LU503210B1 - A live vector vaccine of Baculovirus based on SARS-CoV-2 S protein and the application - Google Patents

Abstract

The present invention relates to the technical field of gene recombination and vaccine, and more particularly to baculovirus live vector vaccine based on SARS-CoV-2 S protein and application thereof. According to the invention, CMV promoter is constructed to replace baculovirus promoter and two kinds of baculovirus with unmodified promoters to obtain recombinant viruses vAc-S and vAc-S-CMV, this invention introduces S gene and expresses S protein on the surface of baculovirus, in order to explore the cellular immunity and humoral immune response in mice caused by injection of the two kinds of recombinant baculovirus, therefore, the invention provides a new idea for the development of SARS-CoV-2 vaccine.

Description

DESCRIPTION LU503210

À live vector vaccine of Baculovirus based on SARS-CoV-2 5 protein and the apphestion

Technical field

The present invention relates to the technical field of gene recombination and vaccine, and more particularly to baculovirus five vector vaccine based on SARS-CoV-2 S protein and application thereof

Background

Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory distress syndrome coronavirus 2 (SARS-CoV-23, which is a new coronavirus disease discovered in Wuhan,

China at the end of December 2019. On January 30, 2020, the World Health Organization declared

SARS-CoOV-2 a global epidemic, and it is still prevalent in the world, At present, there is no drug that can effectively treat it, sc using vaccine is still the best solution to deal with the threat incurred by the virus. At present, it is mainly RNA vaccine and inactivated vaccine clinically.

Transportation and preservation conditions are the restriction of RNA vaccine, and it is easy inactivate during transportation and preservation. Inactivated vaccines have a short duration and poor immune efficacy. Therefore, it is still necessary to find a safe and efficient vaccine preparation method, which can not only enrich the types of vaccines, but also effectively prevent the current severe epidemic situation,

Baculovirus (BV) 15 an envelope virus with a circular double-stranded DNA genome, which can be divided into three species. At present, the most studied one is Autographa californica multiple nuclear polyhedrosis virus (ACMNPV} AcMNPV is widely used because it has the following advantages: 1} it can be translated and decorated correctly; 2) It can accommodate the insertion of multiple genes or a large fragment; 3) ft is safe and can not infect humans, 43

Baculovirus can infect a variety of mammalian cells, making it a a potential candidate for gene therapy. Baculovirus has the characteristics of non-toxicity and high transduction efficiency to human and mammalian cells, so the expression vector constructed by baculovires has great advantages in vaccine development, foreign protein expression and gene therapy.

This invention mainly focuses on the immune research of structural protein S protein bU503210

SARS-CoV-2, and uses modified and unmodified baculoviruses as vectors to express S protein in mice, 80 as fo sindy its inuninne response in mice, providing theoretical basis for clinical research of vaccines.

In the present invention, we constructed CMY promoter to replace baculovirus promoter and two kinds of baculoviruses with unmodified promoters to express human SARS-CoV-2 S gene to obtain recombinant viruses vAc-S and vAc-S-CMYV, and exploring the cellular and humoral immune responses in mice caused by direct injection of these two recombinant baculoviruses into mice.

Summary

The main purpose of the present invention 15 to construct a sate and efficient vaccine targeting

SARS-CoY-2, using modified and unmodified baculoviruses as vectors to express $ protein in mice, which could become a candidate method to prepare SARS-CoV-2 vaccine. According to this invention, the recombinant transfer vectors pFB-S-CMV and pFB-S are provided. The artificial chromosome recombinant plasmids cÂcFB-S and cÂcFHE-S-CMYV of SARS-Cov-2 bacteria were provided. The recombinant baculovirus vAc-s-CMV and vAc-s are provided. To provide the construction method of recombinant virus SARS-CoV-2 8. Provide the application of recombinant SARS-CoV-2 virus. The purpose of the invention is realized by the following technical scheme:

The purpose of the invention is realized by the following technical scheme:

