Patent application title: YEAST STRAIN FOR THE PRODUCTION OF PROTEINS WITH TERMINAL ALPHA-1,3-LINKED GALACTOSE
Inventors:
Natarajan Sethuraman (Hanover, NH, US)
Natarajan Sethuraman (Hanover, NH, US)
Robert C. Davidson (Enfield, NH, US)
Robert C. Davidson (Enfield, NH, US)
Terrance A. Stadheim (Lyme, FR)
Stefan Wildt (Lebanon, NH, US)
IPC8 Class: AA61K3900FI
USPC Class:
4241841
Class name: Drug, bio-affecting and body treating compositions antigen, epitope, or other immunospecific immunoeffector (e.g., immunospecific vaccine, immunospecific stimulator of cell-mediated immunity, immunospecific tolerogen, immunospecific immunosuppressor, etc.)
Publication date: 2011-04-14
Patent application number: 20110086054
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Patent application title: YEAST STRAIN FOR THE PRODUCTION OF PROTEINS WITH TERMINAL ALPHA-1,3-LINKED GALACTOSE
Inventors:
Stefan Wildt
Robert C. Davidson
Natarajan Sethuraman
Terrance A. Stadheim
Agents:
Assignees:
Origin: ,
IPC8 Class: AA61K3900FI
USPC Class:
Publication date: 04/14/2011
Patent application number: 20110086054
Abstract:
Lower eukaryotic host cells have been engineered to produce glycoprotein
having at least one terminal α-galactosyl epitope. The
glycoproteins are useful for the production of highly antigenic
glycoprotein compositions with advantages for the production of vaccines.Claims:
1. A yeast or filamentous fungus host cell that can produce recombinant
glycoproteins having N-glycans that have at least one terminal
α-galactosyl epitope.
2. The host cell of claim 1 wherein said host cell is impaired in initiating α-1,6 mannosyltransferase activity with respect to the glycan on the glycoprotein.
3. The host cell of claim 1 wherein the host cell is diminished or depleted in dolichy-P-Man:Man5GlcNAc2-PP-dolichyl α-1,3 mannosyltransferase activity.
4. The host cell of claim 1 wherein said host cell expresses a mannosidase activity selected from the group consisting of an α-1,2 mannosidase I activity, mannosidase II activity, mannosidase IIx activity and class III mannosidase activity.
5. The host cell of claim 1 wherein said host cell expresses an N-acetyl glucosamine transferase I (GnT) activity a β-galactosyl transferase (β-GalT) activity, and a UDP-galactose-4 epimerase activity.
6. The host cell of claim 1 wherein the host cell is selected from the group consisting of Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia minuta (Ogataea minuts, Pichia lindneri), Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica, Pichia sp., Saccharomyces cerevisiae, Saccharomyces sp., Hansenula polymorpha, Kluyveromyces sp., Kluyveromyces lactis, Candida albicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporium luchnowense, Fusarium sp., Fursarium gramineum, Fusarium venenatum, Physcomitrella patens and Neurospora crassa.
7. The host of claim 1 wherein the host expresses a fusion protein comprising a catalytic domain of an α-galactosyltransferase linked to a targeting peptide that targets the fusion protein to the ER or Golgi of the host cell.
8. The host cell of claim 1, wherein the host cell produce glycoproteins having a N-glycan comprising a terminal α-galactosyl epitope, wherein the predominant N-glycan has a structure selected from the group consisting of: (α-Gal)(β-Gal)GlcNAcMan5GlcNAc2; (α-Gal)(β-Gal)2GlcNAc2Man3GlcNAc2; and (α-Gal)2(β-Gal)2GlcNAc2Man3GlcNAc2.
9. The host cell of claim 1, wherein the host cell has been engineered to produce a vaccine protein.
10. The host cell of claim 9, wherein the vaccine protein is selected from the group consisting of an anti-viral vaccine glycoprotein, an anti-bacterial vaccine glycoprotein and an anti-cancer vaccine glycoprotein.
11. A method for producing a host cell capable of producing N-glycans that have terminal α-galactosyl residues, comprising: (a) providing a recombinant yeast or filamentous fungus host cell capable of producing N-glycans that have at least one terminal α-galactose on the non-reducing end of the N-glycan; and (b) introducing a nucleic acid encoding an fusion protein comprising the catalytic domain of an α-galactosyltransferase linked to a targeting peptide that targets the fusion protein to the ER or Golgi of the host cell into the host cell to provide the host cell producing N-glycans that have terminal α-galactosyl residues.
12. (canceled)
13. The host cell of claim 1, wherein the host cell produces human-like glycoproteins, the host cell comprising a nucleic acid encoding a fusion protein capable of transferring an α-galactose residue onto a terminal β-galactose residue of an N-linked oligosaccharide branch of an N-glycan having a trimannose core of a glycoprotein produced by the host cell, wherein the N-linked oligosaccharide branch has a structure selected from the group consisting of: Galβ1,4-GlcNAcβ1,2-Manα1,3; Galβ1,4-GlcNAcβ1,4-Manα1,3; Galβ1,4-GlcNAcβ1,2-Manα1,6; Galβ1,4-GlcNAcβ1,4 Manα1,6; and Galβ1,4-GlcNacβ1,6-Manα1,6
14. A recombinant glycoprotein produced by a yeast or filamentous fungal host cell, wherein the N-glycans of said glycoprotein comprises a predominant N-glycan comprising a terminal α-galactosyl epitope.
15. The recombinant glycoprotein of claim 14, wherein the predominant N-glycan has a structure selected from the group consisting of (α-Gal)(β-Gal)GlcNAcMan5GlcNAc2; (α-Gal)(β-Gal)2GlcNAc2Man3GlcNAc2; and (α-Gal)2(β-Gal)2GlcNAc2Man3GlcNAc2.
16. A vaccine composition comprising the glycoprotein of claim 14.
17. The vaccine composition of claim 16, wherein the predominant N-glycan is selected from the group consisting of (α-Gal)(β-Gal)GlcNAcMan5GlcNAc2; (α-Gal)(β-Gal)2GlcNAc2Man3GlcNAc2; and (α-Gal)2(β-Gal)2GlcNAc2Man3GlcNAc2.
Description:
FIELD OF THE INVENTION
[0001] The present invention relates to the field of molecular biology, in particular the invention is concerned with yeast strains genetically engineered to produce N-glycans with the predominant terminal sugar structure Gal-α1,3-Gal-β1,4-GlcNAc-R
BACKGROUND OF THE INVENTION
[0002] All mammals except humans and certain other primates contain glycoproteins that have terminal alpha-1,3-galactosyl (alpha-gal) glycan structures resulting from the activity of the enzyme alpha-1,3-galactosyl transferase (Galili et al, J Biol Chem, 263:33, 1988). The enzyme adds a galactose residue to terminally located beta-1,4-linked galactose residues. Beta-1,4-linked galactose residues are found at the termini of many N-glycans of mammals, including those of humans.
[0003] The human immune system has adapted natural immunity to quickly respond to the presence of terminal alpha-gal residues. Approximately one percent of circulating IgGs are directed against the alpha-1,3-galactose epitope (Galili et al, Blood, 82:8, 1993). Antigens that exhibit this epitope are recognized by circulating antibodies, resulting in complement activation, and the efficient activation of antigen-presenting cells via an Fcγ receptor-mediated pathway and the stimulation of a cytotoxic T-cell response. Targeting an immune complex to antigen presenting cells has been shown to reduce the amount of antigen required to elicit a T-cell response.
[0004] Current recombinant vaccines generally suffer from a lack of specific immunogenic response. Furthermore, current vaccines often require general immune stimulators known as adjuvants to elicit a sustained cytotoxic T-cell response. In a limited demonstration, a protein-based vaccine with N-glycans containing terminal α-1,3-galactose has been reported to improve the immunogenicity of such a molecule. (Abdel-Motal et al, J Virology, 80:14, 2006; Abdel-Motal et al, J Virology, 81:17, 2007.) This may be because humans have a high level of circulating antibodies directed against α-1,3-galactose residues. Moreover, because these proteins may be able to stimulate antigen presentation via antibody-directed Fc-gamma mediated signaling, they may reduce the need for non-specific adjuvants. The current state of the art only allows for production of such a vaccine by producing the protein with terminal sialic acid structures (e.g. NANA), then removing the NANA residues in vitro by enzymatic digest to expose the β-1,4 linked galactose residues, and subsequently adding terminal α-1,3-galactose through enzymatic addition (see, Galili, U.S. Pat. No. 6,361,775). This process is expensive, cumbersome, and not easily scalable. Attempts have been made to produce alpha-gal epitopes on viral proteins such as influenza virus hemagglutinin; Henion et al, Vaccine, 15:1174-1182 (1997); gp120; Abdel-Motal et al., J. Virology, 80:6943-6951 (2006); and on cancer cells; Unfer et al., Cancer Res., 63:987-993 (2003).
SUMMARY OF THE INVENTION
[0005] Accordingly, one aim of the present invention is the development of further protein expression systems for yeasts and filamentous fungi, such as Pichia pastoris, based on improved vectors and host cell lines for the production of effective protein-based vaccines with increased immunogenicity.
[0006] The present invention provides improved methods and materials for the production of such vaccines using genetically engineered host strains of yeast and filamentous fungi. The host strains have been genetically modified for the production of proteins having human like N-glycosylation.
[0007] The present invention can be used to improve the current state of the art of protein-based vaccines. A glycosylated protein vaccine produced in the genetically engineered strains described herein can be expected to elicit an elevated immune response through improved antigen presentation compared to a vaccine with terminal β-1,4 galactose, terminal sialic acid, or terminal mannose. This technology is applicable to any number of vaccines that can be developed as recombinant protein-based molecules.
[0008] Recent developments allow the production of fully humanized therapeutics in lower eukaryotic host organisms, yeast and filamentous fungi, such as Pichia pastoris. Gerngross, U.S. Pat. No. 7,029,872, the disclosure of which is hereby incorporated by reference. The present inventors have developed further modifications to produce host organisms which can be used for commercial scale production of vaccines in which the protein produced contains at least one terminal α-1,3-galactose glycoform.
[0009] The present inventors have found that vaccine glycoproteins having terminal α-1,3-galactose can be obtained from recombinant host cells by modifying the glycosylation machinery present in the cells. The inventors surprisingly found that beneficial results were obtained by replacing the host cell's endogenous genes encoding glycosylation proteins, with heterologous genes encoding glycosylation enzymes such that N-glycans having terminal α-1,3-galactose residues are present on the antigenic glycoprotein produced by the host cell.
[0010] In preferred embodiments of the present invention, one can modify the genome of lower eukaryotic cells, such as yeast and filamentous fungi, for example, Pichia pastoris. The resulting transformed lower eukaryotic host cell is able to produce antigenic vaccine glycoproteins with terminal α-1,3-galactose residues on a sufficiently high proportion of N-glycans such that the antigenicity/immunogenicity of the resulting vaccine glycoproteins is improved. The present invention has additional advantages in that lower eukaryotic host cells, such as Pichia pastoris, are able to produce vaccine glycoproteins at high yield, with the predominant species of glycoprotein having terminal α-1,3-galactose residues with improved antigenicity compared to production of the vaccine protein in lower eukaryotic host cells retaining their endogenous glycosylation machinery or other standard protein production hosts such as insect cells or mammalian cell lines like CHO or NS0.
[0011] In a particular embodiment, a codon optimized (for P. pastoris) open reading frame encoding an S. scrofa alpha-1,3-galactosyl transferase (Genebank: P50127) is engineered into a P. pastoris yeast strain. In a preferred embodiment, the enzyme is engineered to be specifically localized to the yeast Golgi to optimize its activity. Particular targeting sequences can be chosen by screening fusion protein constructs for the most active fusion proteins. In an exemplified embodiment, the S. scrofa alpha-1,3-galatosyl transferase is fused to a ScMnn2p transmembrane Golgi targeting localization domain.
[0012] Embodiments of this invention include a lower eukaryotic host cell, more preferably a yeast or filamentous fungal host cell, which has been engineered to produce glycoproteins having a predominant N-glycan comprising a terminal α-galactosyl epitope.
[0013] Embodiments of this invention include a lower eukaryotic host cell, more preferably a yeast or filamentous fungal host cell, wherein the host cell produces glycoproteins having an N-glycan comprising a terminal α-galactosyl epitope, wherein the predominant N-glycan is selected from the group consisting of (α-Gal)(β-Gal)GlcNAcMan5GlcNAc2; (α-Gal)(β-Gal)2GlcNAc2Man3GlcNAc2; and (α-Gal)2(β-Gal)2GlcNAc2Man3GlcNAc2.
[0014] Embodiments of this invention include a lower eukaryotic host cell, more preferably a yeast or filamentous fungal host cell, wherein the host cell has been engineered to produce a vaccine protein.
[0015] Embodiments of this invention include a lower eukaryotic host cell, more preferably a yeast or filamentous fungal host cell, wherein the vaccine glycoprotein is derived from an anti-viral vaccine protein, an anti-bacterial vaccine protein or an anti-cancer vaccine protein.
[0016] Embodiments of this invention include a lower eukaryotic host cell, more preferably a yeast or filamentous fungal host cell, wherein the anti-viral vaccine protein is from an influenza virus. More preferably, the influenza proteins are hemagglutinin (HA) or neuraminidase (NA) glycoproteins.
[0017] Embodiments of this invention include a lower eukaryotic host cell, more preferably a yeast or filamentous fungal host cell, wherein the anti-viral vaccine protein is from a herpes simplex virus. More preferably, herpes envelope glycoproteins gB, gC, and gD are used.
[0018] Embodiments of this invention include a lower eukaryotic host cell, more preferably a yeast or filamentous fungal host cell, wherein the anti-viral vaccine protein is from respiratory syncytial virus (RSV). More preferably viral vaccine glycoproteins are selected from the group consisting of RSV hemagglutinin glycoprotein (H); and fusion glycoprotein F.
[0019] Embodiments of this invention include a lower eukaryotic host cell, more preferably a yeast or filamentous fungal host cell, wherein the anti-bacterial vaccine glycoprotein is derived from a bacterial vaccine protein. More preferably the bacterial vaccine protein is an integral membrane protein, or is an outer membrane protein, or is an outer surface protein, or is from Mycobacterium, or is from Salmonella, or is from Borrelia, or is from Haemophilus.
[0020] Embodiments of this invention include a lower eukaryotic host cell, more preferably a yeast or filamentous fungal host cell, wherein the anti-cancer vaccine glycoprotein is derived from an epitopic peptide derived from a cancer cell such as a Her-2 epitope, a neu epitope or a prostate stem cell antigen (PSCA) epitope.
BRIEF DESCRIPTION OF THE FIGURES
[0021] FIG. 1. A comparison of the N-glycosylation machinery between yeast and mammals. Both produce a Man8GlcNAc2 precursor following protein folding and ER maturation. N-glycosylation pathways differ in the Golgi with mammals trimming mannose and adding additional sugars such as GlcNAc and galactose to produce complex N-glycans. In contrast, yeast add additional mannose with various linkages, including an outer chain, which can be comprised of dozens of mannose residues.
[0022] FIG. 2. Comparison of the terminal sugars found on N-glycans between humans and most other mammals. Most mammals exhibit a minor percentage of terminal α-1,3-linked galactose, a structure completely lacking from the N-glycans of humans.
[0023] FIG. 3. Stepwise modification of the yeast N-glycosylation machinery. Humanization of yeast N-linked glycans results in a series of yeast strains capable of producing human/mammalian intermediate N-glycan structures. A yeast strain capable of producing such a GS5.0 intermediate N-glycan, (β-Gal)2GlcNAc2Man3GlcNAc2, can be modified to produce a uniform GS5.9 N-glycan, (α-Gal)2(β-Gal)2GlcNAc2Man3GlcNAc2, by engineering of an active, properly localized α-GalT in the absence of a sialyl transferase.
