein and an AB5 holotoxin structured fimbria-toxin protein were generated (FIG.1). The monomeric fimbria-toxin MEFA gene consisted of a native LT B subunit gene segment (eltB, without the signal peptide sequence) and a chimeric LT A subunit gene segment, with the two segments fused as a single gene. The chimeric LT A segment had eight epitope segments substituted with foreign (heterologous) functional epitope segments of F4 (two epitopes), F18 (two epitopes), STa toxoid STaN11S(two copies), STb, and Stx2e A. This monomeric fimbria-toxin MEFA gene was cloned into vector pET28α and expressed by E. coli BL21 (DE3), the resultant strain was designated as 9719. The AB5 holotoxin-structured fimbria-toxin MEFA genes were constructed by substituting the counterpart of mutant eltAB genes (with the 192 amino acid residue mutated to code LT toxoid LTR192G) with the chimeric A1 segment of the monomeric fimbriae-toxin MEFA gene. The resultant AB5holotoxin- structured fimbria-toxin MEFA genes possessed the eltAB cistron structure between the chimeric A subunit gene and the B subunit gene (eltB), as well as the native signal sequences of each subunit gene, leading to eltAB-like genes for an LT-like holotoxin-structured fimbria-toxin MEFA protein. The AB5 fimbria-toxin MEFA genes were cloned into vector pBR322 and expressed by an avirulent F4- (1836-2) or F18-fimbrial (8532) E. coli isolate respectively. These two vaccine strains (9938, 9957) were generated. The cloning of the genes was confirmed with DNA sequencing. The expression of the fimbria-toxin MEFA monomer protein (~ 41 kDa) was verified by Western blot with antibodies specific to CT (LT homolog) and F18. The expression of outer-membrane secreted LT-like holotoxin-structured fimbria-toxin MEFA protein from vaccine strain 9938 or 9957 was affirmed in GM1 ELISAs with polyclonal antibodies specific to CT, K88, or F18. Since F4-fimbrial vaccine strain 9938 and F18-fimbrial vaccine strains 9957 had nearly identical growth curve and viability, the two strains were mixed at an equal volume prepared as the cellular component of a PWD vaccine candidate. By further combining this cellular vaccine component with the acellular component (fimbria-toxin MEFA monomer protein), a two-component PWD vaccine candidate, PWDVax, was generated and intended to be used in a heterologous prime-boost immunization schedule, intramuscular injection with acellular component fimbria-toxin MEFA monomer protein as the primary and followed with oral administration of cellular component live product as the booster. 7950-112226-02 Example 3 Heterologous prime-boost immunization with two-component PWDVax Pig serum samples collected 14 days after the prime, intramuscular immunization with the fimbrial- toxin MEFA monomer protein, showed a significant rise of IgG responses to F4 and F18 fimbriae and toxins LT and STb (FIG.2). The ELISA OD650values specific to F4, F18, LT, and STb from the sera of the immunized piglets were 1.68 ± 0.33, 1.59 ± 0.35, 1.61 ± 0.33, and 1.53 ± 0.27, respectively, significantly higher than the antigen-specific OD values from the control sera, 1.42 ± 0.27 (p < 0.001), 1.34 ± 0.19 (p < 0.001), 1.35 ± 0.21 (p < 0.001), and 1.33 ± 0.20 (p < 0.05). The OD value to Stx2e also rose from the sera of the immunized piglets after the primary immunization, though not significantly great compared to the OD value from the control pig sera (1.03 ± 0.21 versus 0.86 ± 0.24). No response to STa was detected from the sera after the injection of the monomeric fimbria-toxin protein. Pig fecal samples collected 14 days after the oral booster inoculation with the PWDVax cellular component, two live strains expressing the GM1-binding holotoxin-structured fimbria-toxin MEFA, showed a significant elevation of secretory IgA antibody responses to all target antigens, F4 and F18 fimbriae and LT, STb, STa, and Stx2e toxins (FIG.3A). The ELISA OD650 values to F4, F18, LT, STa, STb, and Stx2e secretory IgA response were 0.67 ± 0.42, 0.59 ± 0.37, 0.63 ± 0.44, 0.68 ± 0.38, 0.56 ± 0.40, and 0.56 ± 0.38 respectively from the fecal samples collected from the piglets 14 days after oral booster immunization. These OD values specific to each antigen were significantly different than the OD values from the fecal samples of the control piglets, 0.26 ± 0.05 (p<0.0001), 0.31 ± 0.13 (p<0.05), 0.20 ± 0.06 (p<0.0001), 0.31 ±0.06 (p<0.0001), 0.18 ± 0.03 (p<0.001), and 