The genome of SARS-CoV-2 is about 29 9kbp in length, it comprises a 5' cap structure,

ORFla and ORFIL, ppla, and ppiab is encoded by ORFTa and ORFIb, and further hydrolyzed into 16 nonstructural proteins {nsps), and the remaining ORF region encodes 8 accessory proteins {3a, 3b, p6, Ta, Tb, Sb, 9b and orf14) and 4 structural protems §, E, MN, 3'poly (A3 tail, S protein is a type I fusion protein, whose main function 18 to recognize the receptor and mediate the virus to enter the host cell. 8 protein of SARS-CoWV-2 Is composed of two subunits SI and 52. Flynn protease can cleave S protein into S1 and 52 subunits, and S1 contains receptor binding domain {RH} that binds to cell receptor, which is responsible for the binding of virus to receptor angiotensin-converting enzyme 2(ACE2} S protein is highly conservative, which makes it thJ503210 target of vaccine development.

The invention has that beneficial effects as follows:

In this invention, we construct an unmodified baculovirus VAC-S and a modified baculoviruses vAc-5-CMYV that the mammalian promoter CMV replaces the original promoter to express human SARS-CoV-2 S genes, and the vaccine has high viros inhibition rate and good safety performance, thus providing a new idea and method for the prevention and treatment of

COVID-IS.

Brief Description Of The Figures

Fig. 1 illustrates a schematic diagram of construction of recombinant baculovirus vector.

Fig, 2 tlusirates the fragment of recombinant plasmid obtained by double enzyme digestion of Ken Land Xhoi L

Fig. 3 Ulustrates the recombinant plasmid was transformed into competent cells of DHIOBac, and the recombinant cÂc-5/cAc-5-CMYV Bacmids was identified by colony PCR after screening with blue and white spots.

Fig. 4 illustrates the expression of $ protein in virus-transduced BHK cells and sf9 cells.

Fig. S illustrates on the 0, 1415 28% 42 52% days after immunization, the IgG of SARS CoV- 2 anti-& protein in the serum of mice was detected by ELISA. Among them, the specific antibody in PBS group remained ai a low level, and the antibody in vAc-S and vÂc-5S-CMV groups increased significantly 28 days after immunization. On the 52% day, the antibody levels of vAc-5 and vAc-S-CMV immunization groups were 30 times higher than those of PBS control group, and there was no significant difference between the groups. The antibody level of vAc-S was slightly lower, but increased after the second booster. vAc-WT, an empty carrier control group, detected a certain antibody level which was 3 2 times higher than that of PRX group after immunization for 28 days and 42 days, and 1.4 times higher than that of irmmunization group after 52 days.

Fig. 6 Ulustrates the IFN-y produced in CD4+ positive cells of vAC-S-CATV immunized group was higher than that of IL-4.

Fig. 7 illustrates the level of IFN-y produced by CD8+ positive cells in vAc-S-CMRU503210 immunized group was higher than that of IL-4.

Fig. 8 illustrates 10 days after the last immunization, the serum of mice immunized with PBS,

VAC-WT, vAc-§ and vAc-S-CMV was tested by ELISA using pseudovirus systems. If can be seen that the sera of mice immunized with vAc-S and vAc-S-CMV all produced effective neutralizing antibodies, and the titer of neutralizing antibodies produced by vAc-S immunized mice was higher than that produced by vAc-S-CMV immunized mice.

Description of the present invention

The following embodiments are provided for the sole purpose of iflustrating the principle of the present invention, they are in no way intended to limit or narrow the scope of the invention.

Embodiment 1 transformation of transfer vector pFastBac Dual (FIG. 13 pFastBac Dual was digested with BarnH T and Smal L and the gel extraction fragment was enzymatically linked with pT-CMV vector fragment digested by the same enzyme at 4°C overnight to oblain pFB-CMY, which was transformed into Ecol DHSo, and the positive clone was identified by enzyme digestion, and named pFB-CMYV. 5 gene was cloned into pFB-CMV plasnud by using Kpn {and Xhol 1 sites, and the recombinant transfer vector pFB-S-CMV was constructed.

