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KR-102963468-B1 - Modified S1 subunit of coronavirus spike protein

KR102963468B1KR 102963468 B1KR102963468 B1KR 102963468B1KR-102963468-B1

Abstract

The present invention relates particularly to a recombinant avian coronavirus spike protein or a fragment thereof comprising a mutation to cysteine at the 267th amino acid position. In addition, the present invention relates to an immunogenic composition comprising an avian coronavirus having such a spike protein.

Inventors

  • 크래머-쿠엘 아니카
  • 슈테판 토마스 민
  • 필립 한스-크리스티안

Assignees

  • 베링거잉겔하임베트메디카게엠베하

Dates

Publication Date
20260513
Application Date
20200506
Priority Date
20190510

Claims (20)

  1. An avian coronavirus spike protein, wherein at least a portion of the S1 subunit is derived from an avian coronavirus having restricted cell or tissue tropism, the amino acid position at 267 is cysteine, the amino acid sequence of SEQ ID NO. 1 is used to determine the position numbering in the spike protein, and the cysteine at the amino acid position at 267 results in the extended cell or tissue tropism of the avian coronavirus.
  2. A recombinant avian coronavirus spike protein comprising a mutation to cysteine at the 267th amino acid position, wherein the amino acid sequence of SEQ No. 1 is used to determine position numbering in said spike protein, and said mutation to cysteine at the 267th amino acid position results in extended cell or tissue orientation of said avian coronavirus.
  3. In claim 1, the avian coronavirus spike protein, wherein the avian coronavirus is an infectious bronchitis virus (IBV).
  4. An avian coronavirus spike protein according to claim 1, wherein the cysteine at amino acid position 267 is introduced by mutation.
  5. The avian coronavirus spike protein of claim 1, wherein the avian coronavirus infects and/or replicates in at least one cell line selected from the list consisting of DF-1 (Douglas Foster), PBS-12, PBS-12SF (PBS-12 serum free), BHK21 (baby hamster kidney), HEK 293T (human embryonic kidney), Vero (Verda Reno), MA104, RK13 (rabbit kidney), LMH (leghorn male hepatoma), MDCK (Madin-Darby canine kidney), MDBK (Madin-Darby bovine kidney), PK15 (porcine kidney), PK2A (porcine kidney), SF9, SF21, and SF+ ( Spodoptera frugiperda ).
  6. An avian coronavirus spike protein according to claim 1, wherein the 267th amino acid position is located within the S1 subunit of the spike protein.
  7. In claim 1, the spike protein is an avian coronavirus spike protein that is not derived from the IBV Beaudette strain.
  8. In paragraph 3, the IBV spike protein is derived from IBV having a genotype or serotype or strain selected from the list consisting of Arkansas, Brazil, California, Connecticut, Delaware, Netherlands, Florida, Georgia, Gray, Holte, Iowa, Italy-02, JMK, LDT3, Maine, H52, H120, M41, Pennsylvania, PL84084, Qu, QX, Q1, SE 17, variant 2 and 4/91.
  9. In paragraph 3, the IBV spike protein is selected from a list of genotypes consisting of GI-2 to 27, GII-1, GIII-1, GIV-1, GV-1 and GVI-1.
  10. In claim 3, the IBV spike protein comprises or includes a sequence having at least 80%, 85%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98%, or 99.99% sequence identity with the amino acid sequence shown in SEQ ID NO. 2, 3, 4, 5, 6, 7, 8, or 77.
  11. In paragraph 3, the IBV spike protein, wherein at least a portion of the S1 subunit is derived from IBV selected from the list of genotypes or serotypes or variants consisting of Arkansas, Brazil, California, Connecticut, Delaware, Netherlands, Florida, Georgia, Grey, Holte, Iowa, Italy-02, JMK, LDT3, Maine, H52, H120, M41, Pennsylvania, PL84084, Qu, QX, Q1, SE 17, variant 2 and 4/91.
  12. In claim 1, the avian coronavirus spike protein having limited cell or tissue orientation is limited to infection and/or replication in developing eggs and/or primary chicken kidney cells.
  13. A nucleic acid having a sequence encoding the spike protein of claim 1.
  14. A plasmid containing the nucleic acid sequence of claim 13.
  15. A cell containing the plasmid of paragraph 14.
  16. A virus particle comprising the spike protein of claim 1.
  17. An avian coronavirus comprising the spike protein of claim 1.
  18. In paragraph 17, the above-mentioned avian coronavirus is attenuated avian coronavirus.
  19. A cell containing the virus particle of paragraph 16.
  20. Immunogenic composition comprising the spike protein of claim 1.