0.19 ± 0.04 (p<0.001), respectively. The jejunum washes collected at necropsy also showed a significant rise of IgA responses from the immunized piglets (FIG.3B). The ELISA OD650 readings specific to F4, F18, LT, STa, STb, or Stx2e were 0.84 ± 0.56, 0.87 ± 0.53, 0.71 ± 0.59, 0.74 ± 0.57, 1.24 ± 0.62, and 1.48 ± 0.65 respectively from the jejunum washes from the immunized piglets. These OD values were significantly great compared to those in the control piglets 0.35 ± 0.13 (p = 0.001), 0.57 ± 0.21 (p = 0.01), 0.27 ± 0.11 (p < 0.01), 0.45 ± 0.19 (p = 0.02), 0.58 ± 0.21 (p < 0.001), and 0.78 ± 0.49 (p < 0.001), respectively. After oral challenge with F18 ETEC strain 9922 (F18, LT, STa, STb, Stx2e), all 13 control piglets developed diarrhea, 7 piglets with watery diarrhea and 6 with mild diarrhea. In contrast, 9 out of 15 immunized pigs remained healthy, only 1 immunized pig developed watery diarrhea and 4 showed mild diarrhea (Table 2). This led to PWDVax efficacy estimated at 87.5% against watery diarrhea and 66.7% against any diarrhea from F18 ETEC infection. Table 2. Clinical outcomes after challenge with an F18 ETEC strain and post-weaning diarrhea (PWD) vaccine candidate PWDVax 7950-112226-02 Treatment group No. of piglets with clinical outcomes / total no. Efficacy (%) against in the group (%) x had over two logs reduction of F18 ETEC bacterial colonization in small intestines. The CFUs (per gram ileum tissue) of the challenge bacteria from the immunized pigs were 1.1 ± 1.2 (x107), which were significantly fewer than the CFUs from the ileum of the control pigs (3.3 ± 4.3, x109; p < 0.001) (FIG.4A). PCR testing with the primers specific verified colonized bacteria the F18 ETEC challenge strain. The fecal dry matter from the feces collected from the control pigs was significantly less than that of the pigs immunized with PWDVax (FIG.4B). For the feces collected from the control pigs, the dry matter was 16.2 ± 5.6 (%). This was significantly lower than the dry matter from the feces collected from the vaccinated pigs (21 ± 4.3%; p < 0.05). The daily weight gain between the two groups, however, showed no significant differences. Example 4 Challenge Study This example describes representative methods of testing efficacy of fimbria-toxin MEFA vaccines against ETEC infection in pigs. Vaccine Description: The immunogenic component of the experimental vaccines is a fimbria-toxin multiepitope fusion antigen (MEFA) as described in Example 2. The vaccine to be tested consists of two components: (1) MEFA protein mixed with an adjuvant [deletion mutant heat-labile enterotoxin (dmLT; a toxoid, hence, non-toxic / inactivated form of LT)] administered intramuscularly (IM). The IM vaccine dose is 100 μL of MEFA at a concentration of 100 μg / 100 μL mixed with 1 μL of dmLT adjuvant at a concentration of 1 μg / μL. (2) A non-pathogenic, live F4ac+E. coli strain (1836-2) containing a pBR322 derivative plasmid that expresses the MEFA protein (1836-2::MEFA). The parent strain 1836-2 is a wild-type E. coli porcine isolate that naturally expresses F4ac+fimbria, but no enterotoxins. Expression of F4ac+fimbria allows the strain to colonize the small intestines of pigs that have the respective fimbrial receptors. The lack of enterotoxins makes them unable to cause disease. Live bacteria expressing F4ac+fimbriae are needed to colonize the intestine, which in turn is needed for induction of effective mucosal immunity that includes production of secretory IgA. Active production of secretory IgA antibodies specific for F4ac fimbrial antigen, as well as the toxins included in the MEFA, is needed for active immunity of weaned and older pigs against ETEC. Only pigs that have the intestinal 7950-112226-02 receptors for these fimbriae develop severe illness in response to ETEC infection. F4ac+ETEC are one of the most prevalent porcine-specific ETEC organisms worldwide. Vaccination protocol: This study will involve conventionally farrowed pigs (described below). Vaccinations will begin when the pigs are still being nursed by the sow, and will continue when weaned (after removal of the sow from the pigs). When the pigs are 3-5 days old, they will be given the IM vaccine (MEFA antigen mixed with adjuvant). When the pigs are 17-21 days old, the sows will be removed, and the vaccinate group pigs will be given orally 5 x 109CFU of F4ac+1836-2::MEFA in 1.0 mL PBS, whereas control group