SEA LICE ANTIGENS AND VACCINES
20220088160 · 2022-03-24
Inventors
- Ian Robert BRICKNELL (Hampden, ME, US)
- Sarah Elizabeth BARKER (Edinburgh, GB)
- Jessica Lynn PIESZ (Old Town, ME, US)
Cpc classification
Y02A40/81
GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
A61K39/00
HUMAN NECESSITIES
International classification
Abstract
Isolated proteins from caligid copepods, mutant protein from caligid copepods, polynucleotides encoding the same, and antigens and vaccines comprising the same, in particular for the treatment or prevention of caligid copepod infection in fish. Proteins are a mutant of fructose bisphosphate aldolase (FBP), a mutant of glutathione 5-transferase 1, isoform D (GST), peptidyl prolyl cis-trans isomerase 5-precursor (PPIase), glutathione S-transferase 1, isoform D (GST), a mutant of triosephosphate isomerase (TIM) and cystathionine gamma-lyase (CSE).
Claims
1. An antigen comprising one or more protein from the circum-oral gland (COG) or the frontal gland complex (FGC) of a caligid copepod, or a mutant thereof, wherein the protein is selected from the group consisting of: a mutant of fructose bisphosphate aldolase (FBP); a mutant of glutathione S-transferase 1, isoform D (GST); peptidyl prolyl cis-trans isomerase 5-precursor (PPIase); glutathione S-transferase 1, isoform D (GST); a mutant of triosephosphate isomerase (TIM); and cystathionine gamma-lyase (CSE), optionally wherein the caligid copepod is Lepeophtheirus salmonis or Caligus rogercresseyi.
2. (canceled)
3. The antigen according to claim 1, wherein the amino acid sequence of the one or more protein is selection from the group consisting of: SEQ ID NO:1; SEQ ID NO:2; SEQ ID NO:3; SEQ ID NO:4; SEQ ID NO:5; SEQ ID NO:6; and homologues thereof.
4.-5. (canceled)
6. An antigen comprising one or more protein according to claim 1, further comprising one or more protein having the amino acid sequence of one or more of the group consisting of: SEQ ID NO:7; SEQ ID NO:8; SEQ ID NO:9; SEQ ID NO:10; SEQ ID NO:11; SEQ ID NO:12; and homologues thereof.
7. A vaccine against caligid copepod infection in fish, the vaccine comprising an immunologically effective amount of one or more antigen according to claim 1, and a pharmaceutically-acceptable diluent or carrier, and optionally an adjuvant, optionally wherein the caligid copepod is Lepeophtheirus salmonis or Caligus rogercresseyi.
8. A vaccine according to claim 7, wherein each of the one or more antigens is different from the other antigen or antigens in the vaccine.
9. A vaccine according claim 8, wherein the vaccine comprises six or more antigens, wherein one of the six antigens comprises a mutant FBP, one of the six antigens comprises a mutant GST, one of the six antigens comprises PPIase, one of the six antigens comprises GST, one of the six antigens comprises TIM, and one of the six antigens comprises CSE.
10. A vaccine according claim 8, wherein the vaccine comprises six or more antigens, wherein one of the six antigens comprises the amino acid sequence of SEQ ID NO:1, one of the six antigens comprises the amino acid sequence of SEQ ID NO:2 or homologues thereof, one of the six antigens comprises the amino acid sequence of SEQ ID NO:3 or homologues thereof, one of the six antigens comprises the amino acid sequence of SEQ ID NO:4 or homologues thereof, one of the six antigens comprises the amino acid sequence of SEQ ID NO:5 or homologues thereof, and one of the six antigens comprises the amino acid sequence of SEQ ID NO:6 or homologues thereof.
11. (canceled)
12. The vaccine according to claim 7, wherein the fish is a salmonid.
13.-17. (canceled)
18. An antigen comprising a polynucleotide comprising DNA encoding a protein isolated from the circum-oral gland (COG) or the frontal gland complex (FGC) of a caligid copepod, or a mutant thereof, wherein the encoded protein is selected from the group consisting of: a mutant of fructose bisphosphate aldolase (FBP); a mutant of glutathione S-transferase 1, isoform D (GST); peptidyl prolyl cis-trans isomerase 5-precursor (PPIase); native GST; a mutant of triosephosphate isomerase (TIM); and cystathionine gamma-lyase (CSE), optionally wherein the caligid copepod is Lepeophtheirus salmonis or Caligus rogercresseyi.
19. (canceled)
20. The antigen according to claim 18, comprising DNA encoding the amino acid sequence of one or more of the group consisting of: SEQ ID NO:1; SEQ ID NO:2; SEQ ID NO:3; SEQ ID NO:4; SEQ ID NO:5; SEQ ID NO:6; and homologues thereof.
21. The antigen according to claim 18, wherein the DNA comprises the nucleotide sequence of one or more of the group consisting of: SEQ ID NO:13; SEQ ID NO:14; SEQ ID NO:15; SEQ ID NO:16; SEQ ID NO:17; SEQ ID NO:18; SEQ ID NO:19; SEQ ID NO:20; SEQ ID NO:21; and homologues thereof.
22. (canceled)
23. An antigen comprising the according to claim 18, further comprising the polynucleotide sequence of one or more of the group consisting of: SEQ ID NO:22; SEQ ID NO:23; SEQ ID NO:24; SEQ ID NO:25; SEQ ID NO:26; SEQ ID NO:27; SEQ ID NO:28; SEQ ID NO:29; SEQ ID NO:30; SEQ ID NO:31; and homologues thereof.
24. A vaccine against caligid copepod infection in fish, the vaccine comprising an immunologically effective amount of one or more antigen according to claim 18, a pharmaceutically-acceptable diluent or carrier, and optionally an adjuvant, optionally wherein the caligid copepod is Lepeophtheirus salmonis or Caligus rogercresseyi.
25. The vaccine against caligid copepod infection in fish according to claim 24, wherein the vaccine comprises an immunologically effective amount of a combination of two or more antigens, wherein each of the one or more antigens independently comprises the DNA sequence selected from the group consisting of: SEQ ID NO:13; SEQ ID NO:14; SEQ ID NO:15; SEQ ID NO:16; SEQ ID NO:17; SEQ ID NO:18; SEQ ID NO:19; SEQ ID NO:20; SEQ ID NO:21; and homologues thereof. The vaccine may further comprise an immunologically effective amount of a combination of two or more antigens, wherein each of the one or more antigens independently comprises the DNA sequence selected from the group consisting of: SEQ ID NO:22; SEQ ID NO:23; SEQ ID NO:24; SEQ ID NO:25; SEQ ID NO:26; SEQ ID NO:27; SEQ ID NO:28; SEQ ID NO:29; SEQ ID NO:30; SEQ ID NO:31; and homologues thereof.
26. The vaccine according to claim 24, wherein each of the one or more antigens is different from the other antigen or antigens in the vaccine.
27. The vaccine according to claim 26, wherein the vaccine comprises six antigens, wherein one of the six antigens comprises the DNA sequence of SEQ ID NO:13 or homologues thereof, one of the six antigens comprises the DNA sequence of SEQ ID NO:14 or homologues thereof, one of the six antigens comprises the DNA sequence of SEQ ID NO:15 or SEQ ID NO:16 or homologues thereof, one of the six antigens comprises the DNA sequence of SEQ ID NO:17 or SEQ ID NO:18 or homologues thereof, one of the six antigens comprises the DNA sequence of SEQ ID NO:19 or homologues thereof, and one of the six antigens comprises the DNA sequence of SEQ ID NO:20 or SEQ ID NO:21 or homologues thereof.
28. The vaccine according to claim 27, further comprising five antigens, wherein one of the five antigens comprises the DNA sequence of SEQ ID NO:22 or SEQ ID NO:23 or homologues thereof, one of the five antigens comprises the DNA sequence of SEQ ID NO:24 or SEQ ID NO:25 or homologues thereof, one of the five antigens comprises the DNA sequence of SEQ ID NO:26 or SEQ ID NO:27 or homologues thereof, one of the five antigens comprises the DNA sequence of SEQ ID NO:28 or SEQ ID NO:29 or homologues thereof, and one of the five antigens comprises the DNA sequence of SEQ ID NO:30 or SEQ ID NO:31 or homologues thereof.
29. (canceled)
30. The vaccine according to claim 24, wherein the fish is a salmonid.
31.-35. (canceled)
36. A method of treatment or prevention of caligid copepod infection in fish, comprising administering a therapeutic amount of the antigen, or vaccine of any one previous claim, optionally with the co-administration of an adjuvant, optionally wherein the caligid copepod is Lepeophtheirus salmonis or Caligus rogercresseyi.
37. (canceled)
38. The method according to claim 36, wherein the fish is a salmonid.
39. (canceled)
Description
[0066] The invention will now be described by way of example with reference to the drawings in which:
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[0078] For each of
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[0080] Absorbance at 450 nm shown for individual fish (circles, squares or triangles) with line indicating mean±SEM (n=12 fish per group). DM2 cocktail (delivery method 2 cocktail) is a vaccine prime using a cocktail of five recombinant antigens (50 μg) with vaccine boost using cocktail of five recombinant proteins (50 m). DM2 ctrl (delivery method 2 control) is a “prime” using mCherry-His recombinant protein (250 μg) plus flagellin (50 ng) with vaccine boost using mCherry-His recombinant protein (250 m).
EXAMPLES
Example 1—Isolation of Candidate Antigen Peptides from Circum-Oral Glands
[0081] The circum-oral glands (COGs) were visualized in L. salmonis at chalimus stages using 3,3′-diaminobenzidine tetrahydrochloride (DAB). COGs were isolated by microdissection and transferred into microcentrifuge tubes containing protease inhibitor cocktail (AEBSF [4-(2-aminoethyl)benzenesulfonyl fluoride] at 2 mM, Aprotinin at 0.3 μM, Bestatin at 116 μM, E-64 at 14 μM, Leupeptin at 1 μM and EDTA at 1 mM in 100 ml stock solution; Sigma-Aldrich Cat. No. P2714) at a 1 to 10 dilution in cold, sterile crustacean Ringers saline. Ringers saline was prepared by dissolving 0.58 M sodium chloride, 0.013 M potassium chloride, 0.013 M calcium chloride, 0.026 M magnesium chloride, 0.00054 M disodium hydrogen phosphate in 0.05M Tris-HCl, pH 7.5. Tissue was homogenised for two minutes at a frequency of 28 hertz using a TissueLyser II (Qiagen) by adding 100 μl 0.5 mm glass beads (BioSpec Products, catalog number 11079105) to 100 μl of sample. The supernatant was collected by centrifuging homogenate at 10,000×g for 30 minutes at 4° C. Protein concentration was determined using a BCA protein assay kit (Pierce Cat. No. 23227). The COG supernatant yielded 610 μg of protein. Samples were stored at −80° C.
[0082] Protein samples were concentrated with a 3K MWCO concentrator (Pierce) following manufacturer's instructions, and run on a SDS-PAGE gel. Gel slices containing proteins at 40 and 25 kDa were then analysed by nano-LC MS/MS.
[0083] Eight native proteins identified by the nano-LC MS/MS analysis were selected as candidate antigens: fructose bisphosphate aldolase (FBP; Hu et al., 2015; Lorenzatto et al., 2012); triosephosphate isomerase (TIM; Furuya et al. 2011; Saramago et al., 2012); peroxiredoxin-2 (Prx-2; Knoops et al., 2016; Rhee et al., 2016; Wood et al., 2003); enolase (Diaz-Ramos et al, 2012; Wang et al., 2013); and transitionally-controlled tumour protein homolog (TCTP; Gnanasekar et al., 2009; Gnanasekar and Ramaswamy, 2007; Sun et al., 2008; Nagano-Ito et al., 2009 and 2012); Glutathione S-transferase 1, isoform D (GST; Mounsey et al., 2010; Roncalli et al., 2015); Peptidyl-prolyl cis-trans isomerase 5 precursor (PPIase; Kramer et al., 2004; Ren et al., 2005; Devasahayam et al., 2002); Cystathionine gamma-lyase (CSE; Lee et al., 2014; Sun et al., 2009).
Example 2—Production of Recombinant Vaccines from Circum-Oral Glands Peptides
[0084] The glycosylation of our protein targets was examined using NetNGlyc 1.0. The server identified one potential N-linked glycosylation site for both FBP and TIM.
[0085] NetOGlyc 4.0 software identified two potential O-linked glycosylation sites for Prx-2.