[0024] FIGS. 4A & 4B. A GFI5.0 glycoengineered yeast strain expressing α-GalT yields N-glycans with terminal α-gal. Here, a non-optimized mouse α-1,3-GalT was engineered into a non-optimized GFI5.0 humanized P. pastoris yeast strain. This strain expresses the Kringle 3 domain of human plasminogen under control of the strong, inducible AOX1 promoter. Secreted K3 protein was produced by induction in methanol-containing medium, purified by Ni++ affinity chromatography and N-glycans were released by PNGase F digestion and subjected to MALDI-TOF MS. The α-GalT activity can be observed by the appearance of α-Gal containing peaks (GS5.8 and GS5.9) in a MALDI-TOF MS. 2.0, Man5GlcNAc2; 5.0, (β-Gal)2GlcNAc2Man3GlcNAc2; 5.8, (α-Gal)(β-Gal)2GlcNAc2Man3GlcNAc2; 5.9, (α-Gal)2(β-Gal)2GlcNAc2Man3GlcNAc2.
[0025] FIGS. 5A & 5B. Optimization of the upstream glycosylation machinery in a GFI5.0 glycoengineered yeast strain expressing α-GalT increases the uniformity of the N-glycans. Here a mouse α-1,3-GalT, not codon optimized or localization optimized, was engineered into an optimized GFI5.0 humanized P. pastoris yeast strain. This strain expresses rat recombinant EPO under control of the strong AOX1 promoter. Secreted His-tagged rrEPO protein was produced by induction in methanol containing medium, purified by Ni++ affinity chromatography and N-glycans were released by PNGase F digestion and subjected to MALDI-TOF MS. Active α-GalT can be observed by the appearance of α-Gal containing peaks (GS5.8 and GS5.9) in a MALDI-TOF MS. 2.0, Man5GlcNAc2; 5.0, (β-Gal)2GlcNAc2Man3GlcNAc2; 5.8, (α-Gal)(β-Gal)2GlcNAc2Man3GlcNAc2; 5.9, (α-Gal)2(β-Gal)2GlcNAc2Man3GlcNAc2.
[0026] FIGS. 6A & 6B. Comparison of α-1,3-galactosyl transferase proteins reveals a high degree of similarity. Bos taurus, Sus scrofa, Mus musculus, Canis familiaris α-GalT protein sequences were compared by performing ClustalV analysis using Lasergene (DNAstar, Madison, Wis.).
[0027] FIGS. 7A-7C. Expression of a library of α-GalTs and codon optimization for P. pastoris improves α-Gal transfer. Here codon optimized, but not localization optimized, M. musculus α-1,3-GalT, S. scrofa α-1,3-GalT, C. familiaris α-1,3-GalT were engineered into an optimized GFI5.0 humanized P. pastoris yeast strain. This strain expresses rrEPO under control of the strong, inducible AOX1 promoter. Secreted His-tagged rrEPO protein was produced by induction in methanol containing medium, purified by Ni++ affinity chromatography and N-glycans were released by PNGase F digestion and subjected to MALDI-TOF MS. The α-GalT activity can be observed by the appearance of α-Gal containing peaks (GS5.8 and GS5.9) in a MALDI-TOF MS. 2.0, Man5GlcNAc2; 5.0, (β-Gal)2GlcNAc2Man3GlcNAc2; 5.8, (α-Gal)(β-Gal)2GlcNAc2Man3GlcNAc2; 5.9, (α-Gal)2(β-Gal)2GlcNAc2Man3GlcNAc2.
[0028] FIGS. 8A & 8B. Expression of Kringle 3 in a strain with S. scrofa α-1,3-GalT localized with ScMnt11-58 yields similar N-glycans to that observed with rrEPO. Here an ovine α-1,3-GalT, codon optimized, but not localization optimized, was engineered into an optimized GFI5.0 humanized P. pastoris yeast strain. This strain expresses the Kringle 3 domain of human plasminogen under control of the strong, inducible AOX1 promoter. Secreted His-tagged K3 protein was produced by induction in methanol containing medium, purified by Ni++ affinity chromatography and N-glycans were released by PNGase F digestion and subjected to MALDI-TOF MS. The α-GalT activity can be observed by the appearance of α-Gal containing peaks (GS5.8 and GS5.9) in a MALDI-TOF MS. 2.0, Man5GlcNAc2; 5.0, (β-Gal)2GlcNAc2Man3GlcNAc2; 5.8, (α-Gal)(β-Gal)2GlcNAc2Man3GlcNAc2; 5.9, (α-Gal)2(β-Gal)2GlcNAc2Man3GlcNAc2.
[0029] FIG. 9. Optimization of the leader localization of the α-GalT introduced into a GFI5.0 strain increases the amount of terminal α-gal transferred to N-glycans. Here an ovine α-1,3-GalT, codon optimized, and localization optimized with ScMnn21-36, was engineered into an optimized GFI5.0 humanized P. pastoris yeast strain. This strain expresses the Kringle 3 domain of human plasminogen under control of the strong AOX1 promoter. Secreted His-tagged K3 protein was produced by induction in methanol containing medium, purified by Ni++ affinity chromatography and N-glycans were released by PNGase F digestion and subjected to MALDI-TOF MS. The α-GalT activity can be observed by the appearance of α-Gal containing peaks (GS5.9) in a MALDI-TOF MS. In this mature GFI5.9 strain, GS5.9 can be observed as the predominant N-glycan. 2.0, Man5GlcNAc2; 5.9, (α-Gal)2(β-Gal)2GlcNAc2Man3GlcNAc2.
[0030] FIG. 10. Expression of an Influenza HA ectodomain protein in a GFI5.9 glycoengineered P. pastoris yeast strain. This strain contains ovine α-1,3-GalT, codon optimized, but not localization optimized, engineered into an optimized GFI5.0 humanized P. pastoris yeast strain. Expression of His-tagged Influeza HA (A/Hong Kong/1/58 variant) ectodomain was demonstrated by Western blot analysis of culture supernatant, then Ni++ affinity chromatography purified protein was subjected to PNGase F digestion and MALDI-TOF MS analysis. 2.0, Man5GlcNAc2; 5.0, (β-Gal)2GlcNAc2Man3GlcNAc2; 5.8, (α-Gal)(β-Gal)2GlcNAc2Man3GlcNAc2; 5.9, (α-Gal)2(β-Gal)2GlcNAc2Man3GlcNAc2.
[0031] FIG. 11. Expression of an Influenza HA ectodomain protein in a GFI5.9 glycoengineered P. pastoris yeast strain. This strain contains ovine α-1,3-GalT, codon optimized and localization optimized, engineered into an optimized GFI5.0 humanized P. pastoris yeast strain. Expression of His-tagged Influeza HA (A/South Carolina/1/18 variant) ectodomain was demonstrated by Western blot analysis of culture supernatant, then Ni++ affinity chromatography purified protein was subjected to PNGase F digestion and MALDI-TOF MS analysis. 2.0, Man5GlcNAc2; 5.9, (α-Gal)2(β-Gal)2GlcNAc2Man3GlcNAc2.
[0032] FIG. 12. Expression of HSV-2 gD ectodomain protein in an optimized GFI5.9 glycoengineered P. pastoris yeast strain. This strain contains ovine α-1,3-GalT, codon optimized and localization optimized, engineered into an optimized GFI5.0 humanized P. pastoris yeast strain. Expression of His-tagged HSV-2 gD ectodomain was demonstrated by Western blot analysis of culture supernatant, then Ni++ affinity chromatography purified protein was subjected to PNGase F digestion and MALDI-TOF MS analysis. 2.0, Man5GlcNAc2; 5.0, (β-Gal)2GlcNAc2Man3GlcNAc2; 5.8, (α-Gal)(β-Gal)2GlcNAc2Man3GlcNAc2; 5.9, (α-Gal)2(β-Gal)2GlcNAc2Man3GlcNAc2.
[0033] FIG. 13. Expression of HSV-2 gC ectodomain protein in an optimized GFI5.9 glycoengineered P. pastoris yeast strain. This strain contains ovine α-1,3-GalT, codon optimized and localization optimized, engineered into an optimized GFI5.0 humanized P. pastoris yeast strain. Expression of His-tagged HSV-2 gC ectodomain was demonstrated by Western blot analysis of culture supernatant, then Ni++ affinity chromatography purified protein was subjected to PNGase F digestion and MALDI-TOF MS analysis. 2.0, Man5GlcNAc2; 5.0, (β-Gal)2GlcNAc2Man3GlcNAc2; 5.8, (α-Gal)(β-Gal)2GlcNAc2Man3GlcNAc2; 5.9, (α-Gal)2(β-Gal)2GlcNAc2Man3GlcNAc2.
DESCRIPTION OF THE SEQUENCES
[0034] SEQUENCE ID NOs: 1 and 2 are nucleotide sequences of RCD446 and RCD447, primers for the cloning of murine α-1,3-galactosyl transferase lacking the putative transmembrane Golgi localization domain (MmαGalT).
TABLE-US-00001 RCD446 Primer Sequence ID NO. 1 TTGGCGCGCCAACAGCCCAGACGGCTCTTTCTTG RCD447 Primer Sequence ID NO. 2 GGTTAATTAATCAGACATTATTTCTAACCAAATT
[0035] SEQUENCE ID NO: 3 is an amino acid sequence of the ectodomain (i.e. lacking the C-terminal transmembrane domain) of type H3 HA protein from Influenza A (Hong Kong).
TABLE-US-00002 Sequence ID NO. 3 QDLPGNDNSTATLCLGHHAVPNGTLVKTITDDQIEVTNATELVQSSSTGKICNNPHRILD GIDCTLIDALLGDPHCDVFQNETWDLFVERSKAFSNCYPYDVPDYASLRSLVASSGTLEFI TEGFTWTGVTQNGGSNACKRGPGSGFFSRLNWLTKSGSTYPVLNVTMPNNDNFDKLYI WGVHHPSTNQEQTSLYVQASGRVTVSTRRSQQTIIPNIGSRPWVRGLSSRISIYWTIVKPG DVLVINSNGNLIAPRGYFKMRTGKSSIMRSDAPIDTCISECITPNGSIPNDKPFQNVNKITY GACPKYVKQNTLKLATGMRNVPEKQTRGLFGAIAGFIENGWEGMIDGWYGFRHQNSEG TGQAADLKSTQAAIDQINGKLNRVIEKTNEKFHQIEKEFSEVEGRIQDLEKYVEDTKIDL WSYNAELLVALENQHTIDLTDSEMNKLFEKTRRQLRENAEDMGNGCFKIYHKCDNACIE SIRNGTYDHDVYRDEALNNRFQIKGVELKSGYKDWILWISFAISCFLLCVVLLGFIMWAC QRGNIRCNICIGGGHHHHHHHHH HSV-2 G strain gC protein ectodomain Sequence ID NO. 7 LANASPGRTITVGPRGNASNAAPSASPRNASAPRTTPTPPQPRKATKSKASTAKPAPPPK TGPPKTSSEPVRCNRHDPLARYGSRVQIRCRFPNSTRTEFRLQIWRYATATDAEIGTAPSL EEVMVNVSAPPGGQLVYDSAPNRTDPHVIWAEGAGPGASPRLYSVVGPLGRQRLIIEEL TLETQGMYYWVWGRTDRPSAYGTWVRVRVFRPPSLTIHPHAVLEGQPFKATCTAATYY PGNRAEFVWFEDGRRVFDPAQIHTQTQENPDGFSTVSTVTSAAVGGQGPPRTFTCQLTW HRDSVSFSRRNASGTASVLPRPTITMEFTGDHAVCTAGCVPEGVTFAWFLGDDSSPAEK VAVASQTSCGRPGTATIRSTLPVSYEQTEYICRLAGYPDGIPVLEHHGSHQPPPRDPTERQ VIRAIEGRGGGHHHHHHHHH
[0036] SEQUENCE ID NO: 4 is an amino acid sequence of nucleotide the ectodomain (i.e. lacking the C-terminal transmembrane domain) of type H1 HA protein from Influenza A (A/South Carolina/1/18).
TABLE-US-00003 Sequence ID NO. 4 DTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDSHNGKLCKLKGIAPLQLGKCNIAG WLLGNPECDLLLTASSWSYIVETSNSENGTCYPGDFIDYEELREQLSSVSSFEKFEIFPKTS SWPNHETTKGVTAACSYAGASSFYRNLLWLTKKGSSYPKLSKSYVNNKGKEVLVLWG VHHPPTGTDQQSLYQNADAYVSVGSSKYNRRFTPEIAARPKVRDQAGRMNYYWTLLEP GDTITFEATGNLIAPWYAFALNRGSGSGIITSDAPVHDCNTKCQTPHGAINSSLPFQNIHP VTIGECPKYVRSTKLRMATGLRNIPSIQSRGLFGAIAGFIEGGWTGMIDGWYGYHHQNE QGSGYAADQKSTQNAIDGITNKVNSVIEKMNTQFTAVGKEFNNLERRIENLNKKVDDGF LDIWTYNAELLVLLENERTLDFHDSNVRNLYEKVKSQLKNNAKEIGNGCFEFYHKCDDA CMESVRNGTYDYPKYSEESKLNREEIDGVKLESMGVYQIGGGHHHHHHHHH
[0037] SEQUENCE ID NOS: 5 and 6 are amino acid sequences of two different versions of the HSV-2 G strain gD protein ectodomain.
TABLE-US-00004 HSV-2 G strain gD 339 protein ectodomain Sequence ID NO. 5 KYALADPSLKMADPNRFRGKNLPVLDQLTDPPGVKRVYHIQPSLEDPFQPPSIPITVYYA VLERACRSVLLHAPSEAPQIVRGASDEARKHTYNLTIAWYRMGDNCAIPITVMEYTECP YNKSLGVCPIRTQPRWSYYDSFSAVSEDNLGFLMHAPAFETAGTYLRLVKINDWTEITQF ILEHRARASCKYALPLRIPPAACLTSKAYQQGVTVDSIGMLPRFIPENQRTVALYSLKIAG WHGPKPPYTSTLLPPELSDTTNATQPELVPEDPEDSALLEDPAGTVSSQIPPNWHIPSIQD VAPHHAPAAPSNPGGGHHHHHHHHH HSV-2 G strain gD 306NQ protein ectodomain Sequence ID NO. 6 KYALADPSLKMADPNRFRGKNLPVLDQLTDPPGVKRVYHIQPSLEDPFQPPSIPITVYYA VLERACRSVLLHAPSEAPQIVRGASDEARKHTYNLTIAWYRMGDNCAIPITVMEYTECP YNKSLGVCPIRTQPRWSYYDSFSAVSEDNLGFLMHAPAFETAGTYLRLVKINDWTEITQF ILEHRARASCKYALPLRIPPAACLTSKAYQQGVTVDSIGMLPRFIPENQRTVALYSLKIAG WHGPKPPYTSTLLPPELSDTTNATQPELVPEDPEDSALLEDNQGGGHHHHHHHHH
[0038] SEQUENCE ID NO: 7 is an amino acid sequence of the HSV-2 G strain gC protein ectodomain.
