BACULOVIRUS EXPRESSION VECTOR

Abstract

The invention concerns a baculovirus expression vector for recombinantly expressing an FMDV capsid precursor protein under control of a promoter, the expression vector comprising a nucleic acid sequence encoding the FMDV capsid precursor protein and the translational enhancers Syn2 land p10UTR. The invention further relates to a host cell comprising the baculovirus expression vector, a method of producing FMDV virus-like particles (VLPs), and a method of producing a vaccine.

Claims

1. A baculovirus expression vector capable of recombinantly expressing a Foot and mouth disease virus (FMDV) capsid precursor protein under control of a promoter, the expression vector comprising: (i) a nucleic acid sequence encoding the FMDV capsid precursor protein, (ii) a translational enhancer Syn21 located within the 5′ untranslated region (UTR) of the nucleic acid sequence (i) encoding the FMDV capsid precursor protein, and (iii) a translational enhancer P10UTR, located within the 3′UTR of the nucleic acid sequence (i) encoding the FMDV capsid precursor protein.

2. The baculovirus expression vector according to claim 1, wherein the translational enhancer Syn21 has a nucleic acid sequence corresponding to the nucleic acid sequence of SEQ ID NO. 1.

3. The baculovirus expression vector according to claim 1, wherein the translational enhancer P10UTR has a nucleic acid sequence corresponding to the nucleic acid sequence of SEQ ID NO. 2.

4. The baculovirus expression vector according to claim 1, wherein the FMDV is of the A serotype.

5. The baculovirus expression vector according to claim 1, wherein the FMDV is of the O serotype.

6. The baculovirus expression vector according to claim 1, wherein the capsid precursor protein comprises the capsid precursor P1.

7. The baculovirus expression vector according to claim 1, wherein the vector further comprises: (iv) a nucleic acid sequence encoding a protease capable of cleaving the capsid precursor protein into one or more capsid proteins.

8. The baculovirus expression vector according to claim 7, wherein the capsid precursor protein comprises the capsid precursor P1 and the 2A peptide and the protease is 3C.

9. A host cell comprising the baculovirus expression vector according to claim 1.

10. The host cell according to claim 9, which is an insect cell.

11. A method of producing FMDV capsid precursor protein, the method comprising the steps of: (i) infecting a host cell with the baculovirus expression vector according to claim 1, and (ii) harvesting FMDV capsid precursor protein produced by the host cell.

12. A method of producing FMDV virus-like particles (VLPs), the method comprising the steps of: (i) infecting a host cell with the baculovirus expression vector according to claim 7, and (ii) harvesting FMDV VLPs produced by the host cell.

13-14. (canceled)

15. A method of producing a vaccine, which comprises the steps of: (i) producing FMDV VLPs by the method according to claim 12 and (ii) incorporating the FMDV VLPs into a vaccine by addition of a pharmaceutically acceptable carrier.

16. A method of protecting a subject against an infection with FMDV, which comprises the step of expressing an FMDV capsid precursor protein from the baculovirus expression vector according to claim 7 in a host cell to produce a VLP, incorporating the VLP into a vaccine by addition of a pharmaceutically acceptable carrier and administering the vaccine to the subject.

17. (canceled)

Description

FIGURES

[0069] FIG. 1 schematically shows DNA structures for use according to the invention

[0070] FIG. 2 represent in various ways yields when using the invention

[0071] FIG. 3 shows a Western Blot to indicate yield when using the invention

EXAMPLES

[0072] The invention will be further described by way of the following non-limiting examples, which are meant to serve to assist one of ordinary skill in the art in carrying out the invention.

“Opt1” Baculovirus Construct

[0073] The transcription of the gene-of-interest (GOI) in the commercially available standard baculovirus transfer vector, pFastBac® (Thermo Fisher Scientific, Germany) is driven by the polyhedrin (polh) promoter. The resulting mRNA contains a SV40 3′UTR.

[0074] To investigate whether the translation of this mRNA can be improved, the SV40 3′UTR was replaced by the 3′-UTR from the Autographa californica nucleopolyhedrovirus (AcNPV) p10 gene (P10UTR). In addition, the Syn21 translational enhancer was placed in the 5′UTR of the mRNA and just in front of the open reading frame (ORF) encoding the FMDV capsid precursor protein. The resulting modification of the expression cassette was designated “Opt1” (FIG. 1B). Expression of the FMDV capsid precursor protein from the standard expression cassette was compared to expression of the FMDV capsid precursor protein from the “Opt1” expression cassette.

[0075] Cloning of the expression cassette O/TUR/5/09 VP2-S93F used in Example 1 and of the expression cassette A/IRN/7/13 VP2-H93F used in Example 2 was performed by standard cloning procedures well known in the art. The nucleotide sequence of the expression cassette of O/TUR/5/09 VP2-S93F is according to the nucleic acid sequence of SEQ ID NO. 3. The nucleotide sequence of the expression cassette A/IRN/7/13 VP2-H93F is according to the nucleic acid sequence of SEQ ID NO. 4.