[0086] Protein sequencing results from the nano-LC MS/MS analysis were used to blast NCBI database to obtain the complete mRNA coding sequence. As a quality control measure, the NCBI mRNA sequences of FBP, enolase, TIM, TCTP and Prx-2 were validated by performing RACE cDNA synthesis. To perform RACE cDNA synthesis, cDNA was prepared from RNA collected from 10 adult sea lice (RNeasyR Mini kit (Qiagen)). 5′ and 3′-RACE-Ready cDNA was prepared using a SMARTer RACE 5′/3′ cDNA synthesis kit (TaKaRa) for rapid amplification of cDNA ends. Primers were specially designed for each protein to ensure amplification of the 5′ end (5′ RACE PCR) or 3′ end (3′ RACE PCR) of the mRNA (see Table 1 for list of primers used). PCR products were gel extracted using the NucleoSpin Gel and PCR clean up kit (Clontech).
TABLE-US-00003 TABLE 1 RACE primers Pro- tein 5′ RACE cDNA 3′ RACE cDNA FBP GATTACGCCAAGCTTCAGCGCC GATTACGCCAAGCTTGGCTCC TTCTGGATTTCCAACC ACCAGAGAAGAAATTGCTGAAG (SEQ ID NO: 66) (SEQ ID NO: 67) Eno- GATTACGCCAAGCTTAGTGCAG GATTACGCCAAGCTTGCCGTT lase AGACCAACGACGAGATCAGCG GGAGATGAGGGTGGCTTTGCTC (SEQ ID NO: 64) (SEQ ID NO: 65) TIM GATTACGCCAAGCTTACGCTCT GATTACGCCAAGCTTGAGGTT GAATGACCAAGAATCGCCCAT GTTGTTGGAGCCTCACCCTGC (SEQ ID NO: 70) (SEQ ID NO: 71) TCTP GATTACGCCAAGCTTGGAACCG GATTACGCCAAGCTTCTGCTG AACCCAGTTTCGACCAGACG AAGAGGCCATGGATGACTGTGA (SEQ ID NO: 72) (SEQ ID NO: 73) Prx- GATTACGCCAAGCTTACACTCC GATTACGCCAAGCTTGGTCCT 2 ATCGTCTTCCTTGAGCACGCC TGCCTGCTCCACTGACTCCCAT (SEQ ID NO: 68) (SEQ ID NO: 69)
[0087] In-Fusion cloning of RACE products was then performed following the manufacturer's instructions. Single colonies (8-10) were isolated from culture plates and grown overnight in selective media (ampicillin) at 37° C. with shaking (˜180 rpm). Plasmid DNA was isolated from bacterial lysates using a QIAprep Spin Miniprep Kit following the manufacturer's instructions (Qiagen). To determine which clones contained our RACE insert, we analyzed the DNA by restriction digest using EcoRI and HindIII which flank the cloning site. Digested products were visualized on a 1% ethidium bromide gel.
[0088] Clones containing the largest gene specific inserts were sequenced. The mRNA sequencing results for FBP, enolase, TIM, TCTP and Prx-2, and the mRNA sequences for PPIase, GST and CSE used in the present invention, are shown in below (coding region underlined):
TABLE-US-00004 PPIase mRNA (SEQ ID NO: 32): GGGGGCCTGGAGAAATCTTTAGTATAAAATAGAATTCCTTCCCGTCATTGTGGCACCCAATT GCATTAATCCACCCTCTCGATTAATACTCATCATGAAATTCCTTGGGGTTTCCGGAGCAATC ATCCTTGCTCTTACCCTTTTCGTGACCTTCGCCTATGGGGATGACAATTCCAAGGGTCCCAA AGTAACAGAAACAGTCACATTCTCCATATCCATCGGAGGCAAACCCGCTGGTGATATTAAGA TTGGACTGTTTGGAAAGACTGTGCCAAAGACGGTCAAGAACTTTGTTGAGCTCGCAGCAAAG GAAGACAAAGGCGAGGGTTACAAGGGCTCCAAGTTCCATCGCGTCATTAAGGACTTTATGCT CCAAGGAGGTGACTTTACTCGTGGAGACGGAACTGGTGGACGATCCATCTACGGAGAGAGAT TTGCAGATGAAAACTTCAAGCTGAAGCACTACGGGGCTGGATGGTTGTCCATGGCCAATGCC GGAAAAGACACAAATGGATCTCAATTCTTCATCACTACCAAAAAGACCTCATGGCTCGATGG GAAACACGTTGTCTTTGGAAAGATCATTGGGGGCATGGACGTTGTTCGAAAAATTGAAAGGT CCAGTACGGACGGAAGAGATCGTCCTGTTGAGGATGTTGTCATTGAAGCCGCCACGGTTGAA AAACTTGATAAACCACTCAGTGTTCCCAAGGCGGATGCTGATGAATAAATTTGTTTCCTTTT ATTTCTATGTACTCCTATACGATATTGTAAAGGTATATATTCAATCAGCAGTATTTTAAGAA TAAATTACTCTTTTTTTAT GST mRNA (SEQ ID NO: 33): GGGGGAATTCAGTCTAGCTTTAGTTCCGAAGTTATCAATACCCTGACAAGGAAGCTCTCTCG CACCATAAAGTATATCTAAGATGAGCGTTGAAATTTATGGAATGGATATATCAGCTCCTCAT AGGATCGCTACAATGACTGCTGAAGTTGTTGGAGCTCCTTATGAAGTTAAGGATGTTGATAT CTTTAATGGAGGTAGCAAGACACCTGAATTTCTTGAATTGAATCCTCAACACAACATCCCAG TACTTAAGTATAAGGATTTTGTAATGAACGAGAGTAGAGCTATTGCTGGATTCTTGGCCTCA GAATTTGATAAAAGTGGCAAACTTTACCCAACCTGTCCCATGGCCCATGCTCGAGTTAATCA ACGGTTATACTTCGATATGGGAGTTTTTTATAAGGCCTTTGGAGAGTGTGTGTACCCAATAA TGTTTGCCAATGCTGATGTTCCTGCAGAAAAATACGACAAACTCAAAGAAGTTTTAGGATGG GCCAATGATATGGTAAAAGAGACAGGATTTGCTGCTGGTACCGAAGAGATGACAATTGCTGA TATCGCTTGGGTGGCTACATACAGTTCTATAAAGGAAGCTGATGTGATTGACTTAGTTCCTT ACAAAGAATTGGACGCATGGTTTACCAAATGTGTAGCACTTATTCCAAATTATGAGACGTGC AATGGAAAGGGAGCCAAAGGATTTGGAGATTTTTACAAATCCAAAAGGAAAGAATAATCTTT TTACTGTAAATGCAATATTATTTATTACTTGTATGAAATAACAAGTTTTTAATAAATTTTCG AATTATGATAAGTTAATAAATATTTTTAATATATTAACAC CSE mRNA (SEQ ID NO: 34): GGGGGTCAATATAACGTTCTGTTTCAACGTGTTTAAGGAGAGAGGAGTGGATTGATTGATAT ATTGAAGAAATGTCGACTTATCGTGATAATGACCCTCACTACGCTACTCATGCTATCCATGT TGGACAAAATCCGGAGCAATGGAAATCTTTGGCTGTCGTTCCTCATATAACCCTCTCCACAA CCTATAAACAATATCATCCTGGACAACCCAAAGAGTTTGAATACGGAAGAGGTGGAAATCCT ACTCGTAATATCCTCGAGACATGTATGGCCTCTTTGGATGGTGCTAAACATTGTGTGACTTT TGCTTCTGGTTTAGCAGCTTTAGATGCTATGACTACTATTTTGTCATGTGGGGATCACATTG TTGCCATGAATGATTTGTATGGGGGAACTAATCGATTTTTACGACGAGTATCCGCTAAGCAG GGTCTTACGTCAACTTTTGTCGATATTAATCACGAAGAACTTTTTTCTGCATCGTTTCAAGA TAACACAAAAATGGTATGGATTGAAAGCCCTACAAATCCAACATTACGTATTGTAGACATCA AAAAGGCCGTATCCATTGCCAAATCCAAGAATCCCAATATAATTGTTGTAGTTGACAATACA TTTGTGACCTCATACTTTCAACGGCCCTTGGAATTAGGAGCTGATGTTACATACTATTCATG TACAAAGTATATGAACGGTCATTCTGATGTTATTATGGGTGCCGTTTGTATAAACAGTGATG AAATCCACGAAAGAGTTCGATTTGTTCAATATGCTGTGGGTGCTGTTCCTAGTCCTTTTGAT TGCTTTCTCGTAAATCGTAGTCTCAAGACTCTCAAAGTAAGAATGGTCGAACATCAAAAAAA TGCTCTTATTGTTGGAAAATTTTTAGAAGGTCATTCCAAGATTACCAAGGTTATACACCCTG GATTACCATCCCATCCCGACCATGAAATTGTCAAAAAACAACAGTATGGCCATTCTGGCATG GTATCATTTTATCTCAAGGGTGGACTAGAAGAATCCAACAATTTTTTGAAGGCTGTTAAAGT ATTTATACTCGCAGAGTCTCTTGGAGGTTTCGAATCTTTAGCGGAGTTACCTTACTCTATGA CTCATGCTTCTGTTGCAGAAGAGGAACGGGTTGCTCTTGGTGTTACTAATAATCTCATCAGA CTCTCAATTGGACTTGAAAATGCCGATGATCTCTGTGCAGATTTAGATCAAGCTCTTAATAT CGCATGTTCATAATAAATATTATATATTTAACATCGACTTTGAAGATATCTAAATCAAGCAT TTTCATAAATTAATTAACACTTTTTTTACTATTTTTTAATGACTAATAAAACCTATTATTAT TATTTTTAGTTCCAAACGATGAAAAACTGTCTTTAAAGTAAGTTTAATTTAGAGATAGTATC AATTGAAATAAATGATAGTTAACTAT PRX-2 mRNA (SEQ ID NO: 35): GGGGGAGTCTTATATCTGCTACCGGCAAGTGAACTACCTCTGTCATCTCTCTTTGTAATATC CGACTAAGTAACAAAATGAGTCTTCAACCAACGAATGATGCTCCTCAATTCAAGGCTATGGC CGTTGTGAACAAGGAATTCAAGGAGGTGTCACTCAAGGACTATACCGGCAAATACGTGGTTC TCTTTTTCTACCCCTTGGACTTTACCTTTGTTTGCCCCACAGAAATCATTGCCTTTGGAGAT CGGGCTGCAGATTTCCGTAAAATTGGATGTGAGGTCCTTGCCTGCTCCACTGACTCCCATTT TTCTCATCTCCACTGGATCAACACTCCTCGTAAGGAGGGAGGACTTGGGGACATGGACATTC CCCTCATTGCGGATAAGAACATGGAAATTTCTAGAGCCTATGGCGTGCTCAAGGAAGACGAT GGAGTGTCCTTCAGAGGACTTTTCATCATTGACGGCACTCAGAAACTCCGTCAAATCACAAT CAATGATCTTCCTGTCGGAAGATGCGTAGACGAAACCTTAAGACTTGTACAAGCCTTCCAAT ACACAGACGTGCATGGCGAGGTTTGCCCTGCGGGATGGAAGCCAGGAAAGAAGTCTATGAAG CCCAGCAAGGAAGGTGTCTCATCTTACCTCGCAGATGCTGAACAATCAAAGAAATAATACAG AAGATCTCCCCTGTAGTTATTAGTTTCCATACCAATTCTCTCTTTTAATTCATTCGATTGGA CACTGTTACCATGTTCCACTTTTTAATTGTACCTGGTCAGTCAGTGCCCAAGGTCATTGATT GATTAAGTCTATCAAATATTTATGTATTCCCCGGTGTACTAATAGTTTTTAAGATATAAAAT ATACGACTTTTTAATATATT FBP aldolase mRNA (SEQ ID NO: 36): GGGGGGAGTTAGTATAAGAGATCGACAGGCTCTGTTCGCAACACTTGTTCCTAAAGGCAAAT TATCTTAAATCTTAAAAATGGGTCTTGAAGGAATTGTTCCCCCTGGTGTCATCACTGGAGAC AATCTTATTAAGTTGTTCGAATACTGTAGAGACCATAAAGTTGCTCTCCCTGCTTTCAACTG CACGTCTTCTTCAACCATCAATGCAGTTTTGCAAGCAGCACGGGACATTAAATCCCCTGTGA TTGTTCAATTTTCCAATGGTGGAGCTGCTTTTATGGCCGGCAAAGGCATCAAAAATGACGGT CAAAAGGCTAGTGTCCTTGGTGCAATTGCTGGGGCTCAACATGTTCGTTTAATGGCAAAGCA CTATGGTGTTCCTGTAGTTCTTCACTCTGATCACTGTGCTAAAAAACTCCTCCCATGGTTTG ATGGAATGCTTGAAGCTGATGAAGAGTATTTCAAACAAAATGGTGAACCTCTTTTCTCCAGT CACATGCTTGATCTCTCGGAGGAGTTTGATGAAGAAAATATTTCCACTTGTGCAAAATATTT TACTCGCATGACTAAAATGAAAATGTGGTTAGAAATGGAAATTGGAATCACTGGGGGCGAAG