DETAILED DESCRIPTION OF THE INVENTION
[0039] Unless otherwise defined herein, scientific and technical terms and phrases used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include the plural and plural terms shall include the singular. Generally, nomenclatures used in connection with, and techniques of biochemistry, enzymology, molecular and cellular biology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well known and commonly used in the art. The methods and techniques used to make basic genetic constructs of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. See, e.g., Sambrook et al. Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989); Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992, and Supplements to 2002); Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1990); Taylor and Drickamer, Introduction to Glycobiology, Oxford Univ. Press (2003); Worthington Enzyme Manual, Worthington Biochemical Corp., Freehold, N.J.; Handbook of Biochemistry: Section A Proteins, Vol I, CRC Press (1976); Handbook of Biochemistry: Section A Proteins, Vol II, CRC Press (1976); Essentials of Glycobiology, Cold Spring Harbor Laboratory Press (1999).
[0040] All publications, patents and other references mentioned herein are hereby incorporated by reference in their entireties.
[0041] The following terms, unless otherwise indicated, shall be understood to have the following meanings:
[0042] As used herein, the terms "N-glycan" and "glycoform" are used interchangeably and refer to an N-linked oligosaccharide, e.g., an oligosaccharide that is attached by an asparagine-N-acetylglucosamine linkage between and N-acetylglucosamine residue of the oligosaccharide and an asparagine residue of a polypeptide. The predominant sugars found on glycoproteins are glucose (Glc), galactose (Gal), mannose (Man), fucose (Fuc), N-acetylgalactosamine (GalNAc), N-acetylglucosamine (GlcNAc) and sialic acid (e.g., N-acetyl-neuraminic acid (NANA) and N-glycolyl-neuraminic acid (NGNA)). The processing of the sugar groups occurs co-translationally in the lumen of the ER and continues in the Golgi apparatus for N-linked glycoproteins.
[0043] N-glycans have a common pentasaccharide core of Man3GlcNAc2. N-glycans differ with respect to the number of branches (antennae) comprising peripheral sugars (e.g., GlcNAc, galactose, fucose and sialic acid) that are added to the Man3GlcNAc2 ("Man3") core structure which is also referred to as the "trimannose core", the "pentasaccharide core" or the "paucimannose core". N-glycans are classified according to their branched constituents (e.g., high mannose, complex or hybrid). A "high mannose" type N-glycan has five or more mannose residues. A "complex" type N-glycan typically has at least one GlcNAc attached to the α-1,3 mannose arm and at least one GlcNAc attached to the α-1,6 mannose arm of a "trimannose" core. Complex N-glycans may also have galactose ("Gal") or N-acetylgalactosamine ("GalNAc") residues that are optionally modified with sialic acid or derivatives (e.g., "NANA" or "NeuAc", where "Neu" refers to neuraminic acid and "Ac" refers to acetyl). "α-Gal" refers to an α-1,3-linked galactose and "β-Gal" refers to a β-1,4-linked galactose. Complex N-glycans may also have intrachain substitutions comprising "bisecting" GlcNAc and core fucose. Complex N-glycans may also have multiple antennae on the "trimannose core," often referred to as "multiple antennary glycans." A "hybrid" N-glycan has at least one β-GlcNAc attached to the nonreducing end of the α-1,3 mannose arm of the trimannose core and zero or more mannoses on the α-1,6 mannose arm of the trimannose core. The various N-glycans are also referred to as "glycoforms."
[0044] Abbreviations used herein are of common usage in the art, see, e.g., abbreviations of sugars, above. Other common abbreviations include "PNGase", or "glycanase" which all refer to peptide N-glycosidase F (EC 3.2.2.18).
[0045] The term "marker sequence" or "marker gene" refers to a nucleic acid sequence capable of expressing an activity that allows either positive or negative selection for the presence or absence of the sequence within a host cell. For example, the P. pastoris URA5 gene is a marker gene because its presence can be selected for by the ability of cells containing the gene to grow in the absence of uracil. Its presence can also be selected against by the inability of cells containing the gene to grow in the presence of 5-FOA. Marker sequences or genes do not necessarily need to display both positive and negative selectability. Non-limiting examples of marker sequences or genes from P. pastoris include ADE1, ARG4, HIS4 and URA3. For antibiotic resistance marker genes, kanamycin, neomycin, geneticin (or G418), paromomycin and hygromycin resistance genes are commonly used to allow for growth in the presence of these antibiotics.
[0046] "Operatively linked" expression control sequences refers to a linkage in which the expression control sequence is contiguous with the gene of interest to control the gene of interest, as well as expression control sequences that act in trans or at a distance to control the gene of interest.
[0047] The term "expression control sequence" or "regulatory sequences" are used interchangeably and as used herein refer to polynucleotide sequences, which are necessary to affect the expression of coding sequences to which they are operatively linked. Expression control sequences are sequences that control the transcription, post-transcriptional events and translation of nucleic acid sequences. Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (e.g., ribosome binding sites); sequences that enhance protein stability; and when desired, sequences that enhance protein secretion. The nature of such control sequences differs depending upon the host organism; in prokaryotes, such control sequences generally include promoter, ribosomal binding site, and transcription termination sequence. The term "control sequences" is intended to include, at a minimum, all components whose presence is essential for expression, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences.
[0048] The term "recombinant host cell" ("expression host cell", "expression host system", "expression system" or simply "host cell"), as used herein, is intended to refer to a cell into which a recombinant vector has been introduced. It should be understood that such terms are intended to refer not only to the particular subject cell but to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein. A recombinant host cell may be an isolated cell or cell line grown in culture or may be a cell which resides in a living tissue or organism.
[0049] The term "eukaryotic" refers to a nucleated cell or organism, and includes insect cells, plant cells, mammalian cells, animal cells and lower eukaryotic cells.
[0050] The term "lower eukaryotic cells" includes yeast, fungi, collar-flagellates, microsporidia, alveolates (e.g., dinoflagellates), stramenopiles (e.g, brown algae, protozoa), rhodophyta (e.g., red algae), plants (e.g., green algae, plant cells, moss) and other protists. Yeast and filamentous fungi include, but are not limited to: Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia minuta (Ogataea minuta, Pichia lindneri), Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica, Pichia sp., Saccharomyces cerevisiae, Saccharomyces sp., Hansenula polymorphs, Kluyveromyces sp., Kluyveromyces lactis, Candida albicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporium lucknowense, Fusarium sp., Fusarium gramineum, Fusarium venenatum, Physcomitrella patens and Neurospora crassa. Pichia sp., any Saccharomyces sp., Hansenula polymorpha, any Kluyveromyces sp., Candida albicans, any Aspergillus sp., Trichoderma reesei, Chrysosporium lucknowense, any Fusarium sp. and Neurospora crassa.
[0051] The term "peptide" as used herein refers to a short polypeptide, e.g., one that is typically less than about 50 amino acids long and more typically less than about 30 amino acids long. The term as used herein encompasses analogs and mimetics that mimic structural and thus biological function.
[0052] The term "polypeptide" encompasses both naturally-occurring and non-naturally-occurring proteins, and fragments, mutants, derivatives and analogs thereof as indicated by the context of use. A polypeptide may be monomeric or polymeric. Further, a polypeptide may comprise a number of different domains each of which has one or more distinct activities.
[0053] The term "isolated protein" or "isolated polypeptide" is a protein or polypeptide that by virtue of its origin or source of derivation (1) is not associated with naturally associated components that accompany it in its native state, (2) exists in a purity not found in nature, where purity can be adjudged with respect to the presence of other cellular material (e.g., is free of other proteins from the same species) (3) is expressed by a cell from a different species, or (4) does not occur in nature (e.g., it is a fragment of a polypeptide found in nature or it includes amino acid analogs or derivatives not found in nature or linkages other than standard peptide bonds). Thus, a polypeptide that is chemically synthesized or synthesized in a cellular system different from the cell from which it naturally originates will be "isolated" from its naturally associated components. A polypeptide or protein may also be rendered substantially free of naturally associated components by isolation, using protein purification techniques well known in the art. As thus defined, "isolated" does not necessarily require that the protein, polypeptide, peptide or oligopeptide so described has been physically removed from its native environment.
[0054] The term "polypeptide fragment" as used herein refers to a polypeptide that has a deletion, e.g., an amino-terminal and/or carboxy-terminal deletion compared to a full-length polypeptide. In a preferred embodiment, the polypeptide fragment is a contiguous sequence in which the amino acid sequence of the fragment is identical to the corresponding positions in the naturally-occurring sequence. Fragments typically are at least 5, 6, 7, 8, 9 or 10 amino acids long, preferably at least 12, 14, 16 or 18 amino acids long, more preferably at least 20 amino acids long, more preferably at least 25, 30, 35, 40 or 45, amino acids, even more preferably at least 50 or 60 amino acids long, and even more preferably at least 70 amino acids long. A fragment may comprise a domain with a distinctive activity.
[0055] A "modified derivative" refers to polypeptides or fragments thereof that are substantially homologous in primary structural sequence but which include, e.g., in vivo or in vitro chemical and biochemical modifications or which incorporate amino acids that are not found in the native polypeptide. Such modifications include, for example, acetylation, carboxylation, phosphorylation, glycosylation, ubiquitination, labeling, e.g., with radionuclides, and various enzymatic modifications, as will be readily appreciated by those skilled in the art. A variety of methods for labeling polypeptides and of substituents or labels useful for such purposes are well known in the art, and include radioactive isotopes such as 125I, 32P, 35S, and 3H, ligands which bind to labeled antiligands (e.g., antibodies), fluorophores, chemiluminescent agents, enzymes, and antiligands which can serve as specific binding pair members for a labeled ligand. The choice of label depends on the sensitivity required, ease of conjugation with the primer, stability requirements, and available instrumentation. Methods for labeling polypeptides are well known in the art. See, e.g., Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992, and Supplements to 2002) (hereby incorporated by reference).
[0056] The term "chimeric gene" or "chimeric nucleotide sequences" refers to a nucleotide sequence comprising a nucleotide sequence or fragment coupled to one or more heterologous nucleotide sequences. Chimeric sequences are useful for the expression of fusion proteins. Chimeric genes or chimeric nucleotide sequences may also comprise one or more fragments or domains which are heterologous to the intended host cell, and which may have beneficial properties for the production of heterologous recombinant proteins. Generally, a chimeric nucleotide sequence comprises at least 30 contiguous nucleotides from a gene, more preferably at least 60 or 90 or more nucleotides. Chimeric nucleotide sequences which have at least one fragment or domain which is heterologous to the intended host cell, but which is homologous to the intended recombinant protein, have particular utility in the present invention. For example, a chimeric gene intended for use in an expression system using P. pastoris host cells to express recombinant human glycoproteins will preferably have at least one fragment or domain which is of human origin, while the remainder of the chimeric gene will preferably be of P. pastoris origin.
[0057] The term "fusion protein" refers to a polypeptide comprising a polypeptide or fragment coupled to heterologous amino acid sequences. Fusion proteins are useful because they can be constructed to contain two or more desired functional elements from two or more different proteins. A fusion protein comprises at least 10 contiguous amino acids from a polypeptide of interest, more preferably at least 20 or 30 amino acids, even more preferably at least 40, 50 or 60 amino acids, yet more preferably at least 75, 100 or 125 amino acids. Fusions that include the entirety of the proteins of the present invention have particular utility. The heterologous polypeptide included within the fusion protein of the present invention is at least 6 amino acids in length, often at least 8 amino acids in length, and usefully at least 15, 20, and 25 amino acids in length. Fusions also include larger polypeptides, or even entire proteins, such as the green fluorescent protein ("GFP") chromophore-containing proteins having particular utility. Fusion proteins can be produced recombinantly by constructing a nucleic acid sequence which encodes the polypeptide or a fragment thereof in frame with a nucleic acid sequence encoding a different protein or peptide and then expressing the fusion protein. Alternatively, a fusion protein can be produced chemically by crosslinking the polypeptide or a fragment thereof to another protein.
[0058] As used herein, the twenty conventional amino acids and their abbreviations follow conventional usage. See Immunology--A Synthesis (Golub and Gren eds., Sinauer Associates, Sunderland, Mass., 2nd ed. 1991), which is incorporated herein by reference. Stereoisomers (e.g., D-amino acids) of the twenty conventional amino acids, unnatural amino acids such as α-, α-disubstituted amino acids, N-alkyl amino acids, and other unconventional amino acids may also be suitable components for polypeptides of the present invention. Examples of unconventional amino acids include: 4-hydroxyproline, γ-carboxyglutamate, ε-N,N,N-trimethyllysine, ε-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, N-methylarginine, and other similar amino acids and imino acids (e.g., 4-hydroxyproline). In the polypeptide notation used herein, the left-hand end corresponds to the amino terminal end and the right-hand end corresponds to the carboxy-terminal end, in accordance with standard usage and convention.
[0059] The term "region" as used herein refers to a physically contiguous portion of the primary structure of a biomolecule. In the case of proteins, a region is defined by a contiguous portion of the amino acid sequence of that protein.
[0060] The term "domain" as used herein refers to a structure of a biomolecule that contributes to a known or suspected function of the biomolecule. Domains may be co-extensive with regions or portions thereof; domains may also include distinct, non-contiguous regions of a biomolecule.
[0061] As used herein, the term "molecule" means any compound, including, but not limited to, a small molecule, peptide, protein, sugar, nucleotide, nucleic acid, lipid, etc., and such a compound can be natural or synthetic.
[0062] As used herein, the term "comprise" or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.
[0063] As used herein, the term "predominantly" or variations such as "the predominant" or "which is predominant" will be understood to mean the glycan species that has the highest mole percent (%) of total N-glycans that can be identified after the glycoprotein has been treated with PNGase and the released glycans are analyzed by mass spectroscopy, for example, MALDI-TOF MS. In other words, the term "predominant" is defined as an individual entity, such as a specific glycoform, that is present in greater mole percent than any other individual entity. For example, if a composition consists of species A in 40 mole percent, species B in 35 mole percent and species C in 25 mole percent, species A is "predominant" the composition comprises "predominantly" species A.
[0064] As used herein, the term "vaccine protein" or "vaccine glycoprotein" will be understood to mean that the "vaccine protein" or "vaccine glycoprotein" is intended to be utilized as a vaccine, which may be administrable to humans, and which is intended to elicit an immune response to the protein or glycoprotein used as a the "vaccine protein" or "vaccine glycoprotein." The vaccine protein or vaccine glycoprotein may comprise a full-length native protein or glycoprotein, or it may comprise one or more domains isolated from a native protein or glycoprotein. The vaccine protein or vaccine glycoprotein may be utilized in a vaccine together with other vaccine proteins or vaccine glycoproteins, as well as in formulations comprising additional active agents and/or pharmaceutical carriers. The term "vaccine protein" or "vaccine glycoprotein" may also be used interchangeably with the term "target protein" or "target glycoprotein."
[0065] As used herein the term "epitope" refers to the portion of an antigen that is capable of eliciting an immune response or capable of being recognized by an antibody. Epitopes frequently consist of a conjunction of multiple amino acids, carbohydrate moiety(ies) or both. Epitopes that are referred to as linear frequently do not depend on proper folding of a protein. Epitopes that depend on the proper folding of a protein are referred to as conformational because the epitope is only present when the protein is in its properly folded conformation.
[0066] As used herein, the term "α-galactosyl epitope" means a terminal galactose residue linked α-1,3 to a second galactose residue, the second galactose residue being linked β-1,4 to an N-acetyl glucosamine residue. Examples of glycans with an α-galactosyl epitope include the following branched glycan structures (α-Gal)(β-Gal)GlcNAcMan5GlcNAc2; (α-Gal)(β-Gal)2GlcNAc2Man3GlcNAc2; and (α-Gal)2(β-Gal)2GlcNAc2Man3GlcNAc2. See also GS5.7, GS5.8 and GS5.9 in FIG. 3. As used herein the term "adjuvant" refers to a compound or substance capable of increasing the immunogenic response to a vaccine or vaccine protein without having any specific antigenic effect itself. Adjuvants can include bacterial lipopolysaccharide, liposomes, aluminum salts and oils.