pigs will be given a mock vaccination (oral 1.0 mL PBS). Challenge of immunity: Ten to 14 days after oral vaccination or oral mock vaccination, pigs will be challenged by oral inoculation with 8 x 1010CFU of a nalidixic acid-resistant (NalR) F4ac+(LT+STb+) strain 3030-2. This strain is NalRat a concentration of 50 mg / L, which will allow for the challenge organism to be quantified in intestinal cultures at the conclusion of the experiment, as one measure to allow for assessment of immunity. The NalRmutant 3030-2 strain is susceptible to ampicillin and gentamicin. Experimental animals: Pigs expressing intestinal receptors for F4ac+ETEC will be used in this study. DNA / PCR markers are available to identify pigs expressing F4ac receptors (Rasschaert et. al., Vet. Microbiol.123:249-253, 2007). Pigs determined by PCR to have DNA markers for these receptors will be immunized with the vaccine strain and then challenged with NalRF4ac+ETEC strain 3030-2, as noted above. To identify susceptible pigs, tail switch hair root samples from potential parents will be tested. Boars and sows that are PCR-positive for F4ac receptors will be mated. The progeny of these matings will inherit the genes for receptor expression, and can be tested by PCR on hair samples taken at the time of tail docking (see below). Animal experimental design: Experiments will be done one litter at a time. As noted above, challenge studies will use pigs likely to express intestinal receptors for F4ac+ETEC, as these are the animals susceptible to serious illness associated with such types of ETEC infection. Multiparous pregnant sows that have a previous history of raising pigs such that large enough litter sizes may be expected from breeding, and having known, recorded breeding dates will be used. Pig vaccination and challenge: Pregnant sows will farrow their pigs naturally in crates. One litter will be handled at a time. The crates and rooms are representative of clean, conventional commercial swine husbandry conditions. The pigs will be allowed to suckle their dams to obtain colostrum and milk containing maternal antibodies. To mimic pork industry and standard husbandry practices, the following will be done. At birth, the navel cords will be disinfected with iodine. When the pigs are 2-3 days old, the tails will be docked and tail switch hair root samples taken for PCR; ears notched (for individual pig identification); needle teeth clipped; and iron dextran injections given in the neck. When the pigs are 7 days old, they will have non-medicated creep feed introduced to them, and this feed will continue to be given until they are weaned. At weaning, the pigs will be fed a nutritionally complete and balanced weanling pig ration prepared by Envigo Corp. (Indianapolis, IN). Since studies have shown that certain dietary substances interfere with F4+or F4ac+ETEC adhesion to porcine epithelial cells, namely, casein 7950-112226-02 glycomacropeptide, locust bean, exopolysaccharide, wheat bran, pea hulls, and faba (aka fava) bean hulls, we will avoid the inclusion of these in the formulation of the ration. Pigs will be tested for peripheral blood anti-F4 and anti-LT (from opportunistic ETEC infections) antibody titers. When the pigs are 3-5 days old, they will be given the priming (first) dose of vaccine by IM injection. When the pigs are 17-21 days old, the sows will be removed, resulting in the weaning of the pigs. On the day of weaning, the litter will be divided in half at random with one-half remaining in the original pen, and the other half placed into a raised deck pen in an adjacent room. One pen will be designated vaccinates and the other designated controls with the assignments decided by a coin-flip. On the day of weaning (at 17-21 days of age), vaccinates will be orally inoculated with the live bacterial vaccine strain, whereas nonvaccinated controls will be orally given the diluent (PBS, 1 mL). Pre- vaccination fecal and jugular blood samples will be obtained from the pigs immediately prior to oral vaccination, for tests of antibody titers to corresponding MEFA vaccine antigens. Ten to 14 days after the oral vaccination is completed (when the pigs are 27-35 days old), pigs will challenged by oral inoculation with 8 x 1010CFU of NalRstrain 3030-2. Fecal and jugular vein blood samples will be obtained from the pigs immediately prior to challenge, also for testing of antibody titers to the MEFA