RU2851081C2 - Rabdovirus-negative cell line of the insect spodoptera frugiperda and its screening, identification and application - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to the rabovirus-negative cell line of the insect Spodoptera frugiperda WSK-Sf9 with accession number CCTCC C202246. In addition, the invention also relates to the use of the specified cell line for obtaining a recombinant protein based on a baculovirus expression system.

EFFECT: provision of a rabovirus-negative insect cell line suitable for the expression and production of exogenous recombinant proteins, wherein the level of said expression is higher compared to Sf9 cells.

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Description

Field of invention

The invention relates to the fields of invention of genetic engineering and cellular engineering and relates to a new cell line of the insect Spodoptera frugiperda and, in particular, relates to a rhabdovirus-negative cell line of the insect Spodoptera frugiperda and its screening, identification and use.

Prior art

The insect cell expression system is widely used for recombinant protein production and offers many advantages over other expression systems. For example, since insect baculovirus only parasitizes invertebrates, this expression system offers high safety and high recombinant protein expression levels. It can correctly fold and modify the recombinant protein after translation to produce a biologically active protein. Furthermore, the insect cell expression system is adapted to the complex expression of multiple genes, such as virus-like particles, and can be used for large-scale serum-free cultures.

Currently, numerous recombinant protein-based vaccines produced using insect cell expression systems have demonstrated good efficacy and safety and have been approved for listing worldwide, including the cervical cancer vaccine Cervarix, manufactured by GSK, the prostate cancer vaccine Provenge, manufactured by Dendreon, and the influenza vaccine FluBlok, manufactured by ProteinSciences. Furthermore, many recombinant protein-based vaccines have also demonstrated promising potential in preclinical studies.

The Sf9 cell ( Spodoptera frugiperda cell) is the most widely used insect cell in the insect baculovirus expression system for the expression and production of foreign proteins, including antibodies, vaccines, and recombinant proteins. Sf9 cells are derived from the IPLBSF-21 cell line (also called Sf21), which is derived from the ovarian tissue of the fall armyworm ( Spodoptera frugiperda ) pupae, isolated and cultured in 1977. Sf rhabdovirus is a novel negative-strand RNA virus discovered by FDA researchers in 2014 in the Spodoptera frugiperda Sf9 cell line and its parental Sf21 cell line, including information on the genes for the structural proteins N, P, M, G, and L. In addition, an additional X gene sequence of unknown function between G and L was tested. Although Sf rhabdovirus is not integrated into the host cell genome, it has a complete genome and can be packaged into a complete viral particle, which leads to potential risks in the production and use of recombinant proteins, such as vaccines derived from the baculovirus expression system of Sf9 cells.

Brief summary of the invention

According to the invention, the rhabdovirus-negative cell line of the insect Spodoptera frugiperda WSK-Sf9 (abbreviated WSK-Sf9) is obtained as a result of screening and identification, and this cell line can be used to produce recombinant proteins and vaccines based on recombinant proteins based on the baculovirus expression system.

The aim of the invention is to provide a rhabdovirus-negative cell line of the insect Spodoptera frugiperda WSK-Sf9 with the accession number CCTCC C202246. The cell line was selected under the name of the cell line Spodoptera frugiperda WSK-Sf9, derived from Sf9, on February 16, 2022. The collection center is the China Type Culture Collection Center (CTCC), and its address is: Collection Center of Wuhan University, No. 299 Bayi Road, Wuhan District, Wuhan City, Hubei Province, with postal code 430072.

The rhabdovirus-negative Spodoptera frugiperda WSK-Sf9 insect cell line according to the invention is characterized in that the monoclonal cell is screened using the limiting dilution method and verified using various highly sensitive testing methods, such as nested PCR (polymerase chain reaction), next-generation transcriptome sequencing, fluorescence-based quantitative real-time PCR, and TaqMan probe-based real-time PCR, and then the rhabdovirus-negative Spodoptera frugiperda WSK-Sf9 insect cell line is obtained. The cell is tested for sterility, mycoplasma, exogenous virus, and tumor-inducing ability in accordance with pharmacopoeial requirements, and the results show that all indicators meet these requirements, and this cell can be used as a cell matrix for production.

Another aim of the invention is to propose the use of the rhabdovirus-negative cell line of the insect Spodoptera frugiperda WSK-Sf9 for the production of a recombinant protein based on a baculovirus expression system.

The use includes the use of a medicinal product or vaccine based on a recombinant protein based on a baculovirus expression system.

A recombinant protein-based medicinal product includes a cytokine, hormone, recombinant enzyme, or antibody.