[0067] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can also be used in the practice of the present invention and will be apparent to those of skill in the art. All publications and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. The materials, methods, and examples are illustrative only and not intended to be limiting in any manner.
I. General
[0068] The invention provides methods and materials for the expression of vaccine glycoproteins having α-linked galactose from recombinant host cells, which recombinant host cells have been transformed with vectors encoding vaccine proteins. The vaccine glycoproteins result in improved quality of the recombinant glycoprotein produced from the host cell, particularly increased immune response obtained from vaccine glycoproteins produced in the recombinant host cells transformed with these vectors.
[0069] Although the methods are exemplified with respect to expression in lower eukaryotic organisms, particularly yeast, they can also be practiced in higher eukaryotic organisms and bacteria. The methods involve transforming host cells with a nucleic acid molecule which encodes an improved vaccine protein, especially a vaccine glycoprotein, and thereby, when the host cells are transformed with an expression vector encoding a secreted glycoprotein, the α-linked galactose may contributes to improved quality of the recombinant secreted glycoprotein, particularly, increased immune response to the produced antigenic glycoprotein.
[0070] Accordingly, in preferred embodiments, the methods of the present invention may be used in recombinant expression systems using cells which have been engineered for production of the improved secreted vaccine glycoproteins.
II. Expression of Vaccine Proteins
Nucleic Acid Encoding the Vaccine Protein or Glycoprotein
[0071] Glycoproteins described above are encoded by nucleic acids. The nucleic acids can be DNA or RNA, typically DNA. The nucleic acid encoding the glycoprotein is operably linked to regulatory sequences that allow expression and secretion of the glycoprotein. Such regulatory sequences include a promoter and optionally an enhancer upstream, or 5', to the nucleic acid encoding the protein and a transcription termination site 3' or downstream from the nucleic acid encoding the glycoprotein. The glycoprotein can be a fusion protein. For secreted glycoproteins, the nucleic acid typically includes a leader sequence encoding a leader or signal peptide. The leader or signal peptide is responsible for targeting the protein to the appropriate cellular compartments of the secretory pathway for glycosylation and secretion, typically the endoplasmic reticulum or the Golgi apparatus. The nucleic acid also typically encodes a 5' untranslated region having a ribosome binding site and a 3' untranslated region. The nucleic acid is often a component of a vector replicable in cells in which the glycoprotein is expressed. The vector can also contain a marker to allow recognition of transformed cells. However, some cell types, particularly yeast, can be successfully transformed with a nucleic acid lacking extraneous vector sequences.
[0072] Nucleic acids encoding desired glycoproteins can be obtained from several sources. cDNA sequences can be amplified from cell lines known to express the glycoprotein using primers to conserved regions (see, e.g., Marks et al., J. Mol. Biol. 581-596 (1991)). Nucleic acids can also be synthesized de novo based on sequences in the scientific literature. Nucleic acids can also be synthesized by extension of overlapping oligonucleotides spanning a desired sequence (see, e.g., Caldas et al., Protein Engineering, 13, 353-360 (2000)). If desired, nucleic acid sequences can be codon-optimized according to preferred codon usage tables to improve expression in the host cell of the invention (see, e.g., Chang et al., J. Agric. Food Chem. 54:815-822 (2006). Production of active glycoproteins may require proper folding of the protein when it is produced and secreted by the cells. However such folding may not be essential in order to use the protein as a vaccine protein, for example, when the epitopes of interest are linear. The presence of terminal alpha-1,3-galactose residues may be required for immunogenicity of a vaccine glycoprotein, or may enhance the immunogenicity of the vaccine glycoproteins being produced.
III. Viral and Other Vaccine Targets
[0073] Vaccine targets which may be appropriate for the present invention include those to which an antigenic response may be boosted by attachment of an alpha-gal moiety to the end of an antigen. In preferred embodiments, the vaccine protein will comprise a peptide or protein derived from the target. The target is preferably a disease vector, such as a virus, fungus, bacteria, or other microbe. In other embodiments, the target may be a particular type of cell, such as a cancer cell, which expresses a known epitopic peptide which can be used as the target protein. For example, preferred epitopic peptides may be selected from the group consisting of a Her-2 epitope, a neu epitope or a prostate stem cell antigen (PSCA) epitope.
[0074] Preferred viral targets include those viruses known to cause diseases in humans and/or animals, such as influenza viruses (e.g., influenza A, B or HN51); herpes simplex viruses (HSV, e.g., HSV-1 and HSV-2, which is associated with genital herpes); hepatitis viruses, respiratory syncytial virus (RSV), parainfluenza viruses (PIV, e.g., PIV-type III), human papillomaviruses (HPV, e.g., HPV-16, which may be associated with cervical cancer), Epstein-Barr virus, adenoviruses, human immunodeficiency viruses (HIV, which is associated with AIDS), and human cytomegalovirus (CMV), paramoxyviruses (such as those associated with mumps and measles), poxviruses, Reoviridae, (including rotaviruses, reovirus and Colorado Tick Fever virus) and polioviruses.
[0075] The preferred target proteins for viruses include capsid proteins, surface coat proteins, structural proteins, and other proteins which may be accessible to the immune system, especially those proteins which are naturally glycosylated. One or more additional glycosylation sites may be engineered into the DNA sequence encoding the target protein. Where there is no natural glycosylation site, one or more artificial glycosylation sites may be engineered into a DNA sequence encoding the target protein. DNA sequences encoding the target protein may be cloned or synthesized, and may be optimized for expression in the host cells of the present invention.
[0076] The vaccine protein useful in the present invention may comprise all or fragments of one or more of the following viral proteins. One skilled in the art may locate or identify additional suitable viral proteins by reference to the continually expanding viral genomic databases, such as the website of the International Committee on Taxonomy of Viruses: The Universal Virus Database, version 3; http://www.ncbi.nlm.nih.gov/ICTVdb/ICTVdB/ and http://www.virology.net/garryfavwebindex.html#specific.
[0077] In preferred embodiments, one or more viral proteins or glycoproteins may be used together as part of a compound vaccine. The compound vaccine may comprise multiple vaccine proteins from the same target virus, as well as multiple vaccine proteins from distinct target viruses. The following are non-limiting examples of the target viruses and viral proteins which may serve as the vaccine protein in the present invention:
[0078] Influenza Viruses (hemagglutinin (HA) glycoprotein; neuraminidase (NA) glycoprotein; matrix protein; M1 protein; M2 protein; M3 protein; nucleoprotein (NP) peptide; PA protein; PB1 protein and PB2 protein; Rev protein. See McCauley and Mahy, Biochem. J., 211:281-294 (1983); Lamb et al., Proc Natl Acad Sci USA., 78:4170-4174 (1981)).
[0079] Herpesvirus (including Herpes Simplex Viruses and Cytomegaloviruses): Envelope glycoproteins: glycoprotein B (also called VP7); glycoprotein C/VP7.5; glycoprotein D/VP17/18; envelope glycoprotein E; envelope glycoprotein G; envelope glycoprotein H; envelope glycoprotein I; envelope glycoprotein J; envelope glycoprotein K; envelope glycoprotein L; envelope glycoprotein M; envelope glycoprotein N; small capsid protein; tegument proteins: VP11/12; VP13/14; VP22; and VP-5/ICP5; VP19c; VP23; VP26; ICP1-2; ICP35; ICP47. See, Roizman, Proc Natl Acad Sci USA 93: 11307-11312 (1996); Whitley and Roizman, J. Clin. Invest. 110(2): 145-151 (2002); Dunn et al., Proc Natl Acad Sci USA, 100:14223-14228 (2003). Herpes viruses also include Varicella Zoster (cause of chicken pox); and Epstein-Barr virus.
[0080] Flavivirus (which can include Dengue virus; West Nile virus; hepatitis viruses; and GB viruses); Dengue: Protein C; Protein M; Protein E. See, Chambers et al., Annual Review of Microbiology; 44: 649-688 (1990). Hepatitis viruses: (including GB Virus) surface antigens, such as HBsAg; p17e/HBeAg; HBe1; HBe2; p21c/HBcAg; P39/GP42; p24/GP27; GP33/GP36; preS1; pre-S2; S protein; p74 (NS3); p68 (NS5B); p20 (core protein); HDAg-p24; HDAg-p27. See Sells et al., PNAS USA 84:1005-1009 (1987); Heermann et al., J. Virology 52:396-402 (1984); Svitkin et al., J. Virology; 79:6868-6881 (2005); Salfeld et al., J. Virology 63:798-808 (1989); Grottola et al., Liver Transpl. 8:443-448 (2002); Casey et al., PNAS, USA; 90:9016-9020 (1993). GB Virus: envelope proteins: E1 glycoprotein; E2. See: Muerhoff et al., J. Virology 71:6501-6508; Bukh et al., WO2000/075337.
[0081] Paramyxoviridae/Paramoxyvirus; including: Parainfluenzaviruses (PIV), such as human PIV-1 (Respirovirus) and human PIV-2 and -4 (Rubulavirus); Pneumoviruses such as respiratory synctial virus (RSV); Morbilliviruses, such as Mumps Virus and Measles Virus; Henipavirus, such as Hendravirus and Nipahvirus; and Metapneumoviruses. Typically, the genome includes genes encoding both a nucleocapsid (N) protein; and a nucleocapsid phosphoprotein (P), both of which may serve as the viral vaccine protein. See, Collins, U.S. Pat. No. 6,264,957; certain of the paramyxoviruses, also contain a neuraminidase (NA) protein which can serve as the viral vaccine protein in the present invention. The virus frequently also carries hemagglutinin (H) and fusion (F) glycoproteins on its surface, which can comprise viral vaccine proteins.
[0082] Human papillomaviruses (HPV, e.g., HPV-16, which may be associated with cervical cancer), capsid proteins encoded by L1 and L2; E7 antigenic peptide. See Carter et al., J. Virol. 80: 4664-72 (2006); Pastrana et al., Virology 337: 365-72 (2005).
[0083] Filoviruses (including Ebola virus). VP30; VP35; Polymerase L protein; VP40; VP24; and GP/SGP glycoprotein. See Folks, Nature Medicine 4:16-17 (1998); and Feldmann, Virus Research 24:1-19 (1992).
[0084] Adenoviruses: Protein II (Hexon monomer S); Capsid Proteins S (III) and IIIa (Penton Base Proteins); Fiber Protein IV; Proteins VI, VIII, and IX (Hexon Minor Polypeptides). See, Russell, J. General Virology 81:2573-2604 (2000); Roulston et al, Annu Rev Microbiol 53:577-628 (1999); and Yewdell and Bennink; Annu Rev Cell Dev Biol. 15:579-606 (1999).
[0085] Lentiviruses, such as Human immunodeficiency viruses (associated with acquired immunodeficiency syndrome (AIDS)): Envelope glycoproteins (GP120 and GP41); viral capsid protein (p24); Nucleocapsid proteins (p6 and p7); and matrix protein (p17); HIV regulatory proteins (Tat, Rev, Nef). See, Wain-Hobson, S., 1989. HIV genome variability in vivo. AIDS 3: supp 1; 13-9 (1989); and Ratner et al., Nature 313:277-84 (1985).
[0086] Bunyaviridae (including Hantavirus) envelope glycoproteins (glycoproteins GP1; GP2); nucleocapsid proteins (protein. N). See Schmaljohn et al., J. Gen. Virol. 69:1949-1955 (1988).
[0087] Nidoviruses (Coronaviruses and Arteriviruses): (including viruses associated with severe acute respiratory syndrome (SARS)). S (spike glycoprotein); E (envelope protein); M (Membrane glycoprotein); and HE (Haemagglutinin-esterase); N (phosphoprotein). See, Pyre et al., Virology J., http://www.virologyj.com/content/1/1/7 (2004).
[0088] Reoviridae, (including Rotaviruses, reovirus and Colorado Tick Fever virus): σ1, σ2, and σ3; μ1c; VP1, VP2, VP3, VP4, VP6 and VP7). See Joklik, Microbiological Reviews, 45:483-501 (1981).
[0089] Picornaviridae, including Polioviruses: Viral capsid proteins: VP1, VP2; VP3 and VP4. See, Koch and Koch, The Molecular Biology of Poliovirus. Springer-Verlag/Wein (NY, 1985).
IV. Bacterial and Other Vaccine Targets
[0090] Preferred bacterial targets in the present invention include those known to cause diseases in humans and/or animals, such as Mycobacterium (Tuberculosis; leprosy; chronic infections); Haemophilus influenzae (Respiratory infections; meningitis; conjunctivitis; chancroid); Mycoplasma (Atypical pneumonia; urogenital infections); Bacillus (Anthrax; food poisoning); Salmonella (Typhoid fever; enteritis; food poisoning); Clostridium (Tetanus; botulism; gas gangrene; bacteremia); Treponema (syphilis); Borrelia (Relapsing fever; Lyme disease); Ureaplasma (Opportunistic urogenital infections); Staphylococcus (Skin abscesses; opportunistic infections); Streptococcus (Strep throat and other respiratory infections; skin and other abscesses; puerperal fever; opportunistic infections); Leptospira (Leptospirosis); Campylobacter (Urogenital/digestive tract infections); Heliobacter (Peptic ulcers); Pseudomonas (Urinary tract infections; burns; wounds); Legionella (Pneumonia; respiratory infections); Neisseria (Gonorrhea; meningitis; nasopharyngeal infections); Moraxella (Conjunctivitis); Brucella (Brucellosis); Bordetella (Whooping cough); Francisella (Tularemia); Escherichia (Opportunistic infections of colon and other sites); Shigella (Bacterial dysentery); Klebsiella (Respiratory and urinary tract infections); Enterobacter (Opportunistic infections); Serratia (Opportunistic infections); Proteus (Urinary tract infections); Providencia (Wound and burn infections; urinary tract infections); Morganella (Summer diarrhea; opportunistic infections); Yersinia (Plague; mesenteric lymphadenitis; septicemia); Vibrio (Cholera; acute gastroenteritis); Pasteurella (Infections associated with cat- and dog-bite wounds); Calymmatobacterium (Granuloma inguinale); Gardnerella (Vaginitis); Eikenella (Wound infections); Streptobacillus (Infections associated with rat-bites); Bacteroides/fusobacterium (Oral, digestic, respiratory and urogenital infections; wounds and abscesses); Veillonella (oral microbiota and abscesses); Rickettsia (Typhus; Rocky mountain spotted fever; rickettsialpox); Rochalimaea (Trench fever); Coxiella (Q fever); Bartonella (Oroya fever); Chlamydia (Trachoma; inclusion conjunctivitis; non-gonococcal urethritis; parrot fever); Peptococcus (Postpartum septicemia; visceral lesions); Peptostreptococcus (Puerperal fever; pyogenic infections); Lactobacillus (Microflora of digestive tract and vagina); Listeria (Listeriosis); Erysipelothrix (Erysipeloid); Corynebacterium (Diphtheria and skin opportunists); Propionibacterium (Wound infections and diseases); Eubacterium (Oral and other infections); Actinomyces (Actinomycoses); Nocardia (Nocardiosis; mycetoma; abscesses); and Dermatophilus (Skin lesions).
[0091] The preferred target proteins for bacteria include membrane proteins, structural proteins, and other proteins which may be accessible to the immune system, especially those proteins which are naturally glycosylated. One or more additional glycosylation sites may be engineered into the DNA sequence encoding the target protein. Where there is no natural glycosylation site, one or more artificial glycosylation sites may be engineered into a DNA sequence encoding the target protein. The target may also comprise a peptide fused to a polysaccharide or lipid, wherein the polysaccharide or lipid may optimally be derived from a polysaccharide or lipid which naturally surrounds or encapsulates the target bacteria. DNA sequences encoding the target protein may be cloned or synthesized, and may be optimized for expression in the host cells of the present invention.