vaccine antigens. The volume of the blood samples taken pre-vaccination and pre-challenge will each be 1-3 mL; hence, the total amount of blood collected will be 2-6 mL. As noted above, all pigs in a litter (corresponding to one independent experiment) will be challenged with the same strain, and these pigs will be selected on the basis of having receptors for the respective fimbrial type of the challenge strain. Monitoring for clinical illness: All challenged pigs will be monitored for clinical disease (vomiting, diarrhea, dehydration and lethargy), every 4 h or less (if necessary if weight loss is rapid), and euthanized 48 h post-challenge or upon reaching 15% weight loss, whichever occurs first. Fecal samples will be collected weekly after immunization and daily after challenge to test for shedding of the vaccine and challenge strains and inferred presence of intestinal colonization. Pigs will be weighed daily after challenge. Any pig that has any serious complicating illness unrelated to the study, e.g., anorexia, inability to obtain feed / water, unrelated infections, signs of organ dysfunction unrelated to the study, moribund for some reason unrelated to the study, will be euthanized and removed from the study. Any sow that has severe dystocia will either be treated or euthanized at the discretion of the attending veterinarian and removed from the study. A moribund state is defined as extreme depression, body temperature significantly below normal, or nonresponsive or unconscious with no response to external stimuli, such as handling or the toe-pinch withdrawal test. Euthanasia, necropsy, and sample collection: Pigs will be euthanized by the following approved procedures (AVMA, 2020): (1) IM injection of a cocktail of xylazine (2 mg / kg BW) and 30 mg / kg BW) to induce anesthesia; (2) percutaneous intracardiac blood sample (5-10 mL removed) for later testing; (3) intracardiac sodium pentobarbital (86 mg / kg BW; 1 mL / 4.5 kg BW using a 390 mg / mL solution) to induce death; (4) pneumothorax (confirmation of death); (5) exsanguination (confirmation of death); (6) necropsy with tissue sample collection. The blood obtained from the heart will be used to measure total protein level 7950-112226-02 (TP) and serum electrolytes as measurements of dehydration and electrolyte imbalance, and intestinal segments will be collected for bacterial culture and histopathology to assess levels of protection against bacterial colonization following challenge. Sows will be euthanized after the pigs are weaned. Microbiologic analyses: Microbiologic analyses will be performed by a board-certified veterinary microbiologist and staff who are blinded to the sample origin (vaccinate / control). Rectal swab specimens obtained immediately before challenge inoculation will be cultured aerobically and anaerobically by standard procedures to screen for presence of ETEC. These specimens will be inoculated directly on sheep blood agar (5% sheep blood in Trypticase soy agar) and MacConkey agar and streaked for isolation. Isolated colonies will be tested by MALDI-TOF for E. coli and for other enteric pathogens by PCR using a diagnostic panel. Specimens of ileum will be collected aseptically at necropsy, weighed, ground with a tissue grinder in sterile PBS, and serially diluted in sterile peptone water. Serial dilutions will be spread plated onto MAC- NAL agar to determine the CFU of the respective inocula per gram of tissue. One or two lactose-positive NalRbacterial isolates at the highest dilution used for quantification will be identified as to genus and species (E. coli) by MALDI-TOF, and as to the inoculum strain by multiplex PCR for the LT (eltAB), STb (estB), and STa (estA) genes. Clinical pathologic analyses: Pre-necropsy heart blood samples, collected aseptically, will be distributed into sodium heparin tubes for determination of the hematocrit (HCT) and serum separator tubes for determination of serum electrolytes and other analytes. Blood samples in serum separator tubes will be allowed to clot for 20 min and then centrifuged, and the serum was collected and frozen at -20°C until analyzed. Concentrations of urea nitrogen, creatinine, sodium (Na+), potassium (K+), chloride (Cl-), total CO2 (tCO2), albumin, and total protein in serum samples will be measured using an automated chemistry analyzer. Histopathologic and immunohistochemical analyses. At necropsy, the entire