The cytokine includes recombinant human interleukin, recombinant human epidermal growth factor, recombinant human interferon, recombinant human fibroblast growth factor, recombinant human erythropoietin, or recombinant human granulocyte-macrophage colony-stimulating factor.

The hormone includes recombinant human growth hormone, recombinant human insulin, insulin analogue, or recombinant human follicle-stimulating hormone.

The recombinant enzyme includes recombinant human alpha-glucosidase or recombinant human prourokinase.

The antibody includes a monoclonal antibody, a Fab antibody, an scFv antibody, or a nanobody.

The vaccine includes a recombinant protein vaccine or a virus-like particle vaccine.

The recombinant protein-based vaccine includes a SARS-CoV-2 protein-based vaccine, an influenza virus protein-based vaccine, a recombinant syncytial virus protein-based vaccine, a hepatitis B virus protein-based vaccine, or a rabies virus protein-based vaccine, and preferably a SARS-CoV-2 protein-based vaccine.

The virus-like particle vaccine includes a SARS-CoV-2 virus-like particle vaccine (SARS-CoV-2-VLP), a human papillomavirus virus-like particle vaccine (HPV-VLP), an influenza virus-like particle vaccine (HA-VLP), a poliovirus virus-like particle vaccine (PV-VLP), a respiratory syncytial virus virus-like particle vaccine (RSV-VLP), or an enterovirus vesicular stomatitis virus-like particle vaccine (EV71-VLP). Preferably, the virus-like particle vaccine is a SARS-CoV-2 virus-like particle vaccine.

The technical solution for implementing the use of WSK-Sf9 for producing a recombinant protein or vaccine based on the baculovirus expression system is as follows: the Bac-to-Bac insect baculovirus expression system is used to package and produce baculovirus and infect WSK-Sf9 cells to express the target protein, so that the target recombinant protein is obtained through affinity purification technology.

The advantageous effect of the invention is as follows: a monoclonal cell is screened using the limiting dilution method and tested using various highly sensitive testing methods such as nested PCR, next-generation transcriptome sequencing, fluorescence-based quantitative real-time PCR, and TaqMan probe-based real-time PCR, and then a rhabdovirus-negative Spodoptera frugiperda WSK-Sf9 insect cell line is obtained. The cell is tested for sterility, mycoplasma, exogenous virus, and tumor-inducing ability in accordance with pharmacopoeial requirements. The results show that all indicators meet these requirements, and this cell can be used to produce recombinant protein-based products, such as protein vaccines, for clinical use. In addition, according to the invention, the Bac-to-Bac insect baculovirus expression system is used to package and produce baculovirus and infect WSK-Sf9 cells to express the target protein, so that the target recombinant protein is obtained through affinity purification technology or other technical routes.

According to the invention, the rhabdovirus-negative cell line of the insect Spodoptera frugiperda WSK-Sf9, obtained as a result of screening and identification, has the accession number CTCC C202246. This cell line was selected on February 16, 2022. The collection center is the China Type Culture Collection Center (CTCC), and its address is: Collection Center of Wuhan University, No. 299 Bayi Road, Wuhan District, Wuhan City, Hubei Province, with a postal code of 430072. The name and identification characteristics of the selected culture are the cell line Spodoptera frugiperda WSK-Sf9, derived from Sf9.

Description of graphic materials

Fig. 1 shows the result of agorose gel electrophoresis after nested PCR for identifying WSK-Sf9 cells according to the invention;

Fig. 2 shows the morphological characteristics of the rhabdovirus-negative cell line of the insect Spodoptera frugiperda WSK-Sf9 obtained by screening according to the invention;

Fig. 3 shows a karyotype analysis of the rhabdovirus-negative cell line of the insect Spodoptera frugiperda WSK-Sf9, obtained as a result of screening according to the invention;

Fig. 4 shows the growth curve of the rhabdovirus-negative cell line of the insect Spodoptera frugiperda WSK-Sf9, obtained as a result of screening according to the invention;

Fig. 5 shows a continuous subculture curve of the rhabdovirus-negative cell line of the insect Spodoptera frugiperda WSK-Sf9, obtained as a result of screening according to the invention;

Fig. 6 shows a time gradient test of an exogenous recombinant protein expressed by the rhabdovirus-negative Spodoptera frugiperda insect cell line WSK-Sf9 obtained by screening according to the invention.

Detailed description of embodiment examples of the invention

The following will explain the solutions of the present invention with reference to specific embodiments. Those skilled in the art will understand that the following embodiments are intended solely to illustrate the present invention and should not be considered limiting. In the event that specific technologies or conditions are not set forth in the embodiments, technologies or conditions described in the literature in the field or the product specifications take precedence. Unless the manufacturer is indicated on the reagents or apparatus used, all are standard products that can be purchased from commercial reagent companies.