[0092] The vaccine protein useful in the present invention may comprise all or fragments of the target bacterial protein or proteins. One skilled in the art may locate or identify additional suitable bacterial proteins by reference to the continually expanding bacterial genomic databases, such as those available at the website of the Sanger Center (http://www.sanger.ac.uk/Projects/Microbes/).
[0093] In preferred embodiments, one or more bacterial proteins or glycoproteins may be used together as part of a compound vaccine. The compound vaccine may comprise multiple vaccine proteins from the same target bacterium, as well as multiple vaccine proteins from distinct target bacteria, as well as vaccine proteins from target viruses or other epitopic peptides, such as epitopes to cancer cells. The following are non-limiting examples of target bacteria and bacterial proteins, which may be used as the vaccine protein in the present invention.
[0094] Salmonella (Typhoid fever; enteritis; food poisoning). Envelope proteins (envA; envD; envZ/ompB/tppA/tppB) Outer membrane proteins (ompA; ompC; ompD; ompF; ompH; ompR; pefC; pss; rck; spvA; tctA; tctB); Outer membrane porin protein (nmpC); Outer membrane protease E (pgtE); Outer membrane phospholipase A (pldA); Phosphate limitation-inducible outer membrane pore protein (phoE); Spermidine and putrecine transporter (potA); membrane-bound acyl amino acid esterase (apeE); Membrane-bound sensor (arcB); membrane-bound attachment site (atdA). See: Wu et al, J. Bacteriol. 187:4720-4727 (2005); see also: www.salmonell.org/genomics/.
[0095] Mycobacterium (Tuberculosis; leprosy; chronic infections) Integral membrane proteins (amt; arsA; arsB1; arsB2; arsC; betP; chaA; cysT; cysW; dppB; dppC; drrB; drrC). See Camus et al., Microbiology 148:2967-2973 (2002; Cole et al. Nature 393:537-544 (1998).
[0096] Haemophilus influenzae (Respiratory infections; meningitis; conjunctivitis; chancroid). Outer membrane proteins P1; P2 (b/c); P4(e); P5 (d); P6 (PAL; protein g); PCP; OMP26; D15; transferring binding proteins (Tbp); heme:hemopexin binding protein (HxuA). Vaccine proteins may be linked to lipooligosaccharides (LOS) to increase the antigenicity of the vaccine protein. see Foxwell et al., Microbiol Mol Biol Rev, 62:294-308 (1998); GTP-binding protein (lepA); outer membrane receptor-mediated transport energizer protein (TonB); protein-export membrane proteins (SecD: SecF); Hap and HWM1/HMW2 adhesive proteins; IgA1 protease. See: http://cmr.tigr.org/tigr-scripts/CMR/shared/AllGeneList.cgi?sub_org_val=g- hi&feat_type=ORF; Webster et al., J. Histochem Cytochem 54:829-842 (2006); See Fleischmann and Adams, Science, 269:496-512 (1995); Berenson et al., Infection and Immunity, 73:2728-2735 (2005); Green et al., Infect Immun 59:3191-3198 (1991).
[0097] Mycoplasma (Atypical pneumonia; urogenital infections) membrane proteins p52, p67 (pMGA) and p77; Jan et al., Protein Expression and Purification; 7:160-166 (1996); Lipid-associated membrane proteins (LAMPs); See Lo et al., Clinical Infectious Diseases, 36:1246-53 (2003).
[0098] Treponema (syphilis): membrane protein (tmpA); Tp33 protein; membrane antigen, pathogen-specific (tpd); basic membrane protein (tpn39b); outer membrane proteins (tpn50; tmpB; ompH); membrane lipoproteins (tmpC); lipoproteins (tpp15; tpp17; tpn32); flagellar hook protein flgE; flagellar hook-basal body complex protein fliE; flagellar basal body rod proteins flgB; flgC; flgF; flgG; flagellar basal body rod modification protein flgD; flagellar P-ring protein flgI; flagellar protein flgJ; flagellar hook-associated proteins flgK and flgL; flagellar M-ring protein fliF; flagellar protein fliJ. See, McKevitt et al., Infection and Immunity, 73:4445-4450 (2005).
[0099] Borrelia (Relapsing fever; Lyme disease): Outer surface proteins (OspA; OspB; OspC). See, Anderton et al., Infection and Immunity, 72:2035-2044 (2004).
[0100] Brucella (Brucellosis) Outer Membrane protein 31 (Omp31); See Cassataro et al., Infection and Immunity; 73:8079-8088 (2005); Major OMPs Omp25 OMP31 and Omp2b; less abundant OMPs Omp10, Omp16, and Omp19; and smooth lipopolysaccharide (S-LPS). See Cloeckaert et al., Clinical and Diagnostic Laboratory Immunology; 6:627-629 (1999). Acidic-pH-inducible outer membrane protein (Aop). See Brucella: Molecular and Cellular Biology (Lopez-Goni and Ignacio Moriyon, eds.); Horizon Press (2004).
[0101] Streptococcus (Strep throat and other respiratory infections; skin and other abscesses; puerperal fever; opportunistic infections). M1 protein, a collagen-like surface protein; lepA. See Zhang et al., Proteomics; 7:1379-1390 (2007).
[0102] The preferred target vaccine proteins for bacteria may include peptides or proteins which are known to be produced by the bacteria, especially those which may be present on or near the surface of the bacterial outer membrane such that the target peptide or protein may be accessible to antibodies or cytotoxic T-cells which have been adapted to recognize the target peptide or target protein.
V. Host Cells
[0103] Lower eukaryotes such as yeast are preferred for expression of glycoproteins because they can be economically cultured, give high yields, and when appropriately modified are capable of suitable glycosylation. Yeast particularly offers established genetics allowing for rapid transformations, tested protein localization strategies and facile gene knock-out techniques. Suitable vectors have expression control sequences, such as promoters, including 3-phosphoglycerate kinase or other glycolytic enzymes, and an origin of replication, termination sequences and the like as desired.
[0104] Various yeasts, such as K. lactis, Pichia pastoris, Pichia methanolica, and Hansenula polymorpha are preferred for cell culture because they are able to grow to high cell densities and secrete large quantities of recombinant protein. Likewise, filamentous fungi, such as Aspergillus niger, Fusarium sp, Neurospora crassa and others can be used to produce glycoproteins of the invention at an industrial scale. Other suitable hosts include Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia minuta (Ogataea minuts, Pichia lindneri), Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichia methanalica, Pichia sp., Saccharomyces cerevisiae, Saccharomyces sp., Hansenula sp., Kluyveromyces sp., Candida albicans, Aspergillus nidulans, Aspergillus oryzae, Trichoderma reesei, Chrysosporium luchnowense, Fursarium gramineum, Fusarium venenatum and Physcomitrella patens.
[0105] Lower eukaryotes, particularly yeast and filamentous fungi, can be genetically modified so that they express glycoproteins in which the glycosylation pattern is human-like or humanized. Such can be achieved by eliminating selected endogenous glycosylation enzymes and/or supplying exogenous enzymes as described by Gerngross et al., US 20040018590, the disclosure of which is hereby incorporated herein by reference. For example, a host cell can be selected or engineered to be impaired in initiating α-1,6 mannosyltransferase activity (outer chain initiation) with respect to the glycan on a glycoprotein, or is diminished or depleted in dolichy-P-Man:Man5GlcNAc2-PP-dolichyl α-1,3 mannosyltransferase activity, which would otherwise add mannose residues onto the N-glycan on a glycoprotein. Further, such a host cell, particularly a yeast or filamentous fungal host cell, should express or be engineered to express a mannosidase activity such as an α-1,2 mannosidase I activity, mannosidase II activity, mannosidase IIx activity and class III mannosidase activity. Host cells, particularly yeast and fungal hosts, are also engineered to express an N-acetylglucosamine transferase I (GnT) activity such as GnTI, GnTII, GnTIII, GnTIV, GnTV, GnTVI and GnTIX. Finally, enzymes to generate a pool of UDP-galactose, appropriate Golgi membrane transporters and a gene encoding a β-Galactosyl transferase (e.g, a β-GalT), yield a host strain that is capable of transferring a complex-type human N-glycan with terminal β-1,4-galactose. (Bobrowicz et al., Glycobiology; 14:757-66 (2004)).
VI. Introduction of α-GalT into a Glycoengineered Hybrid N-Glycan Producing Yeast Strain
[0106] A glycoengineered P. pastoris strain that secretes proteins with mammalian hybrid N-glycans containing terminal GlcNAc such as PBP3 is generated by elimination of a portion of a yeast type N-glycosylation (such as PpOCH1) and expressing an α-1,2-MNS1 and GnTI enzymes properly localized in the endoplasmic reticulum (Choi et al, PNAS, 2003; Bobrowicz et al., Glycobiology; 14:757-66 (2004)). Further, a strain that secretes hybrid mammalian N-glycans with terminal β-1,4-galactose, such as RDP39-6, is generated by further expression of hβ-GalTI as well as UDP-Galactose 4-epimerase (Davidson et al., US Patent Application 2006/0040353, the disclosure of which is hereby incorporated herein). Expression of UDP-Gal transporter can further enhance β-galactose transfer (Davidson et al., US Patent Application 2006/0040353). To obtain a terminal α-1,3-galactosyl epitope expressing strain, a further plasmid expressing an α-1,3-galactosyl transferase that is properly targeted to the endoplasmic reticulum is transformed into this β-1,4-galactose terminated hybrid N-glycan producing strain. Transformants are selected on standard yeast selective medium such as that containing Nourseothricin or Hygromycin for which the plasmid contains a resistance gene and correct integrants were screened by yeast cell lysate PCR. Recombinants are then screened for functional expression of properly targeted α-1,3-galactosyl transferase by analyzing N-glycans released by PNGase F digest of the secreted reporter protein such as K3 by Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry (MALDI-TOF MS). Correct addition of α-1,3-galactose to the hybrid β-1,4-galactose terminated acceptor N-glycan is identified by masses that are observed consistent with addition of a single α-linked galactose residue to the GFI3.5 N-glycan (β-Gal)GlcNAcMan5GlcNAc2 of RDP39-6 to yield the GFI5.7 (α-Gal)(β-Gal)GlcNAcMan5GlcNAc2 N-glycan.
VII. Vaccine Compositions
[0107] The lower eukaryotic host cells of the present invention can be used to express vaccine proteins, and in preferred embodiments, vaccine glycoproteins. The vaccine protein or glycoprotein can be formulated with other pharmaceutically acceptable active agents and/or inactive excipients to form vaccine glycoprotein compositions. The vaccine glycoproteins compositions of the present invention comprise an N-glycan comprising a terminal α-galactosyl residue. In certain embodiments, N-glycan comprising a terminal α-galactosyl residue is the predominant N-glycan, In preferred embodiments, the predominant N-glycan comprises at least 30 mole percent, preferably at least 40 mole percent and more preferably at least 50 mole percent of the N-glycans present on the glycoprotein in the composition. In particular preferred embodiments, the predominant N-glycan is selected from the group consisting of (α-Gal)(β-Gal)GlcNAcMan5GlcNAc2; (α-Gal)(β-Gal)2GlcNAc2Man3GlcNAc2; and (α-Gal)2(β-Gal)2GlcNAc2Man3GlcNAc2.
[0108] The vaccine compositions of the present invention preferably comprise vaccine glycoprotein which is of viral or bacterial origin. The vaccine glycoprotein is preferably a glycoprotein which is naturally glycosylated. Alternatively, the vaccine glycoprotein may comprise a glycoprotein that has been synthetically produced, and in particular may comprise a glycoprotein which has been genetically engineered to create one or more N-glycosylation sites not otherwise present in the native protein. In certain embodiments, the vaccine compositions may comprise an epitopic peptide derived from cancer cells. In these embodiments, the vaccine composition may comprise an epitopic glycopeptide sequence derived from an epitopic peptide from a cancer cell or tumor cell antigen. In preferred embodiments, the epitopic peptide is selected from the group consisting of a Her-2 epitope, a neu epitope or a prostate stem cell antigen (PSCA) epitope. In certain preferred embodiments, the vaccine composition may comprise a compound vaccine composition, comprising multiple vaccine proteins or vaccine glycoproteins. In such cases, the vaccine proteins or vaccine glycoproteins may comprise multiple vaccine glycoproteins directed to the same target virus, bacterium or other epitopic peptide; or may comprise vaccine glycoproteins directed to multiple distinct viruses, bacteria and/or epitopic peptides, such as epitopic peptides directed to a cancer cell or tumor cell.
[0109] In the following examples, viral vaccine glycoproteins are expressed in host cells of the species Pichia pastoris. These examples demonstrate the invention with respect to specific preferred embodiments of the invention, and are not limiting in any manner. The skilled artisan, having read the disclosure and examples herein, will recognize that numerous variants, modifications and improvements to the methods and materials described are possible without deviating from the practice of the present invention.
Examples
I. Cloning of Alpha 1,3-Galactose Transferase
[0110] The gene encoding a Mus Musculus α-1,3-galactosyl transferase but lacking the putative transmembrane Golgi localization domain (MmαGalT) was amplified by polymerase chain reaction using mouse Kidney cDNA (Clontech) as a template and primers RCD446 (TTGGCGCGCCAACAGCCCAGACGGCTCTTTCTTG) (SEQ ID NO: 1) and RCD447 (GGTTAATTAATCAGACATTATTTCTAACCAAATT) (SEQ ID NO: 2). The resulting product was cloned into the pCR2.1 Topo vector (Invitrogen), sequenced, and named pRCD680. The MmαGalT gene was digested from pRCD680 using AscI/PacI and cloned into plasmid pRCD508, a pUC19-based plasmid containing the human β-1,4-galactosyl transferase I catalytic domain flanked by AscI/PacI sites (the human domain was excised in the present construct), the 5' 108 nucleotides of the Saccharomyces cerevisiae MNN2 gene, encoding in-frame the N-terminal transmembrane Golgi localization domain, the Pichia pastoris GAPDH promoter and S. cerevisiae CYC1 transcriptional terminator, flanking regions to knock out the P. pastoris ARG1 gene as an integration site, and the P. pastoris HIS1 gene as a selectable marker. This plasmid was named pRCD683 and yields a Pparg1:H1S1 expression construct containing a fusion gene encoding the yeast localization domain of S. cerevisiae Mnn2p and the catalytic domain of the MmaGalT protein. Plasmid pRCD683 was digested with SfiI to linearize and liberate the pUC19 bacterial sequences for transformation into P. pastoris.
II. Generation of a Suitable Recipient Host Strain for α-GalT
[0111] Non-human N-glycans containing terminal α-1,3-galactose are complex-type N-glycans generated by addition of an α-1,3-linked galactose residue to a mammalian N-glycan intermediate structure containing terminal β-1,4-galactose. These glycans result from competition for the terminal β-1,4-galactose by SialT and α-GalT. To eliminate such competition, a glycoengineered yeast strain was chosen as the starting strain. The strain contained the enzymes needed to produce complex type human N-glycans with terminal β-1,4-galactose, but specifically lacking SialT.