AGGATGGTGTTGACAATACCAATGTGAAAGCGGAGTCTCTTTACACCAAACCCGAACAAGTT TACAACGTGTACAAAACACTCAGCGAAATTGGACCAATGTTTTCCATTGCTGCCGCTTTTGG AAACGTACATGGTGTATACAAGGCAGGTAACGTTGTTCTTTCCCCACATTTGTTGGCTGATC ATCAAAAATACATCAAGGAGCAAATTAACTCCCCACTTGATAAACCCGCCTTCCTTGTCATG CACGGAGGCTCCGGCTCCACCAGAGAAGAAATTGCTGAAGCAGTAAGCAACGGTGTGATCAA AATGAATATTGATACGGATACTCAATGGGCTTACTGGGATGGTCTCAGAAAGTTTTATGAAG AAAAGAAGGAGTATCTTCAAGGACAGGTTGGAAATCCAGAAGGCGCTGACAAGCCAAACAAA AAGTTTTACGATCCACGAGTTTGGGTTCGTGCTGCTGAGGAGTCTATGATTAAGAGAGCCAA TGAATCCTTTGAATCATTAAACGCTGTGAATGTCCTTGGTGACTCCTGGAAACACTAAATAC TTATTATTGGATATTCAGAATGTTTTAATTTCTATTTTGGAACTCCGAACTTACTAGTAATT TATTTCTCTTTTAAAAAATGAATCAGTATATTTATTATTCTGTTTATAAAATTAAGTTATTG TTAATTTCCTTAAATTTATTTATCAAAAATTAGAAATTGTTATACATGAAACATTGACATAA ATCTAAAATTGAAACATTTTATGATTTTGATGTTTATAAATGCTAGATAAGAAGTCATAAAT AAATGTATAATAAATTAAACTTCTTTCGTGATTAATTAACTTGCTAATTAATGCATAATTTT CATTTTTTTGAAGATATGCGCTAAAAAATTATTCAATAAAAATTAAAATAG Enolase mRNA (SEQ ID NO: 37): GCTCCGATTCACTTCTTATTTCTCAACGCTCATCGATACTTTATAAGGCTCAAATTCAAAAT GCCTATTAAACACATTCATGCACGTCAAATCTACGACTCTCGTGGTAACCCTACAGTGGAGG TGGATCTCACCACTGAGCGAGGGATTTTCCGCGCTGCCGTCCCCAGTGGAGCTTCCACAGGG GTTCATGAGGCCCTGGAACTGCGCGACAAGGACTCTACCTGGCACGGGAAGAGTGGTCTCAA GGCTGTGAAGAATGTGAACGACGTCCTTGGGCCCGAGTTGGTGAAGAAGAACCTTGACCCCG TGAAGCAAGAGGAGATCGATGATTTCATGATCAGCCTCGACGGGACGGATAACAAGAGCAAA TTTGGGGCTAATTCTATTTTGGGAATCTCGATGGCTGTGTGCAAGGCTGGTGCCGCCCACAA GGGTGTTCCCCTCTACCGCCATATCGCTGACTTGGCGGGTGTGAAGGAAGTGATGATGCCGG TGCCCGCATTTAATGTCATTAACGGAGGTTCTCATGCTGGAAATAAGTTGGCGATGCAAGAA TTCATGATCCTTCCAACTGGAGCTCCCTCCTTCACTGAAGCCATGAGGATGGGATCTGAAAT CTATCACCATCTCAAGGCTCTTATCAAGAAGAAGTACGGGTTGGATGCTACAGCCGTTGGAG ATGAGGGTGGCTTTGCTCCCAACTTCCAAGCCAACGGCGAGGCTATCGACCTTCTTGTTGGA GCCATTGAAAAGGCTGGATACACTGGAAAAATCAAGATCGGAATGGATGTTGCTGCTTCAGA ATTTTACAAAAATGGAAAGTACGATTTAGATTTCAAAAATGAAGAATCCAAAGAGGCCGATT GGCTAACTTCCGAGGCTCTTGGTGAAATGTACAAAGGATTCATCAAGGATGCACCTGTCATT TCCATTGAAGATCCCTACGATCAAGATGATTGGGAGGGATGGACTGCATTGACATCACAAAC TGACATTCAGATTGTCGGAGATGATCTCACAGTCACAAACCCCAAGCGTATTCAAATGGCTG TTGACAAGAAATCTTGCAACTGCCTCCTCTTGAAAGTAAATCAAATTGGTTCAGTAACTGAA TCTATTCGGGCCCACAATCTTGCTAAGAGCAACGGCTGGGGTACCATGGTCTCTCATAGATC TGGTGAGACAGAGGATTGTTTCATCGCTGATCTCGTCGTTGGTCTCTGCACTGGTCAAATCA AGACTGGAGCTCCTTGCAGATCCGAACGTTTGTCTAAATACAATCAATTGTTGCGTATTGAA GAGGAGTTGGGATCCAACGCTAAATATGTCGGTGACAAGTTCAGAATGCCCTTTTAATGATC TAAAGGGTTGTTTCTTCATTGAAGAAAGTTCATTTCTATAGTCACAATAAATTATTTCATGG TTTTACAAGAAATTCACAGGACGAAAAAACAAAAATCTTAATTTATTGAATTATTTCTATAT GTATTACACGCGTACTCTAAGTAAAACCTTATAAAGGAATATAATTGTAATATAATTTATTG TAATATTTTTTTTTTCATATTTAATTTATATTAAGGGTTGCCATTTAAATATATAAATTCCC CGTTGGTAAAAAAAAAAA TCTP mRNA (SEQ ID NO: 38): GAGGTTGTCGGCTTTCAAGGACCACTCAATTCCTCCCTAGTTCTAATTCACTTTCACTCCGG ACTCTTCCCGTAAACACTCCTGCCTTATACAAAATGAAGATCTTTAAGGACGTATTTTCTGG AGATGAATTATTTTCCGACACCTACAAGTTCAAGTTGTTGGATGATTGCTTGTACGAGGTGT ATGGAAAGTATGTCACACGGACTGAAGGAGATGTGGTTCTTGATGGAGCCAACGCATCTGCT GAAGAGGCCATGGATGACTGTGATTCCTCTTCCACCTCTGGTGTCGATGTTGTCCTTAACCA CCGTCTGGTCGAAACTGGGTTCGGTTCCAAGAAGGACTACACCGTATACCTTAAGGACTACA TGAAGAAGGTAGTGACATATTTAGAAGAAAATGGCAAACAAGCCGAAGTAGATACCTTCAAG ACCAACATCAACAAGGTCATGAAGGAACTTTTACCACGGTTTAAGGATCTTCAATTCTATAC TGGAGAAACGATGGACCCTGAGGCCATGATCATCATGCTTGAATACAAGGAAGTTGATGGAA AGGATATTCCCGTCCTCTACTTTTTTAAACATGGATTAAATGAAGAAAAATTTTAAACATTA GTGTCATCATTCATCTCAATTTCTTATAAATGTTTATATCTACAATATATTTTATATAGATA AAAAAGAATTTCCGTTGACAATAATATGCGAACTACCTAATTAAATTATGTTGTATTCATAT TTCTAATGCGATTTTTGGGAAATTTCTCGTTATAACTAAATTCCATTTTTAACGTACACGTC TGTATATGAATATATGTAAAGTGTTATTTACTTGTAAGAC TIM mRNA (SEQ ID NO: 39): GGGGGAGTTATAAAGCACTACTCGATTGCTAAGTACTTCGCGAGGTTCCTACTAATTGTAAT ATAGTTGAAAAAATACATTCAAAAATGGGTGGAGGAAGAAAATTTTTCGTTGGTGGAAACTG GAAAATGAATGGAGACAAGAAATCTATTGATGGAATCGTAGATTTTTTGAGCAAGGGGGATT TGGACCCAAATTGTGAGGTTGTTGTTGGAGCCTCACCCTGCTATTTGGACTATTCCCGTTCT AAACTTCCTGCCAATATCGGAGTGGCTGCACAAAATTGTTATAAGGTGGCCAAAGGAGCATT TACCGGAGAAATCAGTCCTCAAATGATTAAAGATGTTGGTTGTGAATGGGCGATTCTTGGTC ATTCAGAGCGTAGAAATGTCTTTGGGGAATCTGATGAGCTCATTGGCGAAAAGGTTGCTTTT GCACTTGAGTCTGGTCTCAAAATTATTCCATGCATTGGAGAAAAATTAGACGAACGTGAATC TGGGAAGACTGAGGAGGTCTGCTTTAAGCAACTTAAAGCCATTTCTGACAAAGTATCTGATT GGGATCTTGTCGTCTTAGCTTATGAACCAGTTTGGGCCATTGGAACTGGCAAAACAGCTACA CCTGCTCAGGCTCAAGAAACACATCTTGCTCTTCGTAAATGGCTAAAGGAGAACGTTTCTGA GGAAGTTTCACAAAAAGTGCGAATCCTCTATGGAGGTTCCGTGAGTGCTGGTAATTGCAAGG AACTTGGCACTCAGCCTGATATTGACGGCTTCCTTGTTGGAGGAGCCTCTCTCAAACCTGAC TTTGTTCAAATCATCAACGCTACTAAGTAAACAAAATACTGGATATTCGACTCTTCTATAAT AGTCTTATCATCTCTTTAATGCTCTCACTCATTATTTGATAAATAACGAGGTTAAAATATTA TTTATTTGATTAAACGTAATCTAACGTAATACATATATATTAATTTTCACGAATGCAGAAAA AAAATTATTGCATAAATACGTATTTTACA
[0089] The mRNA sequencing data of the target proteins was aligned and compared with the corresponding NCBI mRNA sequence using the Clustal Omega multiple sequence alignment tool (EMBL-EBI).
[0090] In most cases, mRNA sequence data matched exactly or very closely (only single base pair differences) to the NCBI database, however, for one protein, enolase, an additional isoform was identified (new start codon identified upstream from the start site of the NCBI sequence). Using UniProt (Universal Protein Resource) the sequence matched with 99% identity to Tribolium castaneum, the red flour beetle. Both the red flour beetle (hexapod) and sea louse (crustacean) belong to the clade Pancrustacea in the phylum arthropoda (www.uniprot.org).
[0091] The mRNA sequences of PPIase, CSE and GST were obtained from NCBI (National Center for Biotechnology Information; Bethesda, Md., United States), but not validated by performing RACE cDNA synthesis. The PPIase mRNA sequence has the NCBI accession number BT078668.1. The CSE mRNA sequence has the NCBI accession number BT078138.1. The GST mRNA sequence has the NCBI accession number BT078543.1.
[0092] In addition to the eight native proteins, mutant versions of GST, FBP and TIM were evaluated as vaccine antigen candidates. Mutant nucleotide sequences comprising mutations were generated using standard molecular techniques, such that each mutant produced a single amino acid substitution at the amino acid level. Thus, a mutant GST in which the S at position 67 is replaced with an A (i.e. the GST S67A), a mutant FBP in which the N at position 286 is replaced with an D (i.e. the FBP N286D) and a mutant TIM in which the E at position 166 is replaced with an D (i.e. the TIM E166D) were produced.
[0093] The characteristics of sea lice antigens are provided in Table 2.
TABLE-US-00005 TABLE 2 Characteristics of sea lice antigens Protein Amino acid mRNA length Size (kDa) length (residues) (bp) FBP-aldolase 42.1 364 1539 Prx-2 24 199 888 Enolase 48.9 432 1630 Enolase (short) 31.8 290 1146 TCTP 21.6 172 846 TIM 28.7 249 1021 PPIase 24.6 211 825 CSE 45.2 393 1452 GST 26.2 218 846 GST S67A 26.2 218 846 FBP N286D 42.1 364 1539 TIM E166D 28.7 249 1021
[0094] The edited sequences were used to produce the protein antigens by recombinant protein production in E. coli. The DNA sequence for each protein was codon optimized prior to gene synthesis and cloned into the pET-30a (+) expression vector with N-terminal His tag along with TEV cleavage site. Recombinant plasmids were then transformed into E. coli BL21 (DE3) cells and grown overnight at 37° C. A single colony was selected and inoculated into 1 litre of LB media containing kanamycin and incubated at 200 rpm at 37° C.