Embodiment 2 identification of recombinant plasmid by double restriction enzyme digestion (FIG 23

Kpni G Sul

Xhoi 1 G Sul 1xM Zul

DNA lug ddH OG up to 20ul

After 2 bh at 37°C, the agarose nucleic acid electrophoresis gel runmog was used for verification. 0.3g agarose was dissolved in 30md IxTAE, it was heated in microwave oven tor

Trin with high power until there were no visible particles tn the solution, and it was poured into nucleic acid electrophoresis tank, then comb was inserted into the tank, and it was let stand fbH503210 30min until it was solidified for sample detection

Embodiment 3 Construction of Recombinant Baculovirus (FIG 3}

The recombinant plasmid that identified by double restriction enzyme digestion was transformed into DHi0Bac cells, and the recombinant Bacmids were constructed. The method was briefly described as follows: Two competent cells were taken out from -S0°C and inserted into ice to be meited.

The transformed pFB-S and pFB-S-CMWV(lug) plasnuds were added into competent cells respectively, then the EF tube was put on ice for reaction for 30min, then put into a 42°C water bath for thermal activation for 90s, and immediately put into tee for standing for 2-Smin, 900ul of preheated SOC culture medium was added into the EP tube respectively, and then put into a 37°C shaking table at 220rpm for 4h, then was centrifuged at 12000rpm for Smin, and 800ul of supernatant was discarded, the precipitate was resuspend | and it was streaked on the solid medium containing 100ug/ml X-gal, 40ug/mi IPTG, Tug/miGen”, S0ug Kan" and 10ug Tet”, then the dish was turned upside down, and cultured at 37°C overnight.

Four white spots were selected for pF-8, five white spots and one blue spot were selected for pF-S-CMY bacteria, which were added into LE medium containing resistance of Gen” and Kan” and shaken at 37°C, The next day, M13 universal primers and gene-specific primers were used for

PCR identification. The system is as follows:

Takara Tag enzyme G12Sui 10x Ex Tag Buffer 2.54

ANTP Mixture Qui

PUC/MI3-F lui

PF-S-CMV-R ful

Bacterial liquid ful ddH: OC up to 25ul

PCR reaction system LU503210 34°C 30s 98°C 108 58°C 30s x30 72°C min 72°C Son

The recombinant virus plasmid was successfully identified and transfected into sf@ cells.

After the recombinant baculovirus was oblained by culturing at 26°C for 4-5 days, the virus was amplified and purified by ultracentrifugation at 100,000 ¢ at 4°C for 4 hours. The titer of purified virus was determined by PCIDSO, and it was stored for attacking virus.

Embodiment 4 Recombinant baculovirus transduced BHK cells Western blot was used to detect the expression of S protein in recombinant baculovirus transduced BHE cells (FIG. 4)

A proper amount of BHK cells was put in a 35mm cell culture dish, and cultured at 37°C overnight until the density reached 65%, the culture medium was sucked up, IOUMOI of recombinant baculovirus was added, and supplemented with 300ul with Grace’ for absorption at 37°C for 2h. the virus solution was sucked, and washed with DMEM, 2mi DMEM-+10%4FBS and 10m M sodium butyrate were added, and was cultured at 37°C for 24h, then observed the results. After 24 hours of transduction, BHE cells were collecied according to the method in Part 1, Part 2.4.6. After membrane was transferred, the membrane was sealed in TBST containing 5% skim milk powder at room temperature for 2h, and then incubated overnight at 4°C in S monoclonal antibody of

SARS CoV-2 (diluted at 1:1000) After membrane washing, it was put into goat anti-rabbit anti-

IgG {1:10000, ZSGB-BIO) shaker for incubation for 2h, and finally developed by chemilurminescence.