[0112] A glycoengineered P. pastoris yeast strain was generated in which the typical yeast-type N-glycosylation was modified to instead produce fully sialylated human N-glycans. First, deletion of the yeast gene OCH1 eliminated the enzyme activity responsible for `outer chain` glycosylation (Choi et al, Proc Natl Acad Sci US; 100:5022-7 (2003)). Subsequently, a mannosidase I (MNSI) gene and GlcNAc transferase I (GnTI) gene were engineered into this strain and properly localized to the secretory pathway to efficiently generate mammalian hybrid-type N-glycans (Choi et al, 2003). In a further step, a mannosidase II (MNSII) gene and GlcNAc transferase II (GnTII) gene were engineered into the strain and properly localized to the secretory pathway to efficiently generate mammalian complex-type N-glycans (Hamilton et al, Science; 301:1244-6. (2003)). Finally, by further engineering into this strain enzymes to generate a pool of UDP-galactose, appropriate Golgi membrane transporters and a gene encoding β-Galactosyl transferase (β-GalT), a yeast strain was generated that is capable of transferring a complex-type human N-glycan with terminal β-1,4-galactose. (Bobrowicz et al., Glycobiology; 14:757-66 (2004)). A yeast strain producing predominantly terminal β-1,4-galactose is a suitable host strain to receive a properly localized catalytically active α-GalT.
III. Introduction of α-GalT into a Glycoengineered Yeast Strain
[0113] RDP109 is a his1 mutant glycoengineered P. pastoris strain that secretes proteins with mammalian complex N-glycans containing terminal β-1,4-galactose. RDP109 was generated by elimination of the yeast mannosyltransferase Och1p and phosphomannosyltransferases Mnn4 bp and Pno1p and introduction of secretory pathway localized gene fusions encoding the mammalian enzymes MNS1, GnTI, MNSII, GnTII, β-GalTI, as well as genes encoding Golgi UDP-GlcNAc and UDP-Gal transporters and UDP-Galactose 4-epimerase (See Gerngross, U.S. Pat. No. 7,029,872; Davidson et al., US Patent Application 2006/0040353; and Bobrowicz, US Patent Application 2006/0211085, the disclosure of which are each hereby incorporated herein). RDP109 also expresses the Kringle 3 domain of human Plasminogen (K3) as a secreted reporter protein (Choi et al, Proc Natl Acad Sci US; 100:5022-7 (2003)). Transformants were selected on medium lacking histidine and correct integrants were screened by replica-plating transformants to medium lacking arginine. HIS+/arg- recombinants were then screened for functional expression of properly targeted α-1,3-galactosyl transferase by analyzing N-glycans released by PNGaseF digest of the secreted reporter protein K3 by Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry (MALDI-TOF MS). Two transformants, designated RDP241 and RDP242, were identified in which masses were observed consistent with addition of one and two α-linked galactose residues to the GFI5.0 N-glycan (β-Gal)2GlcNAc2Man3GlcNAc2 of RDP109 to yield the GFI5.8 (α-Gal)(β-Gal)2GlcNAc2Man3GlcNAc2 and GFI5.9 (α-Gal)2(β-Gal)2GlcNAc2Man3GlcNAc2 N-glycans respectively (see FIGS. 4A-4B).
IV. Optimization of Leader Localization Sequence for β-1,4-GalT Via a Combinatorial Library
[0114] The N-glycans produced by strains RDP109, RDP241 and RDP242 include significant amounts of hybrid-type N-glycans (see FIGS. 1, 4A-4B). Therefore, a library of strains was created to screen for an optimal leader localization sequence for human β-1,4-galactosyltransferase I (hβ-GalTI) (similar to strategy employed in Choi et al, 2003). Strain PBP235 is a yeast strain that was glycoengineered to produce complex triantennary mammalian N-glycans with terminal GlcNAc. This strain was generated by introducing localized mMNS1 (m=mouse), hGnTI (h-human), dMNSII (d=drosophila), hGnTII and hGnTIVb gene fusions into a strain in which the yeast N-glycosylation machinery (PpOCH1 (Pp=Pichia pastoris), PpPNO1, PpMNN4B, and PpPBS2) was eliminated. This strain was transformed with a series of constructs containing fusion genes of different leader/localization domains fused in frame to the hβGalTI. Previously (Davidson et al., US 20060040353), a fusion gene encoding the N-terminal 36aa of S. cerevisiae Mnn2p was utilized for localization of hβGalTI as well as for hGnTII, dMNSII, and also here for hGnTIV. This resulted in a significant percentage of hybrid-type glycans and also biantennary N-glycans. We hypothesized that this might be due to competition between hβGalTI and either or all of dMNSII, hGnTII, and hGnTIV. The screening of a library of hβGalTI constructs revealed several in which the hybrid N-glycans and biantennary N-glycans were reduced. One of these constructs that yielded reduced hybrid and biantennary N-glycans contained a fusion gene encoding the N-terminal 58aa of S. cerevisiae Mnt1p (ScMntI1-58) (Sc=S. cerevisiae) fused to hβGalTI.
[0115] Using the data from the combinatorial library screen for hβGalTI leader sequences a strain, RDP699-2, was created which secretes proteins with complex mammalian N-glycans with nearly homogeneous terminal β-1,4-linked galactose (GS5.0, FIGS. 3, 5A). This strain has been glycoengineered to eliminate yeast-type N-glycosylation and expresses localized fusions of mMNSI, hGnTI, dMNSII, hGnTII, and hβGalTI. Based on the combinatorial library screen, the hβGalTI is a fusion gene encoding ScMntI1-58 as a localization domain. Because of the improved leader localization, this strain has significantly reduced hybrid-type N-glycans compared to a previous generation strain such as RDP109, RDP241 and RDP242. Strain RDP699-2 also produces rat Erythropoietin (rEpo) as a secreted reporter protein and is arg-because the PpARG1 gene was been deleted. Plasmid pGLY1443 contains a fusion gene encoding ScMntI1-58 fused to the catalytic domain of mαGalT (from pRCD680) described above and contains PpARG1 as a selectable marker. Plasmid pGLY1443 was transformed into strain RDP699-2 and transformants were selected on medium lacking arginine, resulting in strain RDP1030. Analysis of N-glycans released by PNGaseF digest from RDP1030 by MALDI-TOF revealed the presence of both GFI5.8 (α-Gal)(β-Gal)2GlcNAc2Man3GlcNAc2 and GFI5.9 (α-Gal)2(β-Gal)2GlcNAc2Man3GlcNAc2 N-glycans (FIG. 5B) via addition of one and two α-1,3-galactose residues to the GFI5.0 (β-Gal)2GlcNAc2Man3GlcNAc2 structure. However, the hybrid N-glycans present in FIGS. 4A-4B are nearly undetectable (compare FIGS. 4A-4B with FIGS. 5A-5B), resulting from the improved localization of hβ-3GalTI and presumably from reduced competition of hβGalTI with dMNSII and hGnTII for hybrid substrates.
V. Screening a Library of α-3-GalTs Improves α-Gal Transfer
[0116] P. pastoris strain YGLY1169, a strain similar to RDP699-2, was created and secretes proteins with complex mammalian N-glycans with terminal β-1,4-linked galactose ((β-Gal)2GlcNAc2Man3GlcNAc2 GS5.0, FIG. 3). This strain has been glycoengineered to eliminate yeast-type N-glycosylation and expresses localized fusions of mMNSI, hGnTI, dMNSII, hGnTII, and hβGalTI. Based on the combinatorial library screen, the hβGalTI is a fusion gene encoding ScMntI1-58 as a localization domain. Because of the improved leader localization, this strain has significantly reduced hybrid-type N-glycans compared to a previous generation strain such as RDP109 and is thus similar to RDP699-2 (not shown). This strain is also ura- because the PpURA5 gene has been deleted. Plasmids were created containing gene fusions encoding ScMntI1-58 fused to the catalytic domain of various α-GalTs (FIG. 6A-6B) including those from Mus musculus (pGLY1892), Sus scrofa (pGLY1893) and Canis familiaris (pGLY1894). These plasmids all contain the PpURA5 gene as a selectable marker. Plasmids pGLY1892, 1893, and 1894 were transformed into strain YGLY1169 and transformants were selected on medium lacking uracil, resulting in strains YGLY1783, YGLY1785, and YGLY1787, respectively. These strains were each transformed with plasmid pSH692, containing a gene encoding rEPO as a secreted reporter, and the shBLE gene as a selectable marker. Plasmid pSH692 was transformed into strains YGLY1783, YGLY1785, and YGLY1787 and N-glycans released by PNGaseF digest from the resulting transformants were analyzed from secreted rEPO by MALDI-TOF. The N-glycans were similar to that obtained from strain RDP1030 (FIGS. 7A-7C). However, an incremental improvement in the α-gal transfer was observed from introduction of Sus scrofa α-GalT (SsαGalT) compared to MmαGalT, as judged by the relative intensity of peaks corresponding to GFI5.0 (β-Gal)2GlcNAc2Man3GlcNAc2, GFI5.8 (α-Gal)(β-Gal)2GlcNAc2Man3GlcNAc2 and GFI5.9 (α-Gal)2(β-Gal)2GlcNAc2Man3GlcNAc2, (FIG. 7B).
VI. Optimization of Leader Localization Sequence for SsαGalT Via a Combinatorial Fusion Library Improves α-Gal Transfer
[0117] In order to improve α-galactose transfer, a library of plasmids was created to screen for optimal leader localization sequences for SsαGalT, based on plasmid pGLY2169. Plasmid pGLY2169 contains the gene encoding the catalytic domain of SsαGalT without a yeast localization domain, but instead a pair of restriction sites, NotI and AscI and also contains the NAT gene (encoding for resistance to the aminoglycoside Nourseothricin) as a selectable marker. A library of DNA sequences encoding yeast localization domains (numbered 33-67, 745-756) was cloned into this vector using NotI/AscI and the resulting plasmids named pGLY2169-33-pGLY2169-756. This library of plasmids was transformed into P. pastoris strain RDP1482 and transformants were selected on medium lacking uracil. Strain RDP1482 has been engineered to secrete proteins with complex mammalian N-glycans with terminal β-1,4-linked galactose (GS5.0 (β-Gal)2GlcNAc2Man3GlcNAc2, FIG. 3), and expresses localized fusions of mMNSI, hGnTI, dMNSII, hGnTII, and hβGalTI. Again, based on the combinatorial library screen, the hβGalTI is a fusion gene encoding ScMntI1-58 as a localization domain. Strain RDP1482 has also been engineered to secrete the K3 domain of human plasminogen as a reporter protein under control of the AOX1 promoter. K3 was produced by inducing cultures on methanol as a sole carbon source, and supernatant protein was purified from transformants and N-glycans released by PNGase digest were analyzed by MALDI-TOF. The results indicated that when the ScMntI1-58-SsaGalT fusion gene was expressed, a similar ratio of GFI5.0, GFI5.8, and GFI5.9 N-glycans were observed compared transformants of strain YGLY1785 (FIGS. 8A-8B). However, several leader/localization domain-SsaGalT fusions including revealed a significant increase in α-gal transfer as judged by the ratio of GFI5.0, GFI5.8 and GFI5.9 masses observed. For a single leader-SsαGalT fusion, ScMnn21-36-SsaGalT, GFI5.9 was the only peak that was observed, indicating almost quantitative transfer of α-gal to the β-1,4-galactose substrate (FIG. 9).
VII. Expression of Influenza A HA Protein in a GFI5.9 Strain
[0118] DNA sequences encoding the ectodomain (i.e. lacking the C-terminal transmembrane domain) of several representative Influenza A type H3 HA proteins including HA Hong Kong (pGLY2766) [SEQ ID NO: 3], HA Panama (pGLY2764), HA Sydney (pGLY2765), HA New York (pGLY2762), and HA Moscow (pGLY2763), and several representative type H1 HA proteins including HA Beijing (pGLY2760), HA New Calcedonia (pGLY2759), HA Puerto Rico (pGLY2761), and HA South Carolina (pGLY2767) [SEQ ID NO: 4] were synthesized and cloned (GeneArt, LLC). Each of these plasmids was subcloned into a P. pastoris expression vector containing the AOX1 promoter and ShBLE drug resistance marker and fused in frame with a library of secretion signal peptides, including the S. cerevisiae α-Mating Factor prepro secretion signal. GFI5.9 glycoengineered P. pastoris strain YGLY2229 was transformed with each of the Influenza HA-containing expression plasmids and colonies were selected on minimal medium containing 300 mg/L Zeocin. Several transformants were selected and cultivated in 96 well deep well plates for 72 h at 26 C in liquid medium with glycerol as the sole carbon source, then centrifuged and resuspended in medium with methanol as the sole carbon source and incubated at 26 C for 24 hours. Cells were centrifuged and 7 ul of supernatant was subjected to standard Western blot analysis under non-reducing conditions and probing with a pre-labeled anti-HIS (H3 HIS probe, Santa Cruz) antibody. A band of appropriate size was observed for each HA expressed. Approximately 600 ul of supernatant was subjected to Ni-affinity purification, PNGase digestion to remove N-glycans, and MALDI-TOF MS analysis. N-glycan masses corresponding to GS5.0, GS5.8 and GS5.9 glycoforms were observed for all Influenza HA proteins tested. As an example, N-glycans from Influenza A HA Hong Kong fused in frame with the S. cerevisiae alpha mating factor prepro secretion signal (pGLY2922) expressed in YGLY2229 are shown (FIG. 10).
[0119] Similarly, further optimized GFI5.9 glycoengineered P. pastoris strain YGLY3812 was transformed with each of the Influenza HA-containing expression plasmids and colonies were selected on minimal medium containing 300 mg/L Zeocin. Several transformants were selected and cultivated as above. Following Ni-affinity protein purification, PNGase digestion to remove N-glycans, and MALDI-TOF MS analysis. N-glycan masses corresponding to GS5.0, GS5.8 and GS5.9 glycoforms were observed for all Influenza HA proteins tested in ratios similar to other reporter proteins. As an example, N-glycans from Influenza A HA South Carolina expressed in YGLY3812 are shown (FIG. 11).
VIII. Expression of HSV-2 gD Protein in a GFI5.9 Strain
[0120] DNA sequences encoding two different versions of the HSV-2 G strain gD protein ectodomain were synthesized and cloned and named pGLY2757 and pGLY2758 (GeneArt, LLC) [SEQ ID NOS: 5-6]. The two constructs differed by the length of the C-terminus, one encoding the entire ectodomain, amino acids 26-339 (gD 339, pGLY2757) and a second encoding a shorter version without the C-terminal domain, amino acids 26-306 and including two heterologous amino acids, Asn and Gln, appended to the C-terminus after Leu 306 (gD 306NQ, pGLY2758). Each of these plasmids was subcloned into a P. pastoris expression vector containing the AOX1 promoter and ShBLE drug resistance marker and fused in frame with the S. cerevisiae α-Mating Factor pre secretion signal and named pGLY2960 and pGLY2961, respectively. GFI5.9 glycoengineered P. pastoris strain YGLY3812 was transformed with each of the HSV-2 gD-containing expression plasmids and colonies were selected on minimal medium containing 300 mg/L Zeocin. Several transformants were selected and cultivated in 96 well deep well plates for 72 h at 26 C in liquid medium with glycerol as the sole carbon source, then centrifuged and resuspended in medium with methanol as the sole carbon source and incubated at 26 C for 24 hours. Cells were centrifuged and 7 ul of supernatant was subjected to standard Western blot analysis under reducing conditions and probing with a pre-labeled anti-HIS (H3 HIS probe, Santa Cruz) antibody. A band of appropriate size was observed for both of the versions of gD expressed. Approximately 600 ul of supernatant was subjected to Ni-affinity purification, PNGase digestion to remove N-glycans, and MALDI-TOF MS analysis. N-glycan masses corresponding to GS5.8 and GS5.9 glycoforms were observed for both versions of gD with the GS5.9 glycoform as the predominant form (FIG. 12).