The screening, identification and use of the rhabdovirus-negative Spodoptera frugiperda insect cell line WSK-Sf9 will be further illustrated in the following embodiments, and the invention will be further described with reference to the accompanying drawings.

Example 1: Screening and identification of the Sf-rhabdovirus-negative Spodoptera frugiperda insect cell line WSK-Sf9

The only commercially available Sf-rhabdovirus-negative cells, Sf-RVN (Sf-rhabdovirus-negative Sf9), were separated from Sf9 by Professor Jarvis's group using a limiting dilution method combined with antiviral drug treatment (Maghodia AB, et al. Protein expression and purification. 2016; 122:45-55) and verified for Sf-rhabdovirus negativity by nested PCR for the L gene, and are available from GlycoBac (http://www.glvcobac.com/sf-rvn-cells), which has partnered with Millipore/Sigma Inc. to authorize Millipore/Sigma to market them.

According to the non-patent literature: "Ma H, Nandakumar S, Bae EN, Chin PJ, Khan AS, the Spodoptera frugiperda Sf9 cell line is a heterogeneous population of rhabdovirus-infected and virus-negative cells: Isolation and characterization of cell clones containing rhabdovirus X-gene variants and virus-negative cell clones. According to Virology 2019;536:125-33", the Spodoptera frugiperda Sf9 cell line is a heterogeneous cell population that includes two types of cell populations: Sf-rhabdovirus-infected cells and Sf-rhabdovirus-negative cells. One Sf-rhabdovirus-negative cell population can be obtained by the limiting dilution method. According to the invention, a monoclonal cell is selected by a limiting dilution method, and then Sf-rhabdovirus-negative cells are screened and identified by methods such as nested PCR, next-generation transcriptome sequencing, fluorescence-based quantitative real-time PCR and TaqMan probe-based real-time PCR, etc.

1) The parental Sf9 cell line was purchased from ThermoFisher (Lot No.: 2043331), and these purchased cells were defined as passage 0 (PO). The passage used in this screening experiment was passage 4 (P4), and the cell culture medium was serum-free SIM SF medium (Sino Biological, Inc., MSF1). The suspended Sf9 cells were diluted in a 10-fold gradient and seeded in well plates, and these cells were observed daily for several days. When distinct monoclonal cells became visible, they were transferred to a new well plate for expansion. Then, the corresponding cells were collected, total RNA was isolated, and candidate cells were preliminarily identified using a nested PCR primer (Table 1) for the Sf-rhabdovirus-specific M gene.

2) Sf-rhabdovirus-negative candidate cells were continuously subcultured, and cell samples were collected and frozen at -80°C for further use. After 45 passages of continuous culture, nested PCR technology was used to detect cell samples, and the results showed that the Sf-rhabdovirus M genes in P1-P45 WSK-Sf9 were consistently negative (Fig. 1).

3) Next-generation transcriptome sequencing: 5× ¹⁰⁶ parental Sf9 cells and 5× ¹⁰⁶ WSK-Sf9 cells were individually collected for next-generation sequencing, and the results showed that the RNA information of Sf rhabdovirus genes was detected in the transcriptome of parental Sf9 cells (GenBank: KF947078.1), but not in the transcriptome of WSK-Sf9 cells.

4) Fluorescence-based real-time quantitative PCR: Two pairs of qPCR primers for Sf rhabdovirus M genes were designed (Table 2), Bio-Rad SsoFastEvaGreensupermix kit was used for fluorescence-based qPCR analysis, and the results showed that no fluorescence signal was detected in the P3 generation and P28 generation of WSK-Sf9, which proved that WSK-Sf9 cells were Sf rhabdovirus-negative (Table 3).

Note: The data in Table 3 represent the results of measurements of three replicate samples.

5) Probe-based quantitative PCR: A taqMan probe was designed for the M genes of Sf rhabdovirus (Table 4), and the qPCR results showed that no fluorescence signal was detected when tested in the main cell bank (MCB) and working cell bank (WCB) of WSK-Sf9, which proved that WSK-Sf9 cells were Sf rhabdovirus-negative (Table 5).

Note: The data in Table 5 represent the results of measurements of three replicate samples.

Implementation example 2: Growth characteristics of the Sf-rhabdovirus-negative Spodoptera frugiperda insect cell line WSK-Sf9

1) Culture characteristics of WSK-Sf9: The cell could grow in serum-free medium at 27°C to some extent in adherent form or in suspension, with an average diameter in suspension growth of 16.28±0.34 μm and the morphological characteristics of WSK-Sf9 as shown in Fig. 2.