Embodiment 5 Immunization of animals (FIG.5)

Female BALB/c mice aged 8-12 weeks were purchased from Animal Experimental Center of

Anhui Medical University. They were randomly divided into four groups with 6 animals in each group. vÂc-S or vAC-S-CMY virus, vAc-WT and PBS were injected respectively as controls. 1503210 total of 7 5x 10° virus was injected subcutaneously and intraperitoneally, once every two weeks for 4 tres. Before each injection, blood was taken from mouse inner canthos, and the neutralizing antibody titer and IgG level in serum were measured.

Embodiment 6 Isolation and culture of spleen lymphocytes

Ten days after the last immunization, the mouse spleen was taken out under sterile conditions, and 4mi of lymphocyte separation solution was added into a 35mm cell culture dish. The spleen was homogenized on a 200-mesh nylon net with a sterile syringe piston, then added into a 153ml sterile centrifuge tube 500pl RPM ! 1640 culture solution was added to the surface of the separation solution, and centrifuged with 800g horizontal rotor for 30 min, the cells were sucked at the level of lymphocytes into a 15 mi centrifuge tube, 10m! RPM I 1640 was added, and mixed well, centrifuged with 250g horizontal rotor for 10 rain, the supernatant was discarded, and was re-suspend in 1nd RPMI 1640 containing 10%FBS, and counted with blood counting chamber for tater use.

Embodiment 7 Flow cytometry detection (FIG 6, 7)

After counting the resuspension of isolated spleen lymphocytes, 1710° cells were added to 24-well plate in each well, and the mixture of corresponding specific antigen (2us/ml of $ antigen) and human CD28 antibody (Spg/well} was added as co-stimulator to incubate for 5h, According to the operation instructions of Cytofix/Cytoperm W/Golpi Stop Kit, Golgt stop {D 5ul/ml) was added and blocked at 37°C for 4h, then Tul FITC-CD4 and 12501 APC-CDEs were added, and

Was stained in the dark at 4°C for 30 min, and washed with PBS, the membrane was fixed and permeabilized, intracellular PE-IFN-yand PE/Cy7- IL-4 was added for 30min, S00ui BD 1XPerm/wash buffer was added for resuspending, and centrifuged at 100Grpro for Smun, then the supernatant was discarded, and resuspended in Flow eytometry Staining buffer for computer detection { Beckman, EPICS ALTRA IL USA)

Embodiment & neutralization Embodiment (FIG. 8} 293T-ACF2 celts were inoculated in 96-well plate at 4*10"/mi, and cultured at 37°C until the density reached 40-50%. inactivate the serum of mice to be detected at 36°C for 30min, the serum was diiuted from 1:4 to 1.128 with DMEM, and pseudovirus was added {the final dose is lui pbH503210 well} into the diluted serum to incubated at 37°C for 1h, the cell culture supernatant of 96-well plate was sucked and discarded, the neutralized mixture was added and adsorb at 37°C for 7h, the supernatant was removed, DMEM+10%FBES complete medium was added withi00ul/ well, and incubated in an incubator containing 3% C02 at 37°C for 48h according to the Bivuntian kit, 100ul

Bright-Lumi II firefly luciferase detection reagent was added into each well, and incubated at room temperature for Smin, then used the multifunctional microplate detection system (BioTek,

CYTATION 5) to detect the luciferase activity, and the veutralizing antibody titer was calculated with the highest antibody that inhibited viral infection up to 50%,

Claims (2)

CLAIMS LU503210

1. Live vector vaccine of Baculovirus based on SARS-CoY-2 8 protein is characterized in comprising recombinant transfer vectors pFB-S-CMV; pFB-&; SARS-CoV-2 bacterial artificial chromosome recombinant plasmids cÂAcFB-S , cÂAcFB-S-CMV, recombinant baculovirus vAc-S-CMV vAc-S.

2. Live vector vaccine of Baculovirus based on SARS-CoV-2 S protein according to claim 1, and its application in the field of inhibition of SARS-CoV2 infection.