IX. Expression of HSV-2 gC Protein in a GFI5.9 Strain
[0121] A DNA sequence encoding the HSV-2 G strain gC protein ectodomain was synthesized and cloned and named pGLY3640 [SEQ ID NO: 7] (GeneArt, Inc., Toronto, Calif.). The DNA sequence from this plasmid was subcloned into a P. pastoris expression vector containing the AOX1 promoter and ShBLE drug resistance marker and fused in frame with the S. cerevisiae α-Mating Factor pre secretion signal and named pGLY3653. GFI5.9 glycoengineered P. pastoris strain YGLY3812 was transformed with the HSV-2 gC-containing expression plasmid and colonies were selected on minimal medium containing 300 mg/L Zeocin. Several transformants were selected and cultivated in 96 well deep well plates for 72 h at 26 C in liquid medium with glycerol as the sole carbon source, then centrifuged and resuspended in medium with methanol as the sole carbon source and incubated at 26 C for 24 hours. Cells were centrifuged and 7 ul of supernatant was subjected to standard Western blot analysis under reducing conditions and probing with a pre-labeled anti-HIS (H3 HIS probe, Santa Cruz) antibody. A band of appropriate size was observed. Approximately 600 ul of supernatant was subjected to Ni-affinity purification, PNGase digestion to remove N-glycans, and MALDI-TOF MS analysis. N-glycan masses corresponding to GS5.8 and GS5.9 glycoforms were observed with the GS5.9 glycoform as the predominant form (FIG. 13).
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Sequence CWU
1
11134DNAMus musculus 1ttggcgcgcc aacagcccag acggctcttt cttg
34234DNAMus musculus 2ggttaattaa tcagacatta tttctaacca
aatt 343562PRTInfluenza 3Gln Asp Leu Pro
Gly Asn Asp Asn Ser Thr Ala Thr Leu Cys Leu Gly1 5
10 15His His Ala Val Pro Asn Gly Thr Leu Val
Lys Thr Ile Thr Asp Asp 20 25
30Gln Ile Glu Val Thr Asn Ala Thr Glu Leu Val Gln Ser Ser Ser Thr
35 40 45Gly Lys Ile Cys Asn Asn Pro His
Arg Ile Leu Asp Gly Ile Asp Cys 50 55
60Thr Leu Ile Asp Ala Leu Leu Gly Asp Pro His Cys Asp Val Phe Gln65
70 75 80Asn Glu Thr Trp Asp
Leu Phe Val Glu Arg Ser Lys Ala Phe Ser Asn 85
90 95Cys Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Ser
Leu Arg Ser Leu Val 100 105
110Ala Ser Ser Gly Thr Leu Glu Phe Ile Thr Glu Gly Phe Thr Trp Thr
115 120 125Gly Val Thr Gln Asn Gly Gly
Ser Asn Ala Cys Lys Arg Gly Pro Gly 130 135
140Ser Gly Phe Phe Ser Arg Leu Asn Trp Leu Thr Lys Ser Gly Ser
Thr145 150 155 160Tyr Pro
Val Leu Asn Val Thr Met Pro Asn Asn Asp Asn Phe Asp Lys
165 170 175Leu Tyr Ile Trp Gly Val His
His Pro Ser Thr Asn Gln Glu Gln Thr 180 185
190Ser Leu Tyr Val Gln Ala Ser Gly Arg Val Thr Val Ser Thr
Arg Arg 195 200 205Ser Gln Gln Thr
Ile Ile Pro Asn Ile Gly Ser Arg Pro Trp Val Arg 210
215 220Gly Leu Ser Ser Arg Ile Ser Ile Tyr Trp Thr Ile
Val Lys Pro Gly225 230 235
240Asp Val Leu Val Ile Asn Ser Asn Gly Asn Leu Ile Ala Pro Arg Gly
245 250 255Tyr Phe Lys Met Arg
Thr Gly Lys Ser Ser Ile Met Arg Ser Asp Ala 260
265 270Pro Ile Asp Thr Cys Ile Ser Glu Cys Ile Thr Pro
Asn Gly Ser Ile 275 280 285Pro Asn
Asp Lys Pro Phe Gln Asn Val Asn Lys Ile Thr Tyr Gly Ala 290
295 300Cys Pro Lys Tyr Val Lys Gln Asn Thr Leu Lys
Leu Ala Thr Gly Met305 310 315
320Arg Asn Val Pro Glu Lys Gln Thr Arg Gly Leu Phe Gly Ala Ile Ala
325 330 335Gly Phe Ile Glu
Asn Gly Trp Glu Gly Met Ile Asp Gly Trp Tyr Gly 340
345 350Phe Arg His Gln Asn Ser Glu Gly Thr Gly Gln
Ala Ala Asp Leu Lys 355 360 365Ser
Thr Gln Ala Ala Ile Asp Gln Ile Asn Gly Lys Leu Asn Arg Val 370
375 380Ile Glu Lys Thr Asn Glu Lys Phe His Gln
Ile Glu Lys Glu Phe Ser385 390 395
400Glu Val Glu Gly Arg Ile Gln Asp Leu Glu Lys Tyr Val Glu Asp
Thr 405 410 415Lys Ile Asp
Leu Trp Ser Tyr Asn Ala Glu Leu Leu Val Ala Leu Glu 420
425 430Asn Gln His Thr Ile Asp Leu Thr Asp Ser
Glu Met Asn Lys Leu Phe 435 440
445Glu Lys Thr Arg Arg Gln Leu Arg Glu Asn Ala Glu Asp Met Gly Asn 450
455 460Gly Cys Phe Lys Ile Tyr His Lys
Cys Asp Asn Ala Cys Ile Glu Ser465 470
475 480Ile Arg Asn Gly Thr Tyr Asp His Asp Val Tyr Arg
Asp Glu Ala Leu 485 490
495Asn Asn Arg Phe Gln Ile Lys Gly Val Glu Leu Lys Ser Gly Tyr Lys
500 505 510Asp Trp Ile Leu Trp Ile
Ser Phe Ala Ile Ser Cys Phe Leu Leu Cys 515 520
525Val Val Leu Leu Gly Phe Ile Met Trp Ala Cys Gln Arg Gly
Asn Ile 530 535 540Arg Cys Asn Ile Cys
Ile Gly Gly Gly His His His His His His His545 550
555 560His His4525PRTInfluenza 4Asp Thr Ile Cys
Ile Gly Tyr His Ala Asn Asn Ser Thr Asp Thr Val1 5
10 15Asp Thr Val Leu Glu Lys Asn Val Thr Val
Thr His Ser Val Asn Leu 20 25
30Leu Glu Asp Ser His Asn Gly Lys Leu Cys Lys Leu Lys Gly Ile Ala
35 40 45Pro Leu Gln Leu Gly Lys Cys Asn
Ile Ala Gly Trp Leu Leu Gly Asn 50 55
60Pro Glu Cys Asp Leu Leu Leu Thr Ala Ser Ser Trp Ser Tyr Ile Val65
70 75 80Glu Thr Ser Asn Ser
Glu Asn Gly Thr Cys Tyr Pro Gly Asp Phe Ile 85
90 95Asp Tyr Glu Glu Leu Arg Glu Gln Leu Ser Ser
Val Ser Ser Phe Glu 100 105
110Lys Phe Glu Ile Phe Pro Lys Thr Ser Ser Trp Pro Asn His Glu Thr
115 120 125Thr Lys Gly Val Thr Ala Ala
Cys Ser Tyr Ala Gly Ala Ser Ser Phe 130 135
140Tyr Arg Asn Leu Leu Trp Leu Thr Lys Lys Gly Ser Ser Tyr Pro
Lys145 150 155 160Leu Ser
Lys Ser Tyr Val Asn Asn Lys Gly Lys Glu Val Leu Val Leu
165 170 175Trp Gly Val His His Pro Pro
Thr Gly Thr Asp Gln Gln Ser Leu Tyr 180 185
190Gln Asn Ala Asp Ala Tyr Val Ser Val Gly Ser Ser Lys Tyr
Asn Arg 195 200 205Arg Phe Thr Pro
Glu Ile Ala Ala Arg Pro Lys Val Arg Asp Gln Ala 210
215 220Gly Arg Met Asn Tyr Tyr Trp Thr Leu Leu Glu Pro
Gly Asp Thr Ile225 230 235
240Thr Phe Glu Ala Thr Gly Asn Leu Ile Ala Pro Trp Tyr Ala Phe Ala
245 250 255Leu Asn Arg Gly Ser
Gly Ser Gly Ile Ile Thr Ser Asp Ala Pro Val 260
265 270His Asp Cys Asn Thr Lys Cys Gln Thr Pro His Gly
Ala Ile Asn Ser 275 280 285Ser Leu
Pro Phe Gln Asn Ile His Pro Val Thr Ile Gly Glu Cys Pro 290
295 300Lys Tyr Val Arg Ser Thr Lys Leu Arg Met Ala
Thr Gly Leu Arg Asn305 310 315
320Ile Pro Ser Ile Gln Ser Arg Gly Leu Phe Gly Ala Ile Ala Gly Phe
325 330 335Ile Glu Gly Gly
Trp Thr Gly Met Ile Asp Gly Trp Tyr Gly Tyr His 340
345 350His Gln Asn Glu Gln Gly Ser Gly Tyr Ala Ala
Asp Gln Lys Ser Thr 355 360 365Gln
Asn Ala Ile Asp Gly Ile Thr Asn Lys Val Asn Ser Val Ile Glu 370
375 380Lys Met Asn Thr Gln Phe Thr Ala Val Gly
Lys Glu Phe Asn Asn Leu385 390 395
400Glu Arg Arg Ile Glu Asn Leu Asn Lys Lys Val Asp Asp Gly Phe
Leu 405 410 415Asp Ile Trp
Thr Tyr Asn Ala Glu Leu Leu Val Leu Leu Glu Asn Glu 420
425 430Arg Thr Leu Asp Phe His Asp Ser Asn Val
Arg Asn Leu Tyr Glu Lys 435 440
445Val Lys Ser Gln Leu Lys Asn Asn Ala Lys Glu Ile Gly Asn Gly Cys 450
455 460Phe Glu Phe Tyr His Lys Cys Asp
Asp Ala Cys Met Glu Ser Val Arg465 470
475 480Asn Gly Thr Tyr Asp Tyr Pro Lys Tyr Ser Glu Glu
Ser Lys Leu Asn 485 490
495Arg Glu Glu Ile Asp Gly Val Lys Leu Glu Ser Met Gly Val Tyr Gln
500 505 510Ile Gly Gly Gly His His
His His His His His His His 515 520
5255326PRTHerpes simplex virus 5Lys Tyr Ala Leu Ala Asp Pro Ser Leu Lys
Met Ala Asp Pro Asn Arg1 5 10
15Phe Arg Gly Lys Asn Leu Pro Val Leu Asp Gln Leu Thr Asp Pro Pro
20 25 30Gly Val Lys Arg Val Tyr
His Ile Gln Pro Ser Leu Glu Asp Pro Phe 35 40
45Gln Pro Pro Ser Ile Pro Ile Thr Val Tyr Tyr Ala Val Leu
Glu Arg 50 55 60Ala Cys Arg Ser Val
Leu Leu His Ala Pro Ser Glu Ala Pro Gln Ile65 70
75 80Val Arg Gly Ala Ser Asp Glu Ala Arg Lys
His Thr Tyr Asn Leu Thr 85 90
95Ile Ala Trp Tyr Arg Met Gly Asp Asn Cys Ala Ile Pro Ile Thr Val
100 105 110Met Glu Tyr Thr Glu
Cys Pro Tyr Asn Lys Ser Leu Gly Val Cys Pro 115
120 125Ile Arg Thr Gln Pro Arg Trp Ser Tyr Tyr Asp Ser
Phe Ser Ala Val 130 135 140Ser Glu Asp
Asn Leu Gly Phe Leu Met His Ala Pro Ala Phe Glu Thr145
150 155 160Ala Gly Thr Tyr Leu Arg Leu
Val Lys Ile Asn Asp Trp Thr Glu Ile 165
170 175Thr Gln Phe Ile Leu Glu His Arg Ala Arg Ala Ser
Cys Lys Tyr Ala 180 185 190Leu
Pro Leu Arg Ile Pro Pro Ala Ala Cys Leu Thr Ser Lys Ala Tyr 195
200 205Gln Gln Gly Val Thr Val Asp Ser Ile
Gly Met Leu Pro Arg Phe Ile 210 215
220Pro Glu Asn Gln Arg Thr Val Ala Leu Tyr Ser Leu Lys Ile Ala Gly225
230 235 240Trp His Gly Pro
Lys Pro Pro Tyr Thr Ser Thr Leu Leu Pro Pro Glu 245
250 255Leu Ser Asp Thr Thr Asn Ala Thr Gln Pro
Glu Leu Val Pro Glu Asp 260 265
270Pro Glu Asp Ser Ala Leu Leu Glu Asp Pro Ala Gly Thr Val Ser Ser
275 280 285Gln Ile Pro Pro Asn Trp His
Ile Pro Ser Ile Gln Asp Val Ala Pro 290 295
300His His Ala Pro Ala Ala Pro Ser Asn Pro Gly Gly Gly His His
His305 310 315 320His His
His His His His 3256295PRTHerpes simplex virus 6Lys Tyr
Ala Leu Ala Asp Pro Ser Leu Lys Met Ala Asp Pro Asn Arg1 5
10 15Phe Arg Gly Lys Asn Leu Pro Val
Leu Asp Gln Leu Thr Asp Pro Pro 20 25
30Gly Val Lys Arg Val Tyr His Ile Gln Pro Ser Leu Glu Asp Pro
Phe 35 40 45Gln Pro Pro Ser Ile
Pro Ile Thr Val Tyr Tyr Ala Val Leu Glu Arg 50 55
60Ala Cys Arg Ser Val Leu Leu His Ala Pro Ser Glu Ala Pro
Gln Ile65 70 75 80Val
Arg Gly Ala Ser Asp Glu Ala Arg Lys His Thr Tyr Asn Leu Thr
85 90 95Ile Ala Trp Tyr Arg Met Gly
Asp Asn Cys Ala Ile Pro Ile Thr Val 100 105
110Met Glu Tyr Thr Glu Cys Pro Tyr Asn Lys Ser Leu Gly Val
Cys Pro 115 120 125Ile Arg Thr Gln
Pro Arg Trp Ser Tyr Tyr Asp Ser Phe Ser Ala Val 130
135 140Ser Glu Asp Asn Leu Gly Phe Leu Met His Ala Pro
Ala Phe Glu Thr145 150 155
160Ala Gly Thr Tyr Leu Arg Leu Val Lys Ile Asn Asp Trp Thr Glu Ile
165 170 175Thr Gln Phe Ile Leu
Glu His Arg Ala Arg Ala Ser Cys Lys Tyr Ala 180
185 190Leu Pro Leu Arg Ile Pro Pro Ala Ala Cys Leu Thr
Ser Lys Ala Tyr 195 200 205Gln Gln
Gly Val Thr Val Asp Ser Ile Gly Met Leu Pro Arg Phe Ile 210
215 220Pro Glu Asn Gln Arg Thr Val Ala Leu Tyr Ser
Leu Lys Ile Ala Gly225 230 235