2) Karyotype analysis of WSK-Sf9: Karyotype analysis was performed on the parental Sf9 cells, and the results showed that among the 60 metaphase cells, the number of chromosomes was mainly distributed between 180 and 250, with an average of 215 chromosomes per cell; Meanwhile, karyotype analysis was also performed on WSK-Sf9 cells, and the results showed that among the 60 metaphase cells, the number of chromosomes was mainly distributed between 191 and 538, with an average of 299 chromosomes per cell (Fig. 3). This also means that the Sf-rhabdovirus-negative WSK-Sf9 cell line is different from its parental Sf9 cells.

3) Growth curve of WSK-Sf9 cells: WSK-Sf9 cells in the logarithmic growth phase were diluted to approximately 1× ¹⁰⁶ cells/mL and transferred to 250 mL shake flasks with a hole. The culture volume was 100 mL, and the flasks were placed on a shaker at a constant temperature of 27°C for continuous cultivation. The cell density and viability were counted every 24 hours. The growth curve and viability after three different culture batches are shown in Fig. 4. The cell density reached approximately 1× ¹⁰⁷ cells/mL after 96 hours and remained at this level for 6 days. The highest cell density reached 1.2× ¹⁰⁷ cells/mL, and the cell viability gradually decreased from the 11th day, and the average doubling time was approximately 23 hours.

4) Continuous subculture curve of WSK-Sf9 cells: WSK-Sf9 cells in the logarithmic growth phase were diluted to approximately 1× ¹⁰⁶ cells/mL and transferred to 250 mL shake flasks with an opening. The culture volume was 100 mL, and the flasks were placed on a shaker at a constant temperature of 27°C for continuous cultivation. The cell density and viability were counted every 3 days, and passaging was performed. Usually, after 3 days of culture, the cell density could reach 6-8× ¹⁰⁶ cells/mL with a viability of more than 98%. The continuous subculture growth curve and viability are shown in Fig. 5. After 100 consecutive passages, the cell growth characteristics remained stable.

Case Study 3: Safety Testing of the Sf-Rhabdovirus-Negative Spodoptera Frugiperda Insect Cell Line WSK-Sf9

The following tests were performed on WSK-Sf9 cells according to Part III of China Pharmacopoeia (2020 Edition).

1) Species identification: DNA barcoding method was used to identify WSK-Sf9 cells, and the results showed that WSK-SF9 cells were derived from Spodoptera frugiperda ;

2) Sterility test: The membrane filtration method was adopted to carry out the sterility test, and the results showed that the culture of WSK-Sf9 cells was sterile;

3) Mycobacterium test: The culture method was adopted to test for the presence of mycobacterium, and the results showed that the test for the presence of mycobacterium was negative;

4) Mycoplasma test: The culture method, indicator cell culture method and step-PCR method were adopted to test the presence of mycoplasma, and the results showed that the test for mycoplasma was negative;

5) Spiroplasma test: Fluorescence-based PCR method was adopted to test for the presence of Spiroplasma, and the results showed that the test for the presence of Spiroplasma was negative;

6) Test for the presence of exogenous virus:

(1) The in vitro cell observation method, erythrocyte adsorption test, and hemagglutination inhibition test were adopted to test the subculture of various cells, such as marmoset-derived Vero cells, human MRC-5 cells, Sf9 cells, BHK-21 cells, Aedes mosquito-derived cells, and Drosophila-derived D.Mel cells, and all of these cells showed normal morphology and were negative in the test.

(2) Suckling mice, adult mice and chick embryos (5-6 day old chick embryos and 9-11 day old chick embryos) were inoculated in vitro, and the results showed that they all met the requirements.

7) Retrovirus test: No virus-like particles were detected by transmission electron microscope, subculture infection test, HEK293 cell inoculation test and chemical reagent-mediated virus test, and the results showed that they all met the requirements.

8) Cells were negative in the bovine-origin virus test and the porcine-origin virus test;

9) Other specific viruses were tested, including baculoviruses, flock house virus variant T.ni (FHVvar), rhabdoviruses, reoviruses, togaviruses, flaviviruses, bunyaviruses, asphaviruses, ascoviruses, iridoviruses, poxviruses, baculoviruses, Poldnaviridae, parvoviruses, birnaviruses, reoviruses, picornaviruses, dicistroviruses, nodaviruses, and tetraviruses; and the results showed that all tests were negative and met the requirements;

10) Testing tumor-forming ability: The experimental mice were inoculated with WSK-Sf9 cells, and the results showed that these cells were not tumor-forming and could meet the requirements.