[0095] The expression DNA sequences were as set out below:
TABLE-US-00006 FBP N286D (SEQ ID NO: 40): ATGCATCATCACCATCACCACGAAAACCTGTATTTTCAGGGCATGGGTCTGGAAGGCATCGT TCCGCCGGGTGTCATTACGGGTGATAACCTGATTAAACTGTTCGAATACTGCCGCGACCACA AAGTGGCACTGCCGGCTTTTAACTGCACCAGCTCTAGTACGATTAATGCAGTGCTGCAGGCG GCCCGTGATATTAAATCTCCGGTTATCGTCCAATTTAGTAACGGCGGTGCAGCTTTCATGGC GGGCAAAGGTATTAAAAATGATGGCCAGAAAGCCTCCGTTCTGGGCGCCATCGCAGGTGCTC AACATGTTCGCCTGATGGCCAAACACTATGGTGTCCCGGTGGTTCTGCATTCTGATCACTGC GCGAAAAAACTGCTGCCGTGGTTCGATGGCATGCTGGAAGCCGACGAAGAATACTTTAAACA GAACGGTGAACCGCTGTTCTCCTCACACATGCTGGATCTGTCGGAAGAATTTGACGAAGAAA ATATCAGCACCTGTGCGAAATATTTCACCCGTATGACGAAAATGAAAATGTGGCTGGAAATG GAAATTGGCATCACGGGCGGTGAAGAAGATGGTGTCGACAACACCAATGTGAAAGCCGAAAG CCTGTATACGAAACCGGAACAGGTCTATAACGTGTACAAAACCCTGTCCGAAATTGGCCCGA TGTTTTCAATCGCGGCCGCATTCGGCAACGTTCATGGTGTCTATAAAGCCGGTAATGTCGTG CTGTCTCCGCATCTGCTGGCTGATCACCAGAAATACATCAAAGAACAAATCAACAGTCCGCT GGACAAACCGGCGTTTCTGGTGATGCATGGCGGTTCGGGTAGCACCCGTGAAGAAATTGCGG AAGCCGTGAGCAACGGTGTTATTAAAATGGACATCGATACCGACACGCAGTGGGCATATTGG GATGGCCTGCGCAAATTCTACGAAGAAAAGAAAGAATACCTGCAGGGCCAAGTTGGTAACCC GGAAGGTGCTGATAAACCGAATAAAAAATTCTATGACCCGCGTGTGTGGGTTCGTGCTGCCG AAGAAAGTATGATCAAACGCGCTAACGAATCCTTTGAATCCCTGAACGCAGTGAATGTGCTG GGTGACAGTTGGAAACACTAATGA GST S67A (SEQ ID NO: 41): ATGCATCACCACCACCACCACGAAAATCTATACTTCCAAGGAATGTCAGTTGAAATCTACGG TATGGACATTAGCGCGCCGCATCGCATCGCGACCATGACCGCGGAAGTGGTTGGTGCGCCGT ACGAAGTGAAGGACGTTGATATTTTCAACGGTGGCAGCAAAACCCCGGAGTTTCTGGAACTG AACCCGCAGCACAACATCCCGGTGCTGAAGTACAAAGACTTCGTTATGAACGAGGCGCGTGC GATTGCGGGTTTCCTGGCGAGCGAATTTGATAAGAGCGGCAAACTGTACCCGACCTGCCCGA TGGCGCATGCGCGTGTGAACCAACGTCTGTACTTCGACATGGGTGTGTTCTATAAGGCGTTT GGCGAGTGCGTTTACCCGATCATGTTTGCGAACGCGGACGTTCCGGCGGAGAAGTATGATAA GCTGAAAGAAGTGCTGGGCTGGGCGAACGACATGGTTAAAGAAACCGGTTTCGCGGCGGGCA CCGAGGAAATGACCATCGCGGATATTGCGTGGGTGGCGACCTACAGCAGCATCAAAGAGGCG GACGTGATTGATCTGGTTCCGTATAAGGAACTGGATGCGTGGTTTACCAAATGCGTTGCGCT GATTCCGAACTATGAGACCTGCAACGGCAAGGGTGCGAAGGGTTTTGGCGATTTCTACAAGA GCAAGCGTAAAGAGTAATGA PPIase (SEQ ID NO: 42): ATGCATCACCACCACCACCACGAAAACCTATACTTCCAAGGAATGAAGTTCCTAGGCGTTAG CGGCGCGATTATCCTGGCGCTGACCCTGTTCGTTACCTTTGCGTACGGTGACGATAACAGCA AGGGCCCGAAAGTGACCGAAACCGTTACCTTTAGCATCAGCATTGGTGGCAAGCCGGCGGGT GACATCAAAATTGGTCTGTTTGGCAAGACCGTGCCGAAGACCGTTAAAAACTTCGTGGAACT GGCGGCGAAAGAGGACAAAGGTGAAGGCTATAAGGGTAGCAAATTCCACCGTGTGATCAAAG ATTTTATGCTGCAGGGTGGCGACTTCACCCGTGGTGATGGCACCGGTGGCCGTAGCATTTAC GGCGAGCGTTTCGCGGACGAAAACTTTAAGCTGAAACACTATGGTGCGGGTTGGCTGAGCAT GGCGAACGCGGGTAAAGATACCAACGGCAGCCAATTCTTTATCACCACCAAGAAAACCAGCT GGCTGGACGGTAAACACGTGGTTTTCGGCAAGATCATTGGTGGCATGGATGTGGTTCGTAAG ATCGAGCGTAGCAGCACCGACGGTCGTGATCGTCCGGTTGAGGATGTGGTTATTGAAGCGGC GACCGTTGAAAAACTGGACAAACCGCTGAGCGTGCCGAAGGCGGATGCGGATGAGTAATGA GST (SEQ ID NO: 43): ATGCACCACCACCACCACCACGAAAATCTATACTTCCAAGGAATGTCAGTAGAAATATACGG TATGGACATTAGCGCGCCGCACCGCATTGCGACCATGACCGCGGAAGTGGTTGGTGCGCCGT ACGAAGTGAAGGACGTTGATATCTTCAACGGTGGCAGCAAAACCCCGGAGTTTCTGGAACTG AACCCGCAGCACAACATCCCGGTGCTGAAGTACAAAGACTTCGTTATGAACGAGAGCCGTGC GATTGCGGGTTTCCTGGCGAGCGAATTTGATAAGAGCGGCAAACTGTACCCGACCTGCCCGA TGGCGCATGCGCGTGTGAACCAACGTCTGTACTTCGACATGGGTGTGTTCTATAAGGCGTTT GGCGAGTGCGTTTACCCGATCATGTTTGCGAACGCGGACGTTCCGGCGGAGAAGTATGATAA GCTGAAAGAAGTGCTGGGCTGGGCGAACGACATGGTTAAAGAAACCGGTTTCGCGGCGGGCA CCGAGGAAATGACCATCGCGGATATTGCGTGGGTGGCGACCTACAGCAGCATCAAAGAGGCG GACGTGATTGATCTGGTTCCGTATAAGGAACTGGATGCGTGGTTTACCAAATGCGTTGCGCT GATTCCGAACTATGAGACCTGCAATGGCAAGGGTGCGAAGGGTTTTGGCGACTTTTACAAGA GCAAGCGTAAGGAGTAATGA TIM E166D (SEQ ID NO: 44): ATGCATCATCATCATCATCACGAAAATCTGTACTTTCAAGGCATGGGCGGCGGTCGCAAATT CTTTGTCGGCGGCAACTGGAAAATGAACGGCGATAAAAAATCTATCGATGGTATCGTGGATT TTCTGAGCAAAGGCGATCTGGATCCGAATTGCGAAGTGGTTGTGGGTGCGAGCCCGTGTTAT CTGGATTACAGCCGTTCTAAACTGCCGGCAAACATTGGTGTGGCCGCACAGAATTGCTATAA AGTTGCGAAAGGCGCCTTCACCGGTGAAATTAGCCCGCAGATGATCAAAGATGTTGGCTGTG AATGGGCAATTCTGGGTCATTCTGAACGTCGCAACGTGTTTGGCGAAAGTGATGAACTGATC GGTGAAAAAGTTGCATTCGCGCTGGAAAGCGGCCTGAAAATTATCCCGTGCATCGGTGAAAA ACTGGATGAACGCGAATCTGGTAAAACGGAAGAAGTGTGTTTTAAACAGCTGAAAGCCATTT CTGATAAAGTAGTGATTGGGATCTGGTTGTGCTGGCGTATGATCCGGTGTGGGCGATTGGTA CCGGTAAAACCGCAACGCCGGCACAGGCACAGGAAACCCACCTGGCACTGCGTAAATGGCTG AAAGAAAACGTTAGCGAAGAAGTGTCTCAGAAAGTTCGCATTCTGTACGGCGGTAGTGTTAG CGCGGGCAATTGCAAAGAACTGGGTACCCAGCCGGATATCGATGGCTTCCTGGTGGGTGGTG CTTCCCTGAAACCGGACTTTGTGCAGATTATCAACGCTACGAAATAATGA CSE (SEQ ID NO: 45): ATGCACCACCACCACCACCACGAAAACCTATACTTCCAAGGAATGAGTACATACCGTGATAA TGACCCGCACTACGCGACCCACGCGATTCACGTTGGTCAGAACCCGGAACAATGGAAGAGCC TGGCGGTGGTTCCGCACATTACCCTGAGCACCACCTACAAGCAGTATCACCCGGGTCAACCG AAAGAATTTGAGTATGGTCGTGGTGGCAACCCGACCCGTAACATCCTGGAAACCTGCATGGC GAGCCTGGACGGTGCGAAACACTGCGTTACCTTTGCGAGCGGCCTGGCGGCGCTGGATGCGA TGACCACCATCCTGAGCTGCGGTGACCACATTGTGGCGATGAACGATCTGTATGGTGGCACC AACCGTTTCCTGCGTCGTGTTAGCGCGAAGCAGGGCCTGACCAGCACCTTTGTGGACATCAA CCACGAGGAACTGTTCAGCGCGAGCTTTCAAGATAACACCAAAATGGTTTGGATTGAAAGCC CGACCAACCCGACCCTGCGTATCGTTGACATTAAGAAAGCGGTGAGCATCGCGAAGAGCAAA AACCCGAACATCATTGTGGTTGTGGACAACACCTTCGTTACCAGCTACTTTCAGCGTCCGCT GGAGCTGGGTGCGGATGTGACCTACTATAGCTGCACCAAGTATATGAACGGTCACAGCGACG TTATTATGGGCGCGGTGTGCATCAACAGCGATGAAATTCACGAGCGTGTTCGTTTCGTTCAA TATGCGGTTGGTGCGGTGCCGAGCCCGTTCGACTGCTTTCTGGTGAACCGTAGCCTGAAGAC CCTGAAAGTTCGTATGGTGGAACACCAAAAGAACGCGCTGATTGTTGGTAAATTTCTGGAGG GCCACAGCAAGATCACCAAAGTGATTCATCCGGGTCTGCCGAGCCACCCGGATCACGAAATC GTTAAGAAACAGCAATACGGTCACAGCGGCATGGTGAGCTTCTATCTGAAAGGTGGCCTGGA GGAAAGCAACAACTTCCTGAAGGCGGTTAAAGTGTTTATCCTGGCGGAGAGCCTGGGTGGCT TTGAAAGCCTGGCGGAGCTGCCGTATAGCATGACCCATGCGAGCGTGGCGGAGGAAGAGCGT GTTGCGCTGGGTGTGACCAACAACCTGATCCGTCTGAGCATTGGCCTGGAAAATGCGGACGA CCTGTGCGCGGACCTGGACCAAGCGCTGAACATCGCGTGCAGCTAATGA Prx-2 (SEQ ID NO: 46): ATGCACCATCACCACCACCACGAAAATCTGTACTTCCAAGGCATGTCACTGCAACCGACGAA CGACGCCCCGCAATTCAAAGCAATGGCAGTGGTTAACAAAGAATTCAAAGAAGTTTCGCTGA AAGATTACACCGGCAAATACGTCGTGCTGTTTTTCTATCCGCTGGACTTTACCTTCGTCTGC CCGACGGAAATTATCGCATTTGGCGATCGTGCGGCCGACTTCCGCAAAATTGGTTGCGAAGT GCTGGCTTGTAGCACCGATTCTCATTTCAGTCATCTGCACTGGATCAACACGCCGCGTAAAG AAGGCGGTCTGGGCGATATGGACATTCCGCTGATCGCAGATAAAAATATGGAAATTTCCCGC