Example 1

[0076] In this Example, the expression level of an FMDV capsid precursor protein of an O serotype from the Opt1 expression cassette is compared to the expression from a standard expression cassette for obtaining high yields.

[0077] Erlenmeyer flasks with 100 ml containing 3.2×10.sup.5 cells/ml of Tni cells were infected at MOI=1 with recombinant baculoviruses containing the P1-2A-3Cpro expression cassette based on O/TUR/5/09 VP2-S93F. The culture was harvested at 4 dpi and the cells were collected by centrifugation and subsequently sonicated in Tris-KCl pH8.0 buffer at one-tenth of the original culture volume.

[0078] Samples were analyzed by Western blotting using the anti-VP0 monoclonal antibody (Loureiro et al., 2018, https://wellcomeopenresearch.org/articles/3-88). Visual inspection of the Western blot suggests that the Opt1 version of the baculovirus vector performs slightly better than the standard one in terms of yield of FMDV-related proteins.

[0079] To quantify the differences between the 2 baculovirus constructs, ELISA was performed using monoclonal antibody INT-FMA-01-08 which detects both intact capsids (75S/146S) and pentameric building blocks of the capsids (12S). For this, serially diluted samples were incubated for 1 h at 37° C. on microtiter plates coated overnight at 4° C. with antibody. After removing the samples and three washes with PBS-Tween, a fixed amount of biotinylated INT-FMA-01-08 antibody was added to plates and incubated for 1 h at 37° C. The biotinylated antibody was removed and plates were washed three times with PBS-Tween, after which peroxidase-conjugated streptavidin was added to the plates followed by chromophoric detection.

[0080] Results of Western blotting are shown in FIG. 2A. Results of ELISA are shown in FIG. 2B. The ELISA shows an increase in the expression of the FMDV-related proteins of the O serotype of 1.4-fold for the Opt1 expression cassette compared to the standard expression cassette. This may not seem substantial but given the fact that a production run typically takes 5 days, this means that the same amount of antigen can be made in 10 days (2 runs) using the Opt1 expression cassette, compared to about 15 days (3 runs) when using the other expression cassette.

Example 2

[0081] In this Example, the expression level of an FMDV capsid precursor protein of an A serotype from the Opt1 expression cassette is compared to the expression from the standard expression cassette.

[0082] Infection of cells and cell culture was performed as described in Example 1, except that the expression cassette based on A/IRN/7/13 VP2-H93F was used.

[0083] Samples were analyzed by Western blotting as described in Example 1. Visual inspection of the Western blot suggests that the Opt1 version of the baculovirus vector performs better than the standard one in terms of yield of FMDV-related proteins.

[0084] Results of Western blotting are shown in FIG. 3 Based on band intensities, it is estimated that the increase in the expression of the FMDV-related proteins of the A serotype is 3-fold for the Opt1 expression cassette compared to the standard expression cassette.

Example 3

[0085] In this Example, the expression level resulting from the Syn21 and P10UTR translational enhancers in the Opt1 expression cassette was compared with a commercial system (TopBac®, Algenex). In the TopBac® expression vector, expression is achieved under the control of the polyhedron (polh) promoter, and a homologous repeated (hr) transcription enhancer sequence operatively cis-linked to p10 chimeric promoters. The TopBac ° expression vector was described to achieve an up to 4-fold increase in the production yield of a recombinant model protein (green fluorescent protein, GFP) with respect to a standard baculovirus vector. (Löpez-Vidal, J. et al, PLoS ONE 10(10): e0140039).

[0086] Infection of cells and cell culture was performed as described in Example 1.

[0087] Cell and supernatant were analyzed by Western blotting as described in Example 1. Visual inspection of the Western blot suggests that the Opt1 version of the baculovirus vector outperforms both the commercial TopBac ° vector in terms of yield of FMDV-related proteins.

[0088] To better quantify the differences between the 2 constructs, ELISA was performed as described in Example 1.

[0089] The ELISA data confirmed the Western blot results in that the Opt1 expression vector provided overall higher protein yields than the TopBac® vector. In fact, the TopBac® vector, although successful for many other proteins like GFP, does not achieve the high level of expression of the FMDV P1-2A-3Cpro cassette of the Opt1 expression vector (see Table 1 below).

TABLE-US-00001 TABLE 1 ELISA results showing expression level resulting from the Opt1 construct compared to a commercial system. Expression levels obtained with Opt1 were set at 100%. ELISA (%) Baculovirus cell culture 30 expression vector cells medium Opt1 100 100 Top-Bac  27  20

CONCLUSIONS

[0090] For two FMDV strains belonging to two different serotypes, O and A, an increase in FMDV protein expression levels was obtained by using the Opt1 baculovirus construct comprising the Syn21 and P10UTR translational enhancers compared to a standard expression system. Surprisingly, an increase in expression level by using the Opt1 baculovirus expression vector was even achieved compared to a commercial system containing other translational enhancers, and which is described in the art to achieve an increase in expression level and protein yield.