240Trp His Gly Pro Lys Pro Pro Tyr Thr Ser Thr Leu Leu Pro Pro Glu
245 250 255Leu Ser Asp Thr
Thr Asn Ala Thr Gln Pro Glu Leu Val Pro Glu Asp 260
265 270Pro Glu Asp Ser Ala Leu Leu Glu Asp Asn Gln
Gly Gly Gly His His 275 280 285His
His His His His His His 290 2957437PRTHerpes simplex
virus 7Leu Ala Asn Ala Ser Pro Gly Arg Thr Ile Thr Val Gly Pro Arg Gly1
5 10 15Asn Ala Ser Asn Ala
Ala Pro Ser Ala Ser Pro Arg Asn Ala Ser Ala 20
25 30Pro Arg Thr Thr Pro Thr Pro Pro Gln Pro Arg Lys
Ala Thr Lys Ser 35 40 45Lys Ala
Ser Thr Ala Lys Pro Ala Pro Pro Pro Lys Thr Gly Pro Pro 50
55 60Lys Thr Ser Ser Glu Pro Val Arg Cys Asn Arg
His Asp Pro Leu Ala65 70 75
80Arg Tyr Gly Ser Arg Val Gln Ile Arg Cys Arg Phe Pro Asn Ser Thr
85 90 95Arg Thr Glu Phe Arg
Leu Gln Ile Trp Arg Tyr Ala Thr Ala Thr Asp 100
105 110Ala Glu Ile Gly Thr Ala Pro Ser Leu Glu Glu Val
Met Val Asn Val 115 120 125Ser Ala
Pro Pro Gly Gly Gln Leu Val Tyr Asp Ser Ala Pro Asn Arg 130
135 140Thr Asp Pro His Val Ile Trp Ala Glu Gly Ala
Gly Pro Gly Ala Ser145 150 155
160Pro Arg Leu Tyr Ser Val Val Gly Pro Leu Gly Arg Gln Arg Leu Ile
165 170 175Ile Glu Glu Leu
Thr Leu Glu Thr Gln Gly Met Tyr Tyr Trp Val Trp 180
185 190Gly Arg Thr Asp Arg Pro Ser Ala Tyr Gly Thr
Trp Val Arg Val Arg 195 200 205Val
Phe Arg Pro Pro Ser Leu Thr Ile His Pro His Ala Val Leu Glu 210
215 220Gly Gln Pro Phe Lys Ala Thr Cys Thr Ala
Ala Thr Tyr Tyr Pro Gly225 230 235
240Asn Arg Ala Glu Phe Val Trp Phe Glu Asp Gly Arg Arg Val Phe
Asp 245 250 255Pro Ala Gln
Ile His Thr Gln Thr Gln Glu Asn Pro Asp Gly Phe Ser 260
265 270Thr Val Ser Thr Val Thr Ser Ala Ala Val
Gly Gly Gln Gly Pro Pro 275 280
285Arg Thr Phe Thr Cys Gln Leu Thr Trp His Arg Asp Ser Val Ser Phe 290
295 300Ser Arg Arg Asn Ala Ser Gly Thr
Ala Ser Val Leu Pro Arg Pro Thr305 310
315 320Ile Thr Met Glu Phe Thr Gly Asp His Ala Val Cys
Thr Ala Gly Cys 325 330
335Val Pro Glu Gly Val Thr Phe Ala Trp Phe Leu Gly Asp Asp Ser Ser
340 345 350Pro Ala Glu Lys Val Ala
Val Ala Ser Gln Thr Ser Cys Gly Arg Pro 355 360
365Gly Thr Ala Thr Ile Arg Ser Thr Leu Pro Val Ser Tyr Glu
Gln Thr 370 375 380Glu Tyr Ile Cys Arg
Leu Ala Gly Tyr Pro Asp Gly Ile Pro Val Leu385 390
395 400Glu His His Gly Ser His Gln Pro Pro Pro
Arg Asp Pro Thr Glu Arg 405 410
415Gln Val Ile Arg Ala Ile Glu Gly Arg Gly Gly Gly His His His His
420 425 430His His His His His
4358368PRTBos taurus alpha 8Met Asn Val Lys Gly Lys Val Ile Leu Ser
Met Leu Val Val Ser Thr1 5 10
15Val Ile Val Val Phe Trp Glu Tyr Ile His Ser Pro Glu Gly Ser Leu
20 25 30Phe Trp Ile Asn Pro Ser
Arg Asn Pro Glu Val Gly Gly Ser Ser Ile 35 40
45Gln Lys Gly Trp Trp Leu Pro Arg Trp Phe Asn Asn Gly Tyr
His Glu 50 55 60Glu Asp Gly Asp Ile
Asn Glu Glu Lys Glu Gln Arg Asn Glu Asp Glu65 70
75 80Ser Lys Leu Lys Leu Ser Asp Trp Phe Asn
Pro Phe Lys Arg Pro Glu 85 90
95Val Val Thr Met Thr Lys Trp Lys Ala Pro Val Val Trp Glu Gly Thr
100 105 110Tyr Asn Arg Ala Val
Leu Asp Asn Tyr Tyr Ala Lys Gln Lys Ile Thr 115
120 125Val Gly Leu Thr Val Phe Ala Val Gly Arg Tyr Ile
Glu His Tyr Leu 130 135 140Glu Glu Phe
Leu Thr Ser Ala Asn Lys His Phe Met Val Gly His Pro145
150 155 160Val Ile Phe Tyr Ile Met Val
Asp Asp Val Ser Arg Met Pro Leu Ile 165
170 175Glu Leu Gly Pro Leu Arg Ser Phe Lys Val Phe Lys
Ile Lys Pro Glu 180 185 190Lys
Arg Trp Gln Asp Ile Ser Met Met Arg Met Lys Thr Ile Gly Glu 195
200 205His Ile Val Ala His Ile Gln His Glu
Val Asp Phe Leu Phe Cys Met 210 215
220Asp Val Asp Gln Val Phe Gln Asp Lys Phe Gly Val Glu Thr Leu Gly225
230 235 240Glu Ser Val Ala
Gln Leu Gln Ala Trp Trp Tyr Lys Ala Asp Pro Asn 245
250 255Asp Phe Thr Tyr Glu Arg Arg Lys Glu Ser
Ala Ala Tyr Ile Pro Phe 260 265
270Gly Glu Gly Asp Phe Tyr Tyr His Ala Ala Ile Phe Gly Gly Thr Pro
275 280 285Thr Gln Val Leu Asn Ile Thr
Gln Glu Cys Phe Lys Gly Ile Leu Lys 290 295
300Asp Lys Lys Asn Asp Ile Glu Ala Gln Trp His Asp Glu Ser His
Leu305 310 315 320Asn Lys
Tyr Phe Leu Leu Asn Lys Pro Thr Lys Ile Leu Ser Pro Glu
325 330 335Tyr Cys Trp Asp Tyr His Ile
Gly Leu Pro Ala Asp Ile Lys Leu Val 340 345
350Lys Met Ser Trp Gln Thr Lys Glu Tyr Asn Val Val Arg Asn
Asn Val 355 360 3659384PRTCanis
familiaris alpha 9Met Ala Asn Gln Leu Val Ser Pro Glu Glu Gly Glu Lys Ile
Met Asn1 5 10 15Val Lys
Gly Lys Val Ile Leu Ser Met Leu Val Val Ser Thr Val Ile 20
25 30Val Val Phe Trp Glu Tyr Ile Asn Ser
Pro Glu Gly Ser Phe Leu Trp 35 40
45Ile Tyr His Ser Lys Asn Pro Glu Val Gly Glu Ser Arg Ile Gln Lys 50
55 60Gly Trp Trp Phe Pro Asn Trp Phe Asn
Asn Gly Thr His Phe Tyr Gln65 70 75
80Glu Glu Glu Asp Ile Asp Asp Glu Asn Glu Gln Gly Glu Glu
Asn Asn 85 90 95Ala Glu
Leu Gln Leu Ser Asp Trp Phe Asn Pro Gln Lys Arg Pro Glu 100
105 110Val Val Thr Val Thr Arg Trp Lys Ala
Pro Val Val Trp Glu Gly Thr 115 120
125Tyr Asn Ser Thr Ile Leu Glu Asn Tyr Tyr Ala Lys Gln Lys Ile Thr
130 135 140Ile Gly Leu Thr Val Phe Ala
Val Gly Arg Tyr Ile Glu His Tyr Leu145 150
155 160Glu Glu Phe Leu Ile Ser Ala Asn Arg Tyr Phe Met
Val Gly His Lys 165 170
175Val Ile Phe Tyr Ile Met Val Asp Asp Val Ser Arg Met Pro Leu Val
180 185 190Glu Leu Gly Pro Leu Arg
Ser Phe Lys Val Phe Glu Ile Glu Pro Glu 195 200
205Lys Arg Trp Gln Asp Ile Ser Met Met Arg Met Lys Thr Ile
Gly Glu 210 215 220His Ile Val Ala His
Ile Gln His Glu Val Asp Phe Leu Phe Cys Met225 230
235 240Asp Val Asp Gln Val Phe Gln Asp Ser Phe
Gly Val Glu Thr Leu Gly 245 250
255Gln Ser Val Ala Gln Leu Gln Ala Trp Trp Tyr Lys Ala Asp Pro Asp
260 265 270Glu Phe Thr Tyr Glu
Arg Arg Lys Glu Ser Ala Ala Tyr Ile Pro Phe 275
280 285Gly Gln Gly Asp Phe Tyr Tyr His Ala Ala Ile Phe
Gly Gly Thr Pro 290 295 300Ile Gln Val
Leu Asn Ile Thr Gln Glu Cys Phe Lys Gly Ile Leu Gln305
310 315 320Asp Lys Lys Asn Asp Ile Glu
Ala Glu Trp His Asp Glu Ser His Leu 325
330 335Asn Lys Tyr Phe Leu Leu Asn Lys Pro Thr Lys Ile
Leu Ser Pro Glu 340 345 350Tyr
Cys Trp Asp Tyr His Ile Gly Leu Pro Ser Asp Ile Lys Thr Val 355
360 365Lys Ile Ser Trp Gln Thr Lys Glu Tyr
Asn Leu Val Arg Asn Asn Ile 370 375
38010371PRTSus scrofa, alpha 10Met Asn Val Lys Gly Arg Val Val Leu Ser
Met Leu Leu Val Ser Thr1 5 10
15Val Met Val Val Phe Trp Glu Tyr Ile Asn Ser Pro Glu Gly Ser Leu
20 25 30Phe Trp Ile Tyr Gln Ser
Lys Asn Pro Glu Val Gly Ser Ser Ala Gln 35 40
45Arg Gly Trp Trp Phe Pro Ser Trp Phe Asn Asn Gly Thr His
Ser Tyr 50 55 60His Glu Glu Glu Asp
Ala Ile Gly Asn Glu Lys Glu Gln Arg Lys Glu65 70
75 80Asp Asn Arg Gly Glu Leu Pro Leu Val Asp
Trp Phe Asn Pro Glu Lys 85 90
95Arg Pro Glu Val Val Thr Ile Thr Arg Trp Lys Ala Pro Val Val Trp
100 105 110Glu Gly Thr Tyr Asn
Arg Ala Val Leu Asp Asn Tyr Tyr Ala Lys Gln 115
120 125Lys Ile Thr Val Gly Leu Thr Val Phe Ala Val Gly
Arg Tyr Ile Glu 130 135 140His Tyr Leu
Glu Glu Phe Leu Ile Ser Ala Asn Thr Tyr Phe Met Val145
150 155 160Gly His Lys Val Ile Phe Tyr
Ile Met Val Asp Asp Ile Ser Arg Met 165
170 175Pro Leu Ile Glu Leu Gly Pro Leu Arg Ser Phe Lys
Val Phe Glu Ile 180 185 190Lys
Ser Glu Lys Arg Trp Gln Asp Ile Ser Met Met Arg Met Lys Thr 195
200 205Ile Gly Glu His Ile Leu Ala His Ile
Gln His Glu Val Asp Phe Leu 210 215
220Phe Cys Met Asp Val Asp Gln Val Phe Gln Asn Asn Phe Gly Val Glu225
230 235 240Thr Leu Gly Gln
Ser Val Ala Gln Leu Gln Ala Trp Trp Tyr Lys Ala 245
250 255His Pro Asp Glu Phe Thr Tyr Glu Arg Arg
Lys Glu Ser Ala Ala Tyr 260 265
270Ile Pro Phe Gly Gln Gly Asp Phe Tyr Tyr His Ala Ala Ile Phe Gly
275 280 285Gly Thr Pro Thr Gln Val Leu
Asn Ile Thr Gln Glu Cys Phe Lys Gly 290 295
300Ile Leu Gln Asp Lys Glu Asn Asp Ile Glu Ala Glu Trp His Asp
Glu305 310 315 320Ser His
Leu Asn Lys Tyr Phe Leu Leu Asn Lys Pro Thr Lys Ile Leu
325 330 335Ser Pro Glu Tyr Cys Trp Asp
Tyr His Ile Gly Met Ser Val Asp Ile 340 345
350Arg Ile Val Lys Ile Ala Trp Gln Lys Lys Glu Tyr Asn Leu
Val Arg 355 360 365Asn Asn Ile
37011371PRTMus musculus alpha 11Met Asn Val Lys Gly Lys Val Ile Leu Leu
Met Leu Ile Val Ser Thr1 5 10
15Val Val Val Val Phe Trp Glu Tyr Val Asn Ser Pro Asp Gly Ser Phe
20 25 30Leu Trp Ile Tyr His Thr
Lys Ile Pro Glu Val Gly Glu Asn Arg Trp 35 40
45Gln Lys Asp Trp Trp Phe Pro Ser Trp Phe Lys Asn Gly Thr
His Ser 50 55 60Tyr Gln Glu Asp Asn
Val Glu Gly Arg Arg Glu Lys Gly Arg Asn Gly65 70
75 80Asp Arg Ile Glu Glu Pro Gln Leu Trp Asp
Trp Phe Asn Pro Lys Asn 85 90
95Arg Pro Asp Val Leu Thr Val Thr Pro Trp Lys Ala Pro Ile Val Trp
100 105 110Glu Gly Thr Tyr Asp
Thr Ala Leu Leu Glu Lys Tyr Tyr Ala Thr Gln 115
120 125Lys Leu Thr Val Gly Leu Thr Val Phe Ala Val Gly
Lys Tyr Ile Glu 130 135 140His Tyr Leu
Glu Asp Phe Leu Glu Ser Ala Asp Met Tyr Phe Met Val145
150 155 160Gly His Arg Val Ile Phe Tyr
Val Met Ile Asp Asp Thr Ser Arg Met 165
170 175Pro Val Val His Leu Asn Pro Leu His Ser Leu Gln
Val Phe Glu Ile 180 185 190Arg
Ser Glu Lys Arg Trp Gln Asp Ile Ser Met Met Arg Met Lys Thr 195
200 205Ile Gly Glu His Ile Leu Ala His Ile
Gln His Glu Val Asp Phe Leu 210 215
220Phe Cys Met Asp Val Asp Gln Val Phe Gln Asp Asn Phe Gly Val Glu225
230 235 240Thr Leu Gly Gln
Leu Val Ala Gln Leu Gln Ala Trp Trp Tyr Lys Ala 245
250 255Ser Pro Glu Lys Phe Thr Tyr Glu Arg Arg
Glu Leu Ser Ala Ala Tyr 260 265
270Ile Pro Phe Gly Glu Gly Asp Phe Tyr Tyr His Ala Ala Ile Phe Gly
275 280 285Gly Thr Pro Thr His Ile Leu
Asn Leu Thr Arg Glu Cys Phe Lys Gly 290 295
300Ile Leu Gln Asp Lys Lys His Asp Ile Glu Ala Gln Trp His Asp
Glu305 310 315 320Ser His
Leu Asn Lys Tyr Phe Leu Phe Asn Lys Pro Thr Lys Ile Leu
325 330 335Ser Pro Glu Tyr Cys Trp Asp
Tyr Gln Ile Gly Leu Pro Ser Asp Ile 340 345
350Lys Ser Val Lys Val Ala Trp Gln Thr Lys Glu Tyr Asn Leu
Val Arg 355 360 365Asn Asn Val
370
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