GCTTATGGTGTCCTGAAAGAAGATGACGGCGTGTCATTTCGTGGTCTGTTCATTATCGACGG CACCCAGAAACTGCGCCAAATTACGATCAATGATCTGCCGGTTGGTCGTTGCGTCGACGAAA CCCTGCGCCTGGTTCAGGCGTTTCAATACACGGATGTGCACGGTGAAGTTTGTCCGGCCGGC TGGAAACCGGGTAAAAAATCTATGAAACCGTCAAAAGAAGGCGTGTCGTCCTACCTGGCAGA TGCTGAACAATCCAAAAAA FBP aldolase (SEQ ID NO: 47): ATGCATCATCACCATCACCACGAAAACCTGTATTTTCAGGGCATGGGTCTGGAAGGCATCGT TCCGCCGGGTGTCATTACGGGTGATAACCTGATTAAACTGTTCGAATACTGCCGCGACCACA AAGTGGCACTGCCGGCTTTTAACTGCACCAGCTCTAGTACGATTAATGCAGTGCTGCAGGCG GCCCGTGATATTAAATCTCCGGTTATCGTCCAATTTAGTAACGGCGGTGCAGCTTTCATGGC GGGCAAAGGTATTAAAAATGATGGCCAGAAAGCCTCCGTTCTGGGCGCCATCGCAGGTGCTC AACATGTTCGCCTGATGGCCAAACACTATGGTGTCCCGGTGGTTCTGCATTCTGATCACTGC GCGAAAAAACTGCTGCCGTGGTTCGATGGCATGCTGGAAGCCGACGAAGAATACTTTAAACA GAACGGTGAACCGCTGTTCTCCTCACACATGCTGGATCTGTCGGAAGAATTTGACGAAGAAA ATATCAGCACCTGTGCGAAATATTTCACCCGTATGACGAAAATGAAAATGTGGCTGGAAATG GAAATTGGCATCACGGGCGGTGAAGAAGATGGTGTCGACAACACCAATGTGAAAGCCGAAAG CCTGTATACGAAACCGGAACAGGTCTATAACGTGTACAAAACCCTGTCCGAAATTGGCCCGA TGTTTTCAATCGCGGCCGCATTCGGCAACGTTCATGGTGTCTATAAAGCCGGTAATGTCGTG CTGTCTCCGCATCTGCTGGCTGATCACCAGAAATACATCAAAGAACAAATCAACAGTCCGCT GGACAAACCGGCGTTTCTGGTGATGCATGGCGGTTCGGGTAGCACCCGTGAAGAAATTGCGG AAGCCGTGAGCAACGGTGTTATTAAAATGAATATCGATACCGACACGCAGTGGGCATATTGG GATGGCCTGCGCAAATTCTACGAAGAAAAGAAAGAATACCTGCAGGGCCAAGTTGGTAACCC GGAAGGTGCTGATAAACCGAATAAAAAATTCTATGACCCGCGTGTGTGGGTTCGTGCTGCCG AAGAAAGTATGATCAAACGCGCTAACGAATCCTTTGAATCCCTGAACGCAGTGAATGTGCTG GGTGACAGTTGGAAACAC Enolase (SEQ ID NO: 48): ATGCATCATCACCATCACCACGAAAACCTGTATTTTCAGGGCATGCCGATTAAACACATCCA TGCCCGCCAAATCTATGACTCCCGTGGTAACCCGACCGTTGAAGTTGACCTGACCACCGAAC GTGGCATTTTTCGTGCCGCGGTGCCGAGCGGTGCATCTACGGGTGTTCATGAAGCTCTGGAA CTGCGCGATAAAGACTCAACCTGGCACGGCAAATCGGGTCTGAAAGCGGTCAAAAACGTGAA TGATGTTCTGGGCCCGGAACTGGTGAAGAAAAACCTGGACCCGGTCAAACAGGAAGAAATTG ATGACTTTATGATCAGCCTGGATGGTACCGACAACAAATCTAAATTCGGCGCAAATAGTATT CTGGGTATCTCCATGGCAGTCTGTAAAGCTGGCGCAGCTCATAAAGGTGTGCCGCTGTATCG TCACATTGCGGATCTGGCCGGCGTCAAAGAAGTGATGATGCCGGTTCCGGCCTTCAACGTCA TTAATGGCGGTAGCCATGCAGGTAATAAACTGGCTATGCAGGAATTTATGATTCTGCCGACC GGTGCCCCGTCATTCACCGAAGCCATGCGCATGGGTTCGGAAATTTATCATCACCTGAAAGC GCTGATTAAGAAAAAATACGGCCTGGATGCAACGGCTGTTGGTGACGAAGGCGGTTTTGCCC CGAACTTCCAAGCGAATGGCGAAGCCATTGATCTGCTGGTTGGTGCAATCGAAAAAGCTGGC TACACCGGTAAAATTAAAATCGGCATGGATGTCGCGGCCTCCGAATTCTACAAAAACGGTAA ATACGATCTGGACTTCAAAAATGAAGAAAGTAAAGAAGCGGATTGGCTGACCAGCGAAGCCC TGGGCGAAATGTACAAAGGTTTCATCAAAGATGCCCCGGTGATTAGCATCGAAGATCCGTAC GACCAGGATGACTGGGAAGGCTGGACCGCACTGACGTCTCAGACCGATATTCAAATCGTGGG TGATGACCTGACCGTTACGAACCCGAAACGTATCCAGATGGCGGTTGATAAAAAATCTTGCA ACTGTCTGCTGCTGAAAGTCAATCAAATTGGCTCAGTGACCGAATCGATCCGTGCGCATAAC CTGGCCAAATCTAATGGCTGGGGTACGATGGTGTCTCACCGCTCCGGCGAAACCGAAGATTG CTTCATTGCAGACCTGGTGGTTGGCCTGTGTACGGGTCAGATCAAAACCGGTGCTCCGTGCC GTAGCGAACGCCTGTCTAAATATAATCAACTGCTGCGCATCGAAGAAGAACTGGGTAGCAAT GCGAAATATGTGGGTGATAAATTCCGTATGCCGTTT TCTP (SEQ ID NO: 49): ATGCACCACCACCATCACCACGAAAATCTGTACTTCCAAGGCATGAAAATCTTCAAAGACGT GTTTAGCGGCGACGAACTGTTCTCGGATACCTACAAATTTAAACTGCTGGATGATTGCCTGT ATGAAGTGTACGGCAAATATGTTACCCGTACGGAAGGCGATGTGGTTCTGGATGGTGCGAAC GCCAGCGCAGAAGAAGCGATGGATGATTGTGATAGCTCTAGTACCTCTGGTGTGGATGTGGT TCTGAATCATCGCCTGGTTGAAACCGGCTTTGGTAGCAAGAAAGATTACACGGTGTATCTGA AAGATTACATGAAGAAAGTGGTTACGTATCTGGAAGAAAACGGCAAACAGGCGGAAGTGGAT ACCTTCAAAACGAACATCAACAAAGTTATGAAAGAACTGCTGCCGCGTTTTAAAGATCTGCA GTTCTACACCGGTGAAACGATGGATCCGGAAGCCATGATTATCATGCTGGAATATAAAGAAG TTGATGGCAAAGACATTCCGGTGCTGTACTTCTTCAAACACGGCCTGAACGAAGAAAAATTC TIM (SEQ ID NO: 50): ATGCATCATCATCATCATCACGAAAATCTGTACTTTCAAGGCATGGGCGGCGGTCGCAAATT CTTTGTCGGCGGCAACTGGAAAATGAACGGCGATAAAAAATCTATCGATGGTATCGTGGATT TTCTGAGCAAAGGCGATCTGGATCCGAATTGCGAAGTGGTTGTGGGTGCGAGCCCGTGTTAT CTGGATTACAGCCGTTCTAAACTGCCGGCAAACATTGGTGTGGCCGCACAGAATTGCTATAA AGTTGCGAAAGGCGCCTTCACCGGTGAAATTAGCCCGCAGATGATCAAAGATGTTGGCTGTG AATGGGCAATTCTGGGTCATTCTGAACGTCGCAACGTGTTTGGCGAAAGTGATGAACTGATC GGTGAAAAAGTTGCATTCGCGCTGGAAAGCGGCCTGAAAATTATCCCGTGCATCGGTGAAAA ACTGGATGAACGCGAATCTGGTAAAACGGAAGAAGTGTGTTTTAAACAGCTGAAAGCCATTT CTGATAAAGTTAGTGATTGGGATCTGGTTGTGCTGGCGTATGACCGGTGTGGGCGATTGGTA CCGGTAAAACCGCAACGCCGGCACAGGCACAGGAAACCCACCTGGCACTGCGTAAATGGCTG AAAGAAAACGTTAGCGAAGAAGTGTCTCAGAAAGTTCGCATTCTGTACGGCGGTAGTGTTAG CGCGGGCAATTGCAAAGAACTGGGTACCCAGCCGGATATCGATGGCTTCCTGGTGGGTGGTG CTTCCCTGAAACCGGACTTTGTGCAGATTATCAACGCTACGAAA
[0096] To evaluate the level of expression of our targets, small-scale cultures (4 ml) were grown to optimize the temperature, expression time, and Isopropyl β-D-1-thiogalactopyranoside (IPTG) concentration. SDS-PAGE and western blot were used to monitor expression over the different conditions. Once the optimum conditions were identified, culture volume was scaled up to 1 L to ensure ≥10 mg of protein per target.
[0097] After IPTG induction, the 1 L culture was spun down to collect cell pellets. Pellets were then lysed with lysis buffer and sonicated. Both supernatant and pellet fractions were collected and evaluated by SDS-PAGE to identify which fractions contained the target protein. For all proteins except for enolase, the proteins were located in the supernatant and therefore were soluble.
[0098] Soluble proteins were purified by adding the supernatant of the cell lysate to several millilitres of Ni-NTA (nickel-nitrilotriacetic acid) resin for high capacity, high performance nickel-IMAC (immobilized metal affinity chromatography), which is used for routine affinity purification of His-tagged proteins.
[0099] For insoluble proteins, pellets from the cell lysate were solubilized with urea, purified by N-column purification under denaturing conditions, and then refolded. Protein fractions were pooled and filter sterilized (0.22 μm).
[0100] To ensure ≥90% purity of the proteins, an additional two steps of purification by densitometric analysis of Coomassie blue stained SDS-PAGE gel was performed. Proteins were further analysed by western blot using primary mouse-anti-His mAb (GenScript, Cat. No. A00186). Protein concentration was determined using the Bradford protein assay with BSA standards (Pierce).