Description

Modified S1 subunit of coronavirus spike protein Sequence list The present application includes a list of sequences according to 37 C.F.R. 1.821-1.825. The list of sequences attached to the present application is incorporated by reference in its entirety by the present application. Infectious bronchitis virus (IBV), an avian coronavirus, is a prototype gamma-coronavirus of the family Coronaviridae within the order Nidovirales . IBV primarily infects the upper respiratory tract epithelium of chickens, causing respiratory diseases generally complicated by secondary bacterial pathogens (Cook et al. 2012. Avian Pathol. 41:239-250). Some IBV strains additionally affect the renal tubules, oviducts, and parts of the gastrointestinal tract, causing pathological lesions and clinical symptoms in these organ systems. These viruses are present globally in both commercial and backyard chickens. Due to high genomic variability, IBV is distinguished into a wide variety of genotypes, serotypes, and protectotypes. IBV is currently considered one of the most economically relevant viral pathogens in the poultry industry. Infectious bronchitis virus is an enveloped virus with a 27.6 kb positive-sense single-stranded RNA genome (Reference [Cavanagh 2007. Vet. Res. 38:281-297]). The first two-thirds of the viral genome contains a large coding region (also denoted as gene 1) divided into two open reading frames 1a and 1b, encoding at least 15 non-structural proteins involved in RNA replication, editing, and transcription. The last one-third of the viral genome codes for the following structural proteins: spike protein (S, encoded by gene 2), envelope protein (E, encoded by gene 3c), membrane protein (M, encoded by gene 4), and nucleocapsid protein (N, encoded by gene 6). Proteins S, E, and M are part of the viral envelope, while protein N forms the ribonucleic acid protein core together with the viral RNA. The coronavirus spike protein determines host species tropism (reference [Kuo et al. 2000. J. Virol. 74:1393-1406]). It is a dimeric or trimer membrane-penetrating protein that is cleaved into two subunits, S1 and S2, by proteolysis. The glycosylated S1 domain forms the 'head' of the spike protein and contains a receptor-binding domain that interacts with 2,3-linked sialic acid on the host cell surface (reference [Promkuntod et al. 2014. Virology. 448:26-32]). The S2 domain includes the ectodomain ('stalk') located in the cytoplasm, the membrane-penetrating domain, and the remainder of the endodomain. The attenuated IBV live vaccine variants H52 and H120, which are widely used to date, were developed in the Netherlands in the 1960s by serial passage of the Massachusetts IBV variant in developing eggs (reference [Bijlenga et al. 2004; Avian Pathol. 33:550-557]). The aforementioned vaccine variants must also be cultured in developing eggs for production. Today, IBV vaccines (both inactivated and live vaccines) are still bred in developing eggs, which is a difficult and costly process. The only cell line-adapted IBV described to date is the Beaudette IBV variant, which replicates efficiently in Vero and BHK cells. The literature [Casais et al., 2003. J. Virol. 77; 9084-9089] suggests that the S protein of Beaudette is a determinant of cell line orientation by generating a recombinant IBV using the ectodomain sequence of the Beaudette spike, which can transfer extended cell line orientation to another IBV (M41). International Publication WO 2011/004146 discloses that the S2 subunit derived from Beaudette is responsible for extended tissue orientation. The sequence within the S2 subunit, which is the heparan-sulfate binding site of Beaudette, has been identified as being responsible for extended cell line orientation. In addition, the literature [Bickerton et al., 2018. Journal of Virology 92 (19)] discloses a 8-amino acid butet-specific motif. However, recombinant IBV containing the butet spike S2 subunit is not suitable as a vaccine. The literature [Ellis et al., 2018. J. Virol. 92 (23)] states that recombinant butet containing a chimeric spike having a heterologous S1 subunit derived from M41 or QX combined with the butet spike S2 subunit does not provide sufficient protection against the S1 allogeneic challenge. In addition, the wild-type Butet does not provide protection against allogeneic test infections, as with other licensed vaccines belonging to the Massachusetts serotype (see references [Hodgson et al., 2004: J Virol 78:13804-13811] or [Geilhausen et al., 1973: Archiv fur die gesamte Virusforschung 40: 285-290]). The literature [Fang et al., 2005, Biochemical and Biophysical Research Communication 336; pages 417 to 423] discloses that the adaptation of the butet for proliferation in Vero cells produced 49 amino acid modifications, 26 of which were located within the spike protein. In summary, replacing the spike protein with a heterologous Butet spike protein to provide an IBV vaccine with extended cell or tissue targetin