In conclusion, the Sf-rhabdovirus-negative Spodoptera frugiperda insect cell line WSK-Sf9 meets all safety tests and cell matrix requirements for biopharmaceutical production.

Example of implementation 4: Exogenous recombinant protein expressed by the Sf-rhabdovirus-negative cell line of the insect Spodoptera frugiperda WSK-Sf9

A baculovirus expression vector containing the RBD structural domain of SARS-CoV-2 was constructed, and WSK-Sf9 cells were used to package the baculovirus expressing the recombinant protein. Sf9 and WSK-Sf9 cells were cultured separately; when the density reached 2.5 × 10 ⁶ /mL, they were infected with the viruses separately at an MOI (multiplicity of infection) of 0.5. Supernatants were collected before infection (0 h) and after infection (24 h, 48 h, 72 h, and 96 h), and detected by Western blotting using anti-His tag antibodies, and the results showed that the expression level of the recombinant protein in WSK-Sf9 cells was up-regulated compared with that in Sf9 cells. Following production and purification in a GMP laboratory and subsequent adjuvant preparation, the recombinant protein can be used to prevent SARS-CoV-2 infection. This demonstrates that the Sf rhabdovirus-negative Spodoptera frugiperda WSK-Sf9 insect cell line can be used to express and produce exogenous recombinant proteins, such as protein vaccines, and that this expression level is higher than that of Sf9 cells.

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</INSDQualifier>

<INSDQualifier id="q19">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

<NonEnglishQualifier_value>synthetic

construction</NonEnglishQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>tgcccaagctaaggaaaggg</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="9">

<INSDSeq>

<INSDSeq_length>20</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..20</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q20">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

<NonEnglishQualifier_value>synthetic

construction</NonEnglishQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>tgacatgtggtctccaaccg</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="10">

<INSDSeq>

<INSDSeq_length>20</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..20</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q21">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

<NonEnglishQualifier_value>synthetic

construction</NonEnglishQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>gtatgcaggtggttgaggct</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="11">

<INSDSeq>

<INSDSeq_length>21</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..21</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q22">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

<NonEnglishQualifier_value>synthetic

construction</NonEnglishQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>ctccttctcctccacccacat</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

</ST26SequenceListing>

<---

Claims (11)

1. Rhabdovirus-negative cell line of the insect Spodoptera frugiperda WSK-Sf9 with accession number CCTCC C202246 for the production of recombinant protein based on the baculovirus expression system. 2. Use of the WSK-Sf9 cell line according to claim 1 for producing a recombinant protein based on a baculovirus expression system. 3. The use according to paragraph 2, wherein the recombinant protein obtained on the basis of the baculovirus expression system is used to produce a medicinal product or vaccine. 4. The use according to claim 3, wherein the medicinal product based on a recombinant protein comprises a cytokine, a hormone, a recombinant enzyme or an antibody. 5. The use according to claim 4, wherein the cytokine comprises recombinant human interleukin, recombinant human epidermal growth factor, recombinant human interferon, recombinant human fibroblast growth factor, recombinant human erythropoietin or recombinant human granulocyte-macrophage stimulating factor. 6. The use according to claim 4, wherein the hormone comprises recombinant human growth hormone, recombinant human insulin, an insulin analogue, or recombinant human follicle-stimulating hormone. 7. The use according to claim 4, wherein the recombinant enzyme comprises recombinant human alpha-glucosidase or recombinant human prourokinase. 8. The use according to claim 4, wherein the antibody comprises a monoclonal antibody, a Fab antibody, an scFv antibody or a nanobody. 9. The use according to claim 3, wherein the vaccine comprises a recombinant protein-based vaccine or a virus-like particle-based vaccine. 10. The use according to claim 9, wherein the recombinant protein-based vaccine comprises a SARS-CoV-2 protein-based vaccine, an influenza virus protein-based vaccine, a recombinant syncytial virus protein-based vaccine, a hepatitis B virus protein-based vaccine or a rabies virus protein-based vaccine, and preferably a SARS-CoV-2 protein-based vaccine. 11. The use according to claim 9, wherein the virus-like particle vaccine comprises a SARS-CoV-2 virus-like particle vaccine, a human papillomavirus virus-like particle vaccine, an influenza virus-like particle vaccine, a poliovirus virus-like particle vaccine, a respiratory syncytial virus virus-like particle vaccine, or an enterovirus vesicular stomatitis virus-like particle vaccine, and preferably a SARS-CoV-2 virus-like particle vaccine.