[0101] Aliquots were prepared in 1×PBS buffer with 10% glycerol (pH 7.4) and stored in −80° C. The expression product of the FBP N286D expression DNA sequence (SEQ ID NO:40) is SEQ ID NO:51, which has the following sequence (TEV protease cleavage site is underlined, and the leading 6His tag is apparent):
TABLE-US-00007 MHHHHHHENLYFQGMGLEGIVPPGVITGDNLIKLFEYCRDHKVALPAFNC TSSSTINAVLQAARDIKSPVIVQFSNGGAAFMAGKGIKNDGQKASVLGAI AGAQHVRLMAKHYGVPVVLHSDHCAKKLLPWFDGMLEADEEYFKQNGEPL FSSHMLDLSEEFDEENISTCAKYFTRMTKMKMWLEMEIGITGGEEDGVDN TNVKAESLYTKPEQVYNVYKTLSEIGPMFSIAAAFGNVHGVYKAGNVVLS PHLLADHQKYIKEQINSPLDKPAFLVMHGGSGSTREEIAEAVSNGVIKMD IDTDTQWAYWDGLRKFYEEKKEYLQGQVGNPEGADKPNKKFYDPRVWVRA AEESMIKRANESFESLNAVNVLGDSWKH
[0102] The expression product of the GST S67A expression DNA sequence (SEQ ID NO:41) is SEQ ID NO:52, which has the following sequence (TEV protease cleavage site is underlined, and the leading 6His tag is apparent):
TABLE-US-00008 MHHHHHHENLYFQGMSVEIYGMDISAPHRIATMTAEVVGAPYEVKDVDIF NGGSKTPEFLELNPQHNIPVLKYKDFVMNEARAIAGFLASEFDKSGKLYP TCPMAHARVNQRLYFDMGVFYKAFGECVYPIMFANADVPAEKYDKLKEVL GWANDMVKETGFAAGTEEMTIADIAWVATYSSIKEADVIDLVPYKELDAW FTKCVALIPNYETCNGKGAKGFGDFYKSKRKE
[0103] The expression product of the PPIase expression DNA sequence (SEQ ID NO:42) is SEQ ID NO:53, which has the following sequence (TEV protease cleavage site is underlined, and the leading 6His tag is apparent):
TABLE-US-00009 MHHHHHHENLYFQGMKFLGVSGAIILALTLFVTFAYGDDNSKGPKVTETV TFSISIGGKPAGDIKIGLFGKTVPKTVKNFVELAAKEDKGEGYKGSKFHR VIKDFMLQGGDFTRGDGTGGRSIYGERFADENFKLKHYGAGWLSMANAGK DTNGSQFFITTKKTSWLDGKHVVFGKIIGGMDVVRKIERSSTDGRDRPVE DVVIEAATVEKLDKPLSVPKADADE
[0104] The expression product of the GST expression DNA sequence (SEQ ID NO:43) is SEQ ID NO:54, which has the following sequence (TEV protease cleavage site is underlined, and the leading 6His tag is apparent):
TABLE-US-00010 MHHHHHHENLYFQGMSVEIYGMDISAPHRIATMTAEVVGAPYEVKDVDIF NGGSKTPEFLELNPQHNIPVLKYKDFVMNESRAIAGFLASEFDKSGKLYP TCPMAHARVNQRLYFDMGVFYKAFGECVYPIMFANADVPAEKYDKLKEVL GWANDMVKETGFAAGTEEMTIADIAWVATYSSIKEADVIDLVPYKELDAW FTKCVALIPNYETCNGKGAKGFGDFYKSKRKE
[0105] The expression product of the TIM E166D expression DNA sequence (SEQ ID NO:44) is SEQ ID NO:55, which has the following sequence (TEV protease cleavage site is underlined, and the leading 6His tag is apparent):
TABLE-US-00011 MHHHHHHENLYFQGMGGGRKFFVGGNWKMNGDKKSIDGIVDFLSKGDLDP NCEVVVGASPCYLDYSRSKLPANIGVAAQNCYKVAKGAFTGEISPQMIKD VGCEWAILGHSERRNVFGESDELIGEKVAFALESGLKIIPCIGEKLDERE SGKTEEVCFKQLKAISDKVSDWDLVVLAYDPVWAIGTGKTATPAQAQETH LALRKWLKENVSEEVSQKVRILYGGSVSAGNCKELGTQPDIDGFLVGGAS LKPDFVQIINATK
[0106] The expression product of the CSE expression DNA sequence (SEQ ID NO:45) is SEQ ID NO:56, which has the following sequence (TEV protease cleavage site is underlined, and the leading 6His tag is apparent):
TABLE-US-00012 MHHHHHHENLYFQGMSTYRDNDPHYATHAIHVGQNPEQWKSLAVVPHITL STTYKQYHPGQPKEFEYGRGGNPTRNILETCMASLDGAKHCVTFASGLAA LDAMTTILSCGDHIVAMNDLYGGTNRFLRRVSAKQGLTSTFVDINHEELF SASFQDNTKMVWIESPTNPTLRIVDIKKAVSIAKSKNPNIIVVVDNTFVT SYFQRPLELGADVTYYSCTKYMNGHSDVIMGAVCINSDEIHERVRFVQYA VGAVPSPFDCFLVNRSLKTLKVRMVEHQKNALIVGKFLEGHSKITKVIHP GLPSHPDHEIVKKQQYGHSGMVSFYLKGGLEESNNFLKAVKVFILAESLG GFESLAELPYSMTHASVAEEERVALGVTNNLIRLSIGLENADDLCADLDQ ALNIACS
[0107] The expression product of the Prx-2 expression DNA sequence (SEQ ID NO:46) is SEQ ID NO:57, which has the following sequence (TEV protease cleavage site is underlined, and the leading 6His tag is apparent):
TABLE-US-00013 MHHHHHHENLYFQGMSLQPTNDAPQFKAMAVVNKEFKEVSLKDYTGKYVV LFFYPLDFTFVCPTEIIAFGDRAADFRKIGCEVLACSTDSHFSHLHWINT PRKEGGLGDMDIPLIADKNMEISRAYGVLKEDDGVSFRGLFIIDGTQKLR QITINDLPVGRCVDETLRLVQAFQYTDVHGEVCPAGWKPGKKSMKPSKEG VSSYLADAEQSKK
[0108] The expression product of the FBP aldolase expression DNA sequence (SEQ ID NO:47) is SEQ ID NO:58, which has the following sequence (TEV protease cleavage site is underlined, and the leading 6His tag is apparent):
TABLE-US-00014 MHHHHHHENLYFQGMGLEGIVPPGVITGDNLIKLFEYCRDHKVALPAFNC TSSSTINAVLQAARDIKSPVIVQFSNGGAAFMAGKGIKNDGQKASVLGAI AGAQHVRLMAKHYGVPVVLHSDHCAKKLLPWFDGMLEADEEYFKQNGEPL FSSHMLDLSEEFDEENISTCAKYFTRMTKMKMWLEMEIGITGGEEDGVDN TNVKAESLYTKPEQVYNVYKTLSEIGPMFSIAAAFGNVHGVYKAGNVVLS PHLLADHQKYIKEQINSPLDKPAFLVMHGGSGSTREEIAEAVSNGVIKMN IDTDTQWAYWDGLRKFYEEKKEYLQGQVGNPEGADKPNKKFYDPRVWVRA AEESMIKRANESFESLNAVNVLGDSWKH
[0109] The expression product of the Enolase expression DNA sequence (SEQ ID NO:48) is SEQ ID NO:59, which has the following sequence (TEV protease cleavage site is underlined, and the leading 6His tag is apparent):
TABLE-US-00015 MHHHHHHENLYFQGMPIKHIHARQIYDSRGNPTVEVDLTTERGIFRAAVP SGASTGVHEALELRDKDSTWHGKSGLKAVKNVNDVLGPELVKKNLDPVKQ EEIDDFMISLDGTDNKSKFGANSILGISMAVCKAGAAHKGVPLYRHIADL AGVKEVMMPVPAFNVINGGSHAGNKLAMQEFMILPTGAPSFTEAMRMGSE IYHHLKALIKKKYGLDATAVGDEGGFAPNFQANGEAIDLLVGAIEKAGYT GKIKIGMDVAASEFYKNGKYDLDFKNEESKEADWLTSEALGEMYKGFIKD APVISIEDPYDQDDWEGWTALTSQTDIQIVGDDLTVTNPKRIQMAVDKKS CNCLLLKVNQIGSVTESIRAHNLAKSNGWGTMVSHRSGETEDCFIADLVV GLCTGQIKTGAPCRSERLSKYNQLLRIEEELGSNAKYVGDKFRMPF
[0110] The expression product of the TCTP expression DNA sequence (SEQ ID NO:49) is SEQ ID NO:60, which has the following sequence (TEV protease cleavage site is underlined, and the leading 6His tag is apparent):
TABLE-US-00016 MHHHHHHENLYFQGMKIFKDVFSGDELFSDTYKFKLLDDCLYEVYGKYVT RTEGDVVLDGANASAEEAMDDCDSSSTSGVDVVLNHRLVETGFGSKKDYT VYLKDYMKKVVTYLEENGKQAEVDTFKTNINKVMKELLPRFKDLQFYTGE TMDPEAMIIMLEYKEVDGKDIPVLYFFKHGLNEEKF
[0111] The expression product of the TIM expression DNA sequence (SEQ ID NO:50) is SEQ ID NO:61, which has the following sequence (TEV protease cleavage site is underlined, and the leading 6His tag is apparent):
TABLE-US-00017 MHHHHHHENLYFQGMGGGRKFFVGGNWKMNGDKKSIDGIVDFLSKGDLDP NCEVVVGASPCYLDYSRSKLPANIGVAAQNCYKVAKGAFTGEISPQMIKD VGCEWAILGHSERRNVFGESDELIGEKVAFALESGLKIIPCIGEKLDERE SGKTEEVCFKQLKAISDKVSDWDLVVLAYEPVWAIGTGKTATPAQAQETH LALRKWLKENVSEEVSQKVRILYGGSVSAGNCKELGTQPDIDGFLVGGAS LKPDFVQIINATK
[0112] The expression products were typically applied as antigens. Antigens may also be applied after 6His tag removal using TEV protease. Thus, the antigens may have a leading G residue. The variants of SEQ ID NOs:27 to 31 produced by TEV protease cleavage or as defined by SEQ ID NOs:1-6 are considered to achieve substantially the same result in substantially the same way as SEQ ID NOs:27 to 31 and as defined by SEQ ID NOs:1-6 with a leading G residue. Polynucleotide antigens encoding the same proteins are also considered to achieve substantially the same result in substantially the same way as their polynucleotide variants.
[0113] Thus, the presence or absence of a His tag or an equivalent standard tag and the present or absence of a TEV cleavage site, an equivalent cleavage site or the post-cleavage remnants thereof, are not considered to affect the antigenic properties of the protein or polynucleotide antigens.
[0114] For DNA vaccine production, each of the five antigens were cloned into the pVAX1™ plasmid vector (Invitrogen). A 3 kb vector was designed to promote high-copy number replication in E. coli and high level expression in most mammalian cell lines.
[0115] TIM was additionally cloned into the pVAC1 vector (InvivoGen). pVAC1 is a DNA vector vaccine plasmid designed to stimulate a humoral immune response via intramuscular injection. Antigenic proteins are targeted and anchored to the cell surface by cloning the gene of interest in frame upstream of the C-terminal transmembrane anchoring domain of placental alkaline phosphatase (InvivoGen). The antigenic peptide produced on the surface of muscle cells is believed to be taken up by antigen presenting cells and processed through the major histocompatibility complex class II pathway (InvivoGen).
[0116] The pVAC1-mcs backbone was selected over pVAC2-mcs for cloning because 1) the gene of interest does not contain a signal peptide even though it is secreted in vivo and 2) the vector induces a humoral immune response. The signal sequence IL-2 and the 3′ glycosyl-phosphatidylinositol (GPI) anchoring domain of human placental alkaline phosphatase directs cell surface expression of the antigenic protein (InvivoGen). The 3737 bp vector contains a Zeocin™ resistance gene and was designed for high-copy number replication in E. coli. The EF1-α gene of the pVAC1 vector ensures high levels of expression in skeletal muscle cells and antigen presenting cells. Furthermore, the SV40 enhancer gene heightens the ability of the plasmid to be transported into the nucleus, especially in non-diving cells (InvivoGen).
[0117] The vectors, pVAX1 and pVAC1, are non-fusion vectors, therefore, the inserts needed to include a Kozak translation initiation sequence (e.g. ANNATGG) containing the initiation codon and a stop codon for proper translation and termination of the gene. Primers were designed using SnapGene software to amplify a region that included the restriction enzyme site, the start codon, and the stop codon of the mRNA sequence of our target proteins. The primers are as set out in Table 3. The primers were used to amplify gene products from L. salmonis cDNA via PCR. PCR products of the expected size were PCR or gel purified, digested with the appropriate restriction enzymes, and then PCR purified again.
TABLE-US-00018 TABLE 3 Primers for amplification Pro- tein 5′ forward 3′ reverse FBP GCTATCAAGCTTAAAATGGGTC TCAGATGGATCCTTAGTGTT TTGAAGGAATTGTTC TCCAGGAGTCACCA (SEQ ID NO: 76) (SEQ ID NO: 77) Eno- TATCGCCTGCAGAAAATGCCTA ATCGTAGCGGCCGCTTAAAA lase TTAAACACATTCATGCACGTC GGGCATTCTGAACTTGTC (SEQ ID NO: 74) (SEQ ID NO: 75) TIM TAGCTGGGTACCTTACTTAGTA CGTATCAAGCTTAAAATGGG GCGTTGATGATTTG TGGAGGAAGAAAATTTTTC (SEQ ID NO: 80) (SEQ ID NO: 81) TCTP GTCATTCTGCAGAAAATGAAGA TCAGTAGCGGCCGCTTAAAA TCTTTAAGGACGTAT TTTTTCTTCATTTAATCCATG (SEQ ID NO: 82) (SEQ ID NO: 83) Prx- TCGACGAAGCTTAAAATGAGTC TCGACTGGTACCTTATTTCT 2 TTCAACCAACGAATG TTGATTGTTCAGCATCTGCG (SEQ ID NO: 78) AG (SEQ ID NO: 79)
[0118] Vectors were linearized with the appropriate restriction enzymes for each insert. Linearized vector and insert were ligated with T4 DNA ligase (Invitrogen) and transformed into E. coli Stellar competent cells (Clontech). Transformants were cultured on LB plates containing 50 μg/ml kanamycin overnight at 37° C.
[0119] Single colonies were isolated and cultured overnight in 5 ml LB media+kanamycin (50 μg/ml) at 37° C. with shaking. Glycerol stocks were prepared and stored at −80° C. for each clone. Plasmid DNA was isolated from bacterial lysates using a QIAprep Spin Miniprep Kit (Qiagen) and then digested with the appropriate restriction enzymes and ran on a 1% ethidium bromide gel. Digested clones showing two bands corresponding to the size of the vector and insert were submitted for sequencing using T7 forward and BGH reverse primers (pVAX1 vector) or pVAC1 forward and pVAC1 reverse primers (pVAC1 vector)—see Table 4 for primer sequences.