small intestine will be dissected free from the mesentery and the length from the pyloric to the ileocecal valves will be measured. Specimens from the small intestine will be obtained from the following locations and fixed in 10% neutral buffered formalin for histopathological examination: duodenum (yellow ink; 5 cm distal to the pyloric valve), jejunum-1 (red ink; one-third of the distance between the pyloric and ileocecal valves), jejunum-2 (blue ink; half the distance between the pyloric and ileocecal valves, jejunum-3 (black ink; two-thirds of the distance between the pyloric and ileocecal valves), and ileum (green ink; 5 cm proximal to the ileocecal valve). Ink markings will help ensure intestinal site identity during histologic examination. Specimens of stomach, duodenum, jejunum, ileum, spiral colon, mesenteric lymph node, lung, liver, spleen, kidney, and brain will also be collected and fixed in formalin, processed for histopathology. Formalin-fixed specimens will be processed, embedded in paraffin, sectioned at 5 µm, and stained by hematoxylin and eosin (H&E) by standard procedures. Tissue sections will be examined by routine light microscopic examination by a board-certified veterinary pathologist that will be blinded to treatment (vaccinate / control). Additional sections of ileum will be cut from the paraffin blocks, stained by an immunohistochemical procedure using rabbit polyclonal antiserum against the O antigen of the respective challenged organism (E. coli O157 for strain 3030-2), and examined microscopically by 7950-112226-02 the pathologist to assess for presence / absence of colonization. Quantification of the amount of colonization may be determined by image analysis if it is determined that such data is necessary or beneficial to the evaluation of vaccine efficacy. PWD vaccine candidate assessment: Serum, fecal suspension, and intestinal washes collected from each piglet (of the vaccinated and control groups) will be titrated for IgG and IgA antibody titers to F4ac, F18ac, LT, STa, STb and Stx2e. Antibody titers will be analyzed for correlation with clinical outcomes and efficacy of this live vaccine strain against PWD. Data analyses: Data analyses will be performed by a veterinary epidemiologist. Data will be considered multilevel and longitudinal since animals will be clustered within litter and / or room (depending on the study phase), and there will be repeated measures within animals during the study period. The proportion of pigs experiencing diarrhea (recorded as pigs having a watery fecal deposition [yes / no]; dependent variable) will be calculated at the animal level as well at the litter / room level for descriptive analysis. Generalized linear mixed models (GLMM) will be fitted in SAS 9.4 using the Proc Glimmix procedure to analyze the effect of treatment (immunized vs. control) and treatment by day interaction (fixed effects) with the proportion of pigs experiencing diarrhea. Models will be fitted for comparisons among conventional pigs. A binomial distribution, logit link, restricted pseudolikelihood estimation and Kenward- Rogers degrees of freedom approximation will be fitted. Litter and / or room will be treated as random intercepts and a random residual term for animal will be included to account for the temporal dependence of the data (repeated measures within pigs) using a first-order autoregressive covariance structure (which accounts for equal spacing between measurements over time). Bonferroni or Scheffe’s simultaneous inference procedures will be used to adjust P-values for multiple comparisons. The effect of treatment and study day on the proportion of pigs experiencing anorexia, colonization and death (binary or binomial outcomes) will be also assessed in separate models, using GLMMs as described above. The effect on weight and total protein changes (continuous outcomes) will be evaluated using linear mixed models in SAS (Proc Mixed). Model-adjusted means and their 95% confidence intervals will be provided in all models. Statistical significance in all model variables will be determined at a P-value of ≤ 0.05. It will be apparent that the precise details of the methods or compositions described may be varied or modified without departing from the spirit of the described aspects of the disclosure. We claim all such modifications and variations that fall within the scope and spirit of the claims below.

