KR-20260064852-A - Vaccinia virus recombinant L1R protein and orthopoxvirus vaccine composition comprising the same

Abstract

The present invention relates to a vaccinia virus recombinant L1R protein and an orthophoxvirus vaccine composition containing the same. More specifically, the invention relates to a recombinant L1R protein from which a transmembrane domain has been removed from a vaccinia virus L1R protein, a recombinant vector for efficiently expressing the said protein, a transformant, a method for producing said recombinant L1R protein, and an orthophoxvirus vaccine composition containing said recombinant L1R protein.

Inventors

• 손은주
• 강향주
• 박소윤
• 김진
• 전혜영
• 송영조
• 유치호
• 김정은

Assignees

• 대한민국(방위사업청장)

Dates

Publication Date

20260508

Application Date

20241029

Claims (18)

1. A gene construct comprising a polynucleotide encoding a vaccinia virus recombinant L1R protein, comprising an amino acid sequence in which amino acid residues at positions 186 through 250 are removed from the amino acid sequence of SEQ ID NO. 1.
2. In paragraph 1, The above recombinant L1R protein is a gene construct comprising the amino acid sequence of SEQ ID NO. 2.
3. In paragraph 1, A gene construct further comprising a polynucleotide encoding an endoplasmic reticulum signal peptide and/or a polynucleotide encoding an endoplasmic reticulum retention signal peptide.
4. In paragraph 3, A gene construct further comprising a polynucleotide encoding a His-tag and/or a polynucleotide encoding a peptide linker.
5. In paragraph 3, The polynucleotide encoding the above endoplasmic reticulum signal peptide (ER signal peptide) comprises the nucleotide sequence of SEQ ID NO. 5 encoding NB (new chaperone binding protein), and is a gene construct.
6. In paragraph 3, The above-mentioned endoplasmic reticulum residual signal peptide is a gene construct comprising the amino acid sequence of HDEL (His-Asp-Glu-Leu), HEEL (His-Glu-Glu-Leu), KDEL (Lys-Asp-Glu-Leu), KEEL (Lys-Glu-Glu-Leu), RDEL (Arg-Asp-Glu-Leu), or REEL (Arg-Glu-Glu-Leu).
7. In paragraph 4, The above peptide linker is a gene construct that is (GGGGS)n (where n is an integer from 1 to 5).
8. In paragraph 4, A gene construct in which the following (i) through (v) are connected sequentially: (i) Polynucleotide encoding an endoplasmic reticulum signal peptide, (ii) a polynucleotide encoding the recombinant L1R protein, (iii) a polynucleotide encoding the above peptide linker, (iv) Polynucleotide encoding the His-tag and (v) Polynucleotide encoding an ER retention signal peptide.
9. In paragraph 8, The above gene construct is a gene construct comprising the nucleotide sequence of SEQ ID NO. 13.
10. A recombinant vector comprising a gene construct of any one of claims 1 to 9.
11. Transformed body transformed with the recombinant vector of paragraph 10.
12. In Paragraph 11, The above-mentioned transformant is a transformant that is a plant body.
13. A method for preparing a vaccinia virus recombinant antigen protein comprising the following (a) and (b): (a) a step of transforming a plant body with the recombinant vector of claim 10; and (b) A step of isolating and purifying vaccinia virus recombinant L1R protein from the above plant.
14. In Paragraph 13, A method for producing a vaccinia virus recombinant antigen protein, wherein the above vaccinia virus recombinant L1R protein is expressed in a water-soluble form in the above plant body.
15. Vaccinia virus recombinant antigen protein containing the amino acid sequence of SEQ ID NO. 2.
16. In paragraph 15, Vaccinia virus recombinant antigen protein produced using the recombinant vector of claim 10.
17. An orthofoxvirus vaccine composition comprising the vaccinia virus recombinant antigen protein of claim 15.
18. In Paragraph 17, A composition in which the above-mentioned orthopoxvirus is a smallpox virus, a vaccinia virus, or a monkeypox virus.