TABLE-US-00019 TABLE 4 Primers for sequencing Vector 5′ forward 3′ reverse pVAX1 TAATACGACTCACTATAGGG TAGAAGGCACAGTCGAGG (“T7”; SEQ ID NO: 84) (“BGH”; SEQ ID NO: 85) pVAC1 ACTTGGTGGGTGGAGACTGAA AGGCACCACAGACCTTCCAGG GAGT (SEQ ID NO: 86) AT (SEQ ID NO: 87)
[0120] Clones containing inserts that shared high sequence similarity with the target sequence and in the correct orientation were selected for large-scale plasmid isolation. Two different kits were used for large-scale DNA vaccine preparation: Invitrogen's PureLink™ HiPure Expi Megaprep kit and Qiagen's QIAfilter plasmid giga kit. Due to the low plasmid yields obtained from the Invitrogen kit, the Qiagen Giga kit was the preferred method of isolation.
[0121] A 500 ml (PureLink™ kit) or 2.5 L culture (Qiagen Giga kit) was prepared following the manufacturer's instructions. Briefly, glycerol stocks of positive clones were used to streak a LB+kanamycin plate. A single colony was selected to inoculate 5 ml LB media+kanamycin and grown for 8 h at 37° C. with shaking (˜180 rpm). One milliliter was then transferred to 5-500 ml aliquots of LB media+kanamycin and grown overnight (12-14 h) for large-scale plasmid isolation the following day. All steps were performed following the manufacturer's instructions. Plasmid DNA was resuspended in nanopure water and the total amount (mg) of plasmid DNA was quantified using the NanoDrop 8000 Spectrophotometer (Thermo Scientific). Aliquots were prepared and stored at −20° C. As a quality control measure all plasmids were ran on a 1% ethidium bromide gel to check for bacterial contamination and insert. All DNA vaccines were re-sequenced before use in vaccine trial.
Example 3—Immunological Response to Circum-Oral Glands Peptide Recombinant Antigens
[0122] To evaluate the ability of the five candidate sea lice antigens identified in Example 1 to produce an immunological response in Atlantic salmon, the fish were vaccinated with five antigens simultaneously and the systemic antibody titer at 600 degree days after vaccination.
Treatment Groups
[0123] In more detail, Atlantic salmon of around 40 g in weight were divided into five treatment groups, each group consisting of two duplicate tanks of six salmon. The treatment groups were as follows: [0124] 1. pVAX1 vector DNA delivering all five antigens prime (i.m.; 10 μg per antigen) with subsequent i.p. boost of recombinant protein cocktail of all five antigens plus Montanide ISA 763A VG (50 μg per antigen; Delivery Method 1; “DM1”); [0125] 2. Recombinant protein cocktail of all five antigens prime plus (i.d.; 50 μg per antigen) plus flagellin (50 ng) with subsequent i.p. boost of recombinant protein cocktail of all five antigens plus Montanide ISA 763A VG (50 μg per antigen; Delivery Method 2; “DM2”); [0126] 3. Empty pVAX1 vector (i.m.) with subsequent i.p. administration of mCherry-His recombinant protein plus Montanide ISA 763A VG (“DM1 ctrl”); [0127] 4. mCherry-His prime (i.d.; 250 m antigen) plus flagellin (50 ng) with subsequent boost of mCherry-His (i.p.; 250 m) and [0128] 5. No vaccine control (“PBS”).
[0129] Thus, treatment groups 3 and 4 received sham treatments that contained none of the five antigens, and treatment group 5 served as a control for any non-specific immune responses to injury at vaccination of naïve fish.
[0130] The control mCherry recombinant protein was produced using the following mRNA (SEQ ID NO:62):
TABLE-US-00020 ATGCATCATCACCATCACCACGAAAACCTGTATTTTCAGGGCATGGTTTC CAAAGGCGAAGAAGACAATATGGCAATCATCAAAGAATTTATGCGTTTCA AAGTCCACATGGAAGGTTCAGTCAATGGCCATGAATTTGAAATTGAAGGC GAAGGTGAAGGCCGTCCGTATGAAGGTACCCAGACGGCAAAACTGAAAGT CACCAAAGGCGGTCCGCTGCCGTTTGCTTGGGATATTCTGTCACCGCAAT TCATGTATGGTTCGAAAGCGTACGTTAAACACCCGGCCGATATCCCGGAC TACCTGAAACTGAGCTTTCCGGAAGGCTTCAAATGGGAACGTGTTATGAA CTTCGAAGATGGCGGTGTGGTTACCGTCACGCAGGATAGCTCTCTGCAAG ACGGTGAATTCATCTACAAAGTGAAACTGCGCGGTACCAATTTCCCGTCT GATGGCCCGGTTATGCAGAAGAAAACCATGGGCTGGGAAGCGAGTTCCGA ACGTATGTACCCGGAAGACGGTGCCCTGAAAGGCGAAATCAAACAGCGCC TGAAACTGAAAGATGGCGGTCATTATGACGCAGAAGTGAAAACCACGTAC AAAGCTAAAAAACCGGTCCAACTGCCGGGCGCATACAACGTGAACATCAA ACTGGATATCACCAGCCACAACGAAGACTACACGATCGTTGAACAATATG AACGTGCGGAAGGTCGTCACTCTACGGGCGGTATGGATGAACTGTACAAA TAATGA
[0131] The recombinant mCherry protein had the following sequence (SEQ ID NO:63):
TABLE-US-00021 MHHHHHHENLYFQGMVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEG EGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPD YLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPS DGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTY KAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYK
[0132] Thus, mCherry may have the sequence recited above, which has a His tag (HHHHHH; SEQ ID NO:88) and a TEV cleavage site (ENLYFQG; SEQ ID NO:89), a TEV cleaved variant sequence, or another tagged or untagged variant sequence.
[0133] A further 12 Atlantic salmon were held in duplicate tanks of 6 fish each. These fish were acclimatized for 25 days in the system prior to sampling for basal level immune responses of the population prior to vaccination. This group served as a control for basal specific antibody responses to the antigens.
[0134] All the immune sampling (i.e. blood and mucus sampling post-vaccination) occurred at 602 degree days post-priming vaccination, which the period after which you can begin to detect specific antibody titers to the vaccine antigens. Degree day was calculated by multiplying the average temperature by the number of days (DD=((T.sub.0+T.sub.1+ . . . )/no. of days)×no. of days).
Experimental Methods
[0135] Atlantic salmon parr approximately 40 g in weight were obtained from the USDA, Franklin, Me. facility. Fish were maintained in a recirculating fresh water flow through system in 100-gallon tanks at a stocking density of 25 kg/m.sup.3 and were fed at a rate of 1.5% body weight per day. Water quality and fish condition were monitored daily.
[0136] After a 25-day acclimation period, Atlantic salmon parr were vaccinated. Atlantic salmon were anaesthetized prior to tagging and vaccination by netting fish into 100 mg/L of MS222 supplemented with 200 mg/L sodium bicarbonate as a buffer to sustain neutral pH. The fish were tagged with elastomer along the jaw line for ease of identification.
[0137] The fish were primed by intramuscular injection of the vaccine at a dose of 10 μg per antigen per fish (DM1), a cocktail recombinant protein vaccine at a dose of 50 μg per antigen per fish in a total volume of 30 μl in sterile phosphate buffered saline with 50 ng ultrapure flagellin from Pseudomonas aeruginosa (InvivoGen; “DM2”; n=48 fish per treatment group; duplicate tanks of 24 fish per group), or with the control formulation as appropriate. Post-tagging and vaccination the fish were returned to their respective housing tanks and monitored continuously until full recovery.
[0138] Two weeks after prime vaccination, the fish were anaesthetized and received a boost vaccination of recombinant proteins intraperitoneally at a dose of 50 μg per protein per fish, adjuvanted with Montanide™ ISA 763 A VG in a total volume of 100 μl (DM1 and DM2).
[0139] To measure the specific antibody response to louse antigens post-vaccination the blood and mucus of 12 Atlantic salmon per treatment group were sampled at 602-degree days for ELISA and dot blot analysis, respectively. Fish were euthanized with a lethal dose of 250 mg/L MS-222 buffered with 100 mg/L sodium bicarbonate. Blood was collected by bleeding the fish via the caudal vein. Blood samples were incubated at 4° C. overnight and serum was isolated by centrifugation at 3716×g for 10 min at 4° C. Serum was isolated and stored at −80° C. until further use. Skin mucus samples were collected by placing the fish in a bag containing 10 ml phosphate buffered saline and massaging the fish for 2 minute each to wash off mucus. Mucus was centrifuged at 3716×g for 10 minutes at 4° C. and the supernatant transferred into sterile tubes and stored at −80° C.
[0140] The efficacies of the vaccines in eliciting a systemic immune response were evaluated for each vaccine candidate. All ELISA's were optimized prior to running serum samples from each fish. Optimal protein concentration, primary, and secondary antibody concentrations were determined for each antigen by running a checkerboard assay (Table 5).
TABLE-US-00022 TABLE 5 Checkerboard assay results for antibody detection of sea louse antigens. Protein Stock μg/ml Coating Primary Secondary TIM 12600 2 μg/ml 1/500 1/500 FBP 12500 2 μg/ml 1/1000 1/1000 Prx-2 11800 2 μg/ml 1/1000 1/2000 TCTP 17900 2 μg/ml 1/500 1/1000 Enolase 620 2 μg/ml 1/500 1/2000
[0141] One hundred microliters of antigen (2 μg per well in carbonate:bicarbonate coating buffer; Sigma) was coated onto the wells of a 96-well polystyrene microtiter plate (Thermo Scientific). Plates were washed with low salt wash buffer (3×) and then blocked overnight at 4° C. with 3% (w/v) casein in deionized water. After three more washes with low salt wash buffer, serum dilutions (1/100) in PBS were added to each well and allowed to incubate overnight at 4° C. (100 μl per well). Plates were washed 5× with high salt wash buffer to remove residual serum and unbound antibodies. Primary antibody, mouse anti salmonid Ig monoclonal (Biorad; cat #MCA2182), was diluted to the appropriate concentration in PBS (Table 5) and added to each well (100 μl/well) and incubated at room temperature for 1 h. Plates were washed with high salt wash buffer (5×) to remove unbound antibody. The secondary antibody, goat anti-mouse IgG peroxidase (Sigma; cat #A4416), was diluted to the appropriate concentration with conjugate buffer (1% (w/v) bovine serum albumin diluted in low salt wash buffer) and added to the wells. After a 1 hr incubation at room temperature followed by 5× wash with high salt wash buffer, 100 μl of the chromogen (TMB) was added to each well and incubated for 10 min at room temperature. The reaction was stopped by adding 50 μl 2 M sulfuric acid to each well. Plates were mixed and the absorbance was recorded at 450 nm using a spectrophotometer. Each plate contained relevant controls: 1) pooled positive serum, 2) pooled negative serum, and 3) no serum controls (PBS). The coefficient of variation of the A450 nm of sample replicates within a plate, and the pooled positive serum between plates was always <20%.
Results
[0142] At 602 degree days after vaccination, Atlantic salmon serum antibody levels were measured to the five sea louse antigens included in the vaccine. ELISA analysis data showed Atlantic salmon responded to all five antigens delivered in the cocktail vaccine with a DNA prime (
[0143] An immunological response was also induced by prime vaccination with 10 μg TIM DNA antigen either in a pVAX1 vector or a pVAC1 vector, following by a boost using 50 μg of TIM recombinant protein.
[0144] Thus, TIM, FBP, Prx-2, TCTP and Enolase each provides an antigen that elicits an immunogenic response in fish.
Example 4—Efficacy of Sea Lice Vaccine Candidates
[0145] The efficacy of sea lice vaccine candidates against Lepeophtheirus salmonis (salmon louse) infection in Atlantic salmon (Salmo salar) was evaluated.
[0146] The specific antibody response was measured across 12 treatments (n=15 fish per treatment). Controls included a control for the His-tag as well as a no injection control (phosphate buffered saline [PBS]). The His-tag control served as a control for the His tag on the bacterially expressed sea louse antigens. PBS served as a control for any non-specific immune responses to injury at vaccination and to allow for the evaluation of sea lice settlement of non-vaccinated fish. An additional 42 fish per treatment were vaccinated and sampled to measure vaccine efficacy post sea lice challenge.