Claims

7950-112226-02 We claim:

1. A fusion protein comprising: (i) the amino acid sequence of SEQ ID NO: 1; or (ii) a backbone protein, wherein the backbone protein comprises a consensus sequence with at least 90% identity to SEQ ID NO: 3 and at least one heterologous epitope.

2. The fusion protein of claim 1, wherein the at least one heterologous epitope comprises 8-16 amino acids.

3. The fusion protein of claim 1, wherein the backbone protein comprises an LT toxoid backbone.

4. The fusion protein of claim 1, wherein the fusion protein further comprises an LT B subunit domain.

5. The fusion protein of claim 4, wherein the LT B subunit domain comprises SEQ ID NO:

23.

6. The fusion protein of claim 1, wherein the at least one heterologous epitope comprises one or more of SEQ ID NOs: 7, 9, 11, 13, 17, 19, or 21.

7. The fusion protein of claim 1, comprising SEQ ID NO:

1.

8. A nucleic acid encoding the fusion protein of claim 1.

9. The nucleic acid of claim 8, wherein the nucleic acid comprises SEQ ID NO:

2.

10. The nucleic acid of claim 8, wherein the nucleic acid encoding the backbone protein comprises a consensus sequence at least 90% identical to SEQ ID NO:

4.

11. The nucleic acid of claim 10, wherein the nucleic acid encoding the backbone protein comprises SEQ ID NO:

4.

12. The nucleic acid of claim 10, wherein the nucleic acid encoding the at least one heterologous epitope comprises one or more of SEQ ID NOs: 8, 10, 12, 14, 18, 20, or 22.

13. A vector comprising the nucleic acid of claim 8.

14. An isolated host cell transformed with the vector of claim 13.7950-112226-02 15. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and the fusion protein of claim 1 or a nucleic acid encoding the fusion protein.

16. A fusion protein comprising: (i) the amino acid sequence of SEQ ID NO: 25; or (ii) a backbone protein, wherein the backbone protein comprises a consensus sequence with at least 90% identity to SEQ ID NO: 29 and at least one heterologous epitope.

17. The fusion protein of claim 16, wherein the at least one heterologous epitope comprises 8-16 amino acids.

18. The fusion protein of claim 16, wherein the backbone protein further comprises an LT toxoid backbone.

19. The fusion protein of claim 16, wherein the fusion protein comprises an LT B domain.

20. The fusion protein of claim 19, wherein the LT B domain comprises a starting methionine and a signal peptide.

21. The fusion protein of claim 19, wherein the LT B domain comprises SEQ ID NO:

27.

22. The fusion protein of claim 16, wherein the at least one heterologous epitope comprises one of more of SEQ ID NOs: 7, 9, 11, 13, 17, 19, and 21.

23. The fusion protein of claim 16, comprising SEQ ID NO:

25.

24. A nucleic acid encoding the fusion protein of claim 16.

25. The nucleic acid of claim 24, wherein the nucleic acid comprises SEQ ID NO:

26.

26. The nucleic acid of claim 24, wherein the nucleic acid encoding the backbone protein comprises a consensus sequence at least 90% identical to SEQ ID NO:

30.

27. The nucleic acid of claim 26, wherein the nucleic acid encoding the backbone protein comprises SEQ ID NO: 30.7950-112226-02 28. The nucleic acid of claim 26, wherein the nucleic acid encoding the at least one heterologous epitope comprises one or more of SEQ ID NOs: 8, 10, 12, 14, 18, 20, and 22.

29. A vector comprising the nucleic acid of claim 24.

30. An isolated host cell transformed with the vector of claim 29.

31. The isolated host cell of claim 30, wherein the host cell is a bacterial cell.

32. The isolated host cell of claim 31, wherein the bacterial cell is an enterotoxigenic Escherichia coli (ETEC) F4 fimbrial cell and / or ETEC F18 fimbrial cell.

33. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and the isolated host cell of claim 30.

34. A method of inducing an immune response in a pig, comprising administering to the pig an effective amount of: a) the pharmaceutical composition of claim 15; and b) the pharmaceutical composition of claim 33, wherein a) is administered to the pig before b).

35. The method of claim 34, wherein a) is administered by parenteral administration.

36. The method of claim 35, wherein the parenteral administration is intramuscular, intradermal, or subcutaneous administration.

37. The method of claim 34, wherein b) is administered orally.

38. The method of claim 34, wherein the pharmaceutical composition of a) further comprises an adjuvant.

39. The method of claim 38, wherein the adjuvant comprises a deletion mutant heat-labile enterotoxin (dmLT).

40. The method of claim 39, wherein the dmLT comprises the amino acid sequence of SEQ ID NO: 31.7950-112226-02 41. The method of claim 34, wherein the isolated host cell of the pharmaceutical composition of b) is a mixture of ETEC F4 fimbrial subtype cell and ETEC F18 fimbrial subtype cell.

42. The method of claim 34, wherein the pharmaceutical composition of a) is administered about 12-18 days before the pharmaceutical composition of b).

43. The method of claim 34, wherein the pharmaceutical composition of a) is administered at 3-5 days post-natal.

44. The method of claim 34, wherein the pharmaceutical composition of b) is administered at 17-21 days post-natal.

45. The method of claim 34, wherein the immune response is a protective response against porcine post- weaning diarrhea and / or Escherichia coli infection.

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