Description

Vaccinia virus recombinant L1R protein and orthopoxvirus vaccine composition comprising the same The present invention relates to a vaccinia virus recombinant protein and an orthofox virus vaccine composition containing the same. Smallpox is an acute disease characterized by fever, blisters, and pustular pathological skin changes, caused by the smallpox virus. It has a very high mortality rate and once accounted for 10% of all causes of death worldwide, including in Korea; however, in 1979, smallpox was declared eradicated globally. Nevertheless, following the report of a single case of monkeypox in the UK on May 7, 2022, cases of infection have been reported in numerous countries including the United States, Brazil, Spain, France, and Colombia, and interest has recently been growing again as the possibility of the smallpox virus being used as a biological terror weapon has become known. In Korea, monkeypox was designated as a Class 2 infectious disease on June 8, 2022, and the crisis level was raised from 'concern' to 'caution' after a Korean national who had visited Germany was confirmed to have monkeypox in June of the same year. Consequently, the need for the development of vaccines to prevent orthofoxviruses, including monkeypox, is emerging. Meanwhile, A17L, A27L, A28L, A33R, B5R, D8L, L1R, and H3 have been reported as vaccinia virus proteins identified as targets of neutralizing antibodies in humans (Non-patent Literature 1). Against this background, the inventors developed a gene construct derived from the major antigen(s) of vaccinia virus and a recombinant vector containing the same for high-efficiency production in plants, and completed the present invention by demonstrating the efficacy of a recombinant protein produced using the said recombinant vector as a vaccine. FIG. 1 shows a gene construct [pTEX-NB:L1RdC:L10H] encoding a vaccinia virus recombinant L1R protein (L1RdC) according to one embodiment of the present invention, the base sequence thereof, and the amino acid sequence of the expressed recombinant L1R protein. FIG. 2 is a band image obtained by performing a Western blot to confirm the expression of vaccinia virus recombinant L1R protein (L1RdC) according to one embodiment of the present invention (T, Total extract; S, Soluble; P, Pellet). Figure 3 shows the results of confirming a protein sample obtained during the process of isolating and purifying vaccinia virus recombinant L1R protein (L1RdC) according to one embodiment of the present invention by electrophoresis followed by Western blot (A) and Coomassie blue staining (B). FIG. 4 shows a gene construct encoding various forms of vaccinia virus recombinant L1R antigens according to one embodiment of the present invention. FIG. 5 shows a gene construct [pTEX-NB:H:CBM3:L1RdC:L10H] encoding a recombinant protein in which CBM3 (Cellulose-binding module 3) is fused to the upper portion of a vaccinia virus recombinant L1R protein (L1RdC) according to one embodiment of the present invention, the base sequence thereof, and the amino acid sequence of the expressed recombinant L1R protein. FIG. 6 shows a gene construct [pTEX-NR:L1RdC:L10H] for targeting vaccinia virus recombinant L1R protein (L1RdC) to chloroplasts according to one embodiment of the present invention, the nucleotide sequence thereof, and the amino acid sequence of the expressed recombinant L1R protein. FIG. 7 shows a gene construct [pTEX-L1RdC:L10H] for targeting vaccinia virus recombinant L1R protein (L1RdC) in the cytoplasm according to one embodiment of the present invention, its nucleotide sequence, and the amino acid sequence of the expressed recombinant L1R protein. FIG. 8 shows a gene construct [pTEX-NB:L1RdC:aghFc] encoding a recombinant protein in which human IgG Fc is fused (deglycosylated) to the C-terminus of a vaccinia virus recombinant L1R protein (L1RdC) according to one embodiment of the present invention, the base sequence thereof, and the amino acid sequence of the expressed recombinant L1R protein. Figure 9 is a band image obtained by performing a Western blot to confirm the expression of vaccinia virus recombinant L1R antigen of each gene construct of Figure 4 (T, Total extract; S, Soluble; P, Pellet). FIG. 10 is a graph showing the change in body weight (A) and survival rate (B) of mice following vaccinia virus challenge inoculation after administration of vaccinia virus recombinant L1R protein vaccine according to one embodiment of the present invention. FIG. 11 is a PRNT result (A) and a graph (B) showing the reduction rate of vaccinia virus plaques compared to a control group (cultured only vaccinia virus) according to the dilution ratio of mouse serum after vaccination with vaccinia virus recombinant L1R protein vaccine according to one embodiment of the present invention. It should be noted that in the following description, only the parts necessary for understanding the embodiments of the present invention are described, and the description of othe