Treatments:
[0147] Vaccine 1: enolase native (SEQ ID NO:9) [0148] Vaccine 2: CSE native (SEQ ID NO:6) [0149] Vaccine 3: TIM E166D (SEQ ID NO:5) [0150] Vaccine 4: Prx-2 native (SEQ ID NO:7) [0151] Vaccine 5: FBP N286D (SEQ ID NO:1) [0152] Vaccine 6: TIM native (SEQ ID NO:12) [0153] Vaccine 7: PPIase native (SEQ ID NO:3) [0154] Vaccine 8: FBP native (SEQ ID NO:8) [0155] Vaccine 9: GST native (SEQ ID NO:4) [0156] Vaccine 10: TCTP native (SEQ ID NO:11) [0157] Vaccine 11: GST S67A (SEQ ID NO:2) [0158] Vaccine 12: vehicle control (phosphate buffered saline; PBS)
[0159] For the prime vaccination, each recombinant protein vaccine contained 100 ng of purified flagellin from Pseudomonas aeruginosa (FLA-PA Ultrapure, InvivoGen) and was adjuvanted (Montanide™ ISA 763 A VG; Seppic™). For the boost vaccination, each vaccine formulation was adjuvanted (Montanide™ ISA 763 A VG; Seppic™)
Vaccine Production
[0160] Recombinant protein vaccines were prepared by inoculating lysogenic broth (LB)-kanamycin (50 μg) agar plates with glycerol stocks of E. coli BL21 (DE3) cells, which contain the pET-30a (+) expression plasmid (Novagen) with gene insert, and growing each vaccine candidate overnight at 37° C. Single colonies were isolated and used to inoculate 2-50 ml flasks of LB with kanamycin (50 μg). Cultures were allowed to grow at 37° C. with shaking for 2-4 hours or until the optical density at 600 nm was reached (0.6 to 0.8). Approximately 16.6 ml of culture media was added to 500 ml of LB with kanamycin (50 μg) in a flask for overnight growth at 200 rpm and 37° C. Once target optical densities were reached (i.e. 0.6 to 0.8), IPTG was added at 1 mM dose to each 500 ml flask and temperature was reduced to 18° C. with shaking at 200 rpm. After 15-18 hr of induction, the optical density was measured (target optical densities of 1-7) and cultures were centrifuged at 10,000×g for 10 min at 4° C. The weight of each pellet was measured in each centrifugation bottle. Based on that weight, the amount of lysis buffer was calculated (2 ml of lysis buffer per 100 mg of cell pellet), and pellets were resuspended with vortexing. DNase was added (2 U per ml of lysis buffer) to each bottle and mixed gently. Pellets were sonicated on ice in 20 second bursts for a total of 4 min and then incubated on ice for 15 min with intermittent mixing followed by centrifugation for 20 min at 10,000×g at 4° C. The supernatant was decanted and added to a nickel-iminodiacetic acid-based protein purification resin (His60 Ni Superflow Resin; Takara), and allowed to incubate for 2 to 24 hours with gently stirring at 4° C.
[0161] Some proteins (e.g. Prx-2 and GST) were shown to have a high affinity for the resin and therefore lower incubation times were preferred (˜2 h). Lower affinity proteins (e.g. FBP and TCTP) were allowed to mix with the resin for at least 24 h. Resin and supernatant (˜250-300 ml) was added to 4-10 ml polypropylene gravity flow purification columns (Thermo Scientific, catalog #29924). Once the resin settled to the bottom of the column, 10 ml of equilibration buffer was added (×2). This was followed by 10 ml of wash buffer (×2). The protein was eluted from the column by adding multiple 10 ml aliquots of elution buffer until protein detection by 280 nm light absorbance was negligible. For high affinity proteins, elution buffer containing 400 mM imidazole was added. For lower affinity proteins, 300 mM imidazole elution buffer was used. The eluate for each protein was combined and concentrated using 20 ml, 5 kDa, MWCO concentrators (GE Healthcare catalog #28-9329-59). Excess imidazole was removed by adding concentrates to PD-10 desalting columns (GE Healthcare). Protein was concentrated briefly again and then filter sterilized with 0.22 μM, 13 mm diameter, PVDF syringe filters (Celltreat® catalog #229742). A sterile 80% glycerol solution was added to each protein aliquot to give 8-10% glycerol per tube prior to storage at −80° C. (Acros Organics CAS 56-81-5). Protein concentration was determined using a Pierce® BCA Protein Assay Kit (Thermo Scientific catalog #23227). Proteins that were difficult to express at the quantities required (e.g. enolase) were produced by enhanced methods known to the skilled person (GenScript® protein expression service).
Atlantic Salmon
[0162] Atlantic salmon post smolts (n=684) approximately 70 to 100 grams in weight were maintained in a recirculating artificial salt water system on a 12:12 hr light:dark cycle in 100-gallon tanks at a stocking density of 25 kg/m.sup.3. Water quality, ammonia, nitrite, and fish condition were monitored daily. Salmon were fed a daily ration of BioTrout 3 mm pellets (Bio-Oregon®) at 1.5% body weight per day and maintained at temperatures of 13±1° C., 32±±1‰ salinity, and 8±1 mg/L dissolved oxygen (means±standard deviations).
Fish Vaccination
[0163] Fish size ranged from 98 to 295 grams at prime vaccination (average size 180 g). There were two vaccine treatments per tank (n=19 fish per antigen) in replicates of three tanks (n=38 fish per tank). During the vaccination phase, fish stocking density was <18.1 kg/m.sup.3. Prior to vaccination, 20 fish were euthanized for mucus and blood collection with a lethal dose of MS-222 (250 mg/L). These fish served as a measure of the basal level of immunity of the fish. Fish to be vaccinated were anaesthetized with 100 mg/L MS-222 and then primed intradermally using a sterile 25-gauge needle and syringe. A 200 μg dose was prepared for the following recombinant proteins: enolase, Prx-2, TIM, FBP and TCTP (n=57 fish per treatment). The number of injections per antigen ranged between two to three 10-0 injections per fish to achieve the target dose. For the PBS control, a single 10 μl dose was injected into each fish (n=57). To distinguish fish between vaccine groups, an elastomer tag (Northwest Marine Technology, Inc.) was injected under the skin along the jawline following the intradermal injection of antigen. Each recombinant protein vaccine contained 100 ng of FLA-PA Ultrapure flagellin from P. aeruginosa (InvivoGen cat #tlrl-pafla). Each vaccine formulation was adjuvanted with Montanide™ ISA 763 A VG (Seppic™). Once primed, fish were returned to their respective treatment tanks to recover.
[0164] Two weeks post-prime vaccination, fish were anesthetized and boosted with an intraperitoneal (i.p.) injection of the recombinant protein vaccines, except for Prx-2 proteins, which was boosted 3 weeks and 4 days post-prime vaccination (n=11). One hundred microliters of a 200 μg dose was prepared for the following recombinant proteins: enolase, Prx-2, TIM, FBP and TCTP (n=57 fish per treatment). Each vaccine formulation was adjuvanted with Montanide™ ISA 763 A VG (Seppic™ Lot #36017Z). One hundred microliters of antigen at the described doses (above) plus Montanide was i.p. injected into each fish. For the PBS control, 100 μl PBS was added with adjuvant. Once boosted, fish were returned to their respective treatment tanks to recover.
[0165] At least three weeks prior to sea lice challenge, Atlantic salmon approximately 240 g in size were cohabitated into eight replicate tanks. Around 5 fish per treatment were transferred into each tank giving a total of 65 fish per tank or a stalking density of 41.3 kg/m.sup.3.
Serum and Mucus Collection
[0166] At 602 degree days, 43 days after boost vaccination and 588 degree days (42 days after boost), 15 fish per treatment were euthanized by exposing fish to an overdose of M-S222 (250 mg/L) to measure specific antibody responses after vaccination (n=180 fish). Serum was collected by bleeding the fish via the caudal vein using a sterile 23-gauge needle with a 3 ml syringe. Samples were processed by incubating samples at 4° C. overnight and then centrifuging the blood at 3000×g for 10 min at 4° C. The supernatant containing the plasma was collected and transferred into 2-1.5 ml microcentrifuge tubes and stored at −80° C. for ELISA analysis. Skin mucus samples were collected by placing each fish into a bag containing 10 ml phosphate buffered saline and massaging the fish for 2 minute each to wash off mucus. Samples were centrifuged for 15 minutes at 1500×g at 4° C. Mucus was transferred into two 1.5 ml microcentrifuge tubes and stored at −80° C. for dot blot analysis.
L. salmonis Challenge
[0167] Two thousand L. salmonis egg strings were collected from gravid females and transferred to a sea lice hatchery. L. salmonis copepodids of similar age (3-4 days old) were pooled and the number of copepodids were calculated by counting ten 1-ml aliquots of lice using a dissecting scope to give the mean number of copepodids per ml of seawater. Infections were performed by reducing the volume of the tank holding the fish to a third of the original volume and copepodids were added to each of the replicate tanks to give an infection density of 80 copepodids per fish. The dissolved oxygen was monitored continuously throughout the 1-hour bath infection to maintain dissolved oxygen at 8.5±1.0 mg/L (means±standard deviation). After one hour, the tank water level was restored. Dissolved oxygen was monitored for another 1.5 hours before turning the flow back on to each tank. Fish were monitored for an additional hour to ensure dissolved oxygen and flow rate were maintained in each tank at the appropriate levels.
[0168] To evaluate vaccine efficacy against salmon louse attachment, Atlantic salmon (n=42 fish per treatment; n=504) were challenged with L. salmonis copepodids 980 or 994 degree days after boost vaccination). Eight to eleven days after sea lice challenge, the salmon were exposed to an overdose of MS-222 to perform sea lice counts. Blind counts of the chalimus stages were recorded from the skin and gills of each fish for each treatment using a dissecting microscope and forceps. To reduce count variation, the same four individuals manually counted the number of lice on each fish. After counts were completed, the length (mm) and weight (g) of each fish was recorded.
Data Analysis
[0169] The relative intensity (RI), which is the total number of lice per gram body weight [RI=total number lice/total weight (g)], was calculated for each individual fish subject to a vaccine treatment (Myksvoll et al., 2018). The RI values between vaccine treatments were compared. The average relative intensity (ARI=average number of lice/average weight [g]) was calculated to determine the percent change in lice intensity between vaccinated treatments and the PBS control (Myksvoll et al., 2018). Using these values, the % change was calculated (ARI PBS control−ARI vaccine antigen)/(ARI PBS control)×100).
Results
[0170] The data from the sea lice vaccine trial showed that vaccination with recombinant protein antigens identified from the circum-oral glands of the chalimus stages reduced the number of chalimus per fish caused by the sea lice challenge.
[0171] FBP N286D and GST S67A were shown to be the most protective of the tested antigens, as shown in the RI values reported in Table 6 and
TABLE-US-00023 TABLE 6 Mean relative intensity of sea lice post vaccination and challenge with L. salmonis. Antigen RI (mean ± SEM) PBS control 0.164 ± 0.016 FBP N286D 0.106 ± 0.011 GST S67A 0.107 ± 0.009 Prx-2 0.114 ± 0.012 PPIase 0.114 ± 0.009 FBP 0.117 ± 0.014 GST 0.143 ± 0.013 TIM E116D 0.122 ± 0.010 TCTP 0.125 ± 0.010 Enolase 0.132 ± 0.013 TIM 0.135 ± 0.010 CSE 0.150 ± 0.015
[0172] The percent reduction in chalimus counts ranged from 9.1% to 33.0% (Table 7).
TABLE-US-00024 TABLE 7 Percent reduction of L. salmonis chalimus stages after vaccination. Antigen Reduction (%) FBP N286D 33.0 GST S67A 31.1 Prx-2 28.9 PPIase 27.5 FBP 25.1 GST 20.9 TIM E116D 20.6 TCTP 19.1 Enolase 17.9 TIM 13.1 CSE 9.1
[0173] Atlantic salmon were vaccinated with 11 different L. salmonis candidate antigens and challenged with the infective stage of the parasite. Using the average relative intensity, the percent change between the PBS control and candidate vaccine was calculated.
[0174] The antigens had no negative effect on the growth of the vaccinated fish.
[0175] Thus, vaccination with the L. salmonis antigens identified from the circum-oral glands of the chalimus stages reduced the relative intensity of chalimus infestation on Atlantic salmon.
[0176] The immunogenicity of the candidate antigens was assessed by western blot. Data showed that the pooled serum samples from vaccinated and sea lice challenged fish contained antibodies to the sea lice vaccine antigens. Protein bands of the correct sizes were detected on the nitrocellulose membrane after development (FBP, 42.1 kDa; TCTP, 21.6 kDa; enolase, 48.9 kDa; TIM, 28.7 kDa; Prx-2, 24.0; PPIasse, 24.6 kDa; CSE, 45.2 kDa; GST 26.2, kDa; GST S67A, 26.2 kDa; FBP N2867D, 42.1 kDa; and TIM E166D, 28.7 kDa). These results suggest that the monovalent recombinant protein vaccines, Prx-2, FBP, Enolase, TIM, TIM E166D, FBP N286D, GST, GST S67A, PPIase, CSE, and TCTP induced an antibody response in the host. Furthermore, the results show that the antigenic response to the vaccines by the host was protective upon secondary challenge with sea lice.
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