Method for preparing corona virus-like particles and vaccine comprising the same

Abstract

This invention provides a method for preparing coronavirus virus-like particles and a vaccine comprising the same. The method includes: constructing a recombinant baculovirus carrying a coronavirus spike protein gene; transducing the recombinant baculovirus into C6 / 36 cells; culturing the transduced C6 / 36 cells in serum-free medium to produce coronavirus virus-like particles, wherein the obtained coronavirus virus-like particles contain the coronavirus spike protein. Furthermore, the coronavirus spike protein gene may be derived from SARS-CoV, MERS-CoV, or SARS-CoV-2. Further, this invention also provides a vaccine comprising the coronavirus virus-like particles prepared by the above method, which can be administered via nasal drops or intramuscular injection.

Description

Technical Field

[0001] This invention relates to the technical fields of virology and vaccinology, and particularly to a method for preparing coronavirus-like particles (VLPs) and their applications. Specifically, this invention provides a novel method for producing coronavirus-like particles using recombinant baculovirus and serum-free culture medium, and the adjuvant-free vaccine prepared thereby is effective via nasal drops or intramuscular injection. Prior Technology

[0002] The coronavirus disease (COVID-19) pandemic has had a significant impact on global health and the economy. Although several vaccines have been developed, there is still room for improvement, particularly in inducing mucosal immunity and responding to viral mutations.

[0003] Traditional vaccines are primarily administered via intramuscular injection; however, many pathogens, including SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus), mainly enter the human body through mucosal surfaces. Therefore, developing mucosal immune vaccines that can induce antibody secretion in the respiratory tract is crucial for controlling SARS-CoV-2 and its variants.

[0004] Virus-like particles are excellent candidates for vaccinology due to their ideal antigenicity and safety. For example, the NVX-CoV2373 intramuscular injection vaccine (Novavax, USA) is an artificial nanoparticle vaccine against SARS-CoV-2, which has shown good safety and efficacy in clinical trials.

[0005] Intranasal administration has become an attractive method for delivering mucosal vaccines. Compared to intramuscular injection, intranasal vaccines may have advantages in inducing sterilization immunity against mucosal pathogens. However, the development of intranasal vaccines faces challenges such as the mucus barrier, epithelial barrier, and lack of compatible immune adjuvants.

[0006] In existing technologies, the production of coronavirus virus-like particles typically requires the co-expression of multiple structural proteins in mammalian or insect cells. This approach may increase the complexity and cost of production. Furthermore, many existing VLP production systems use serum-containing culture media, which may introduce additional variables and potential safety issues.

[0007] The inventors of this invention, recognizing the limitations and shortcomings in current coronavirus vaccine development, particularly the challenges in inducing mucosal immunity, addressing viral mutations, and the complexity of the production process, have developed this invention. In the prior art, the production of coronavirus virus-like particles (VLPs) typically requires the co-expression of multiple structural proteins in mammalian or insect cells, increasing production complexity and cost. Furthermore, many existing VLP production systems use serum-containing culture media, potentially introducing additional variables and safety issues. While intranasal vaccines show promise in inducing mucosal immunity, they still face challenges such as the mucus barrier, epithelial barrier, and lack of compatible immune adjuvants. Therefore, the inventors of this invention have diligently researched and developed a simplified method for producing VLPs while maintaining high efficiency and safety, and explored the possibility of adjuvant-free intranasal administration, ultimately completing the present invention's method for preparing coronavirus virus-like particles and a vaccine containing them. This invention provides a method for producing VLPs using a serum-free culture medium and a single-protein expression system, which not only simplifies the production process but also improves product consistency and safety. Furthermore, the adjuvant-free nasal drop vaccine formulation of the present invention is expected to induce a strong mucosal immune response, providing a new method for controlling SARS-CoV-2 and its variants. Summary of the Invention

[0008] The main objective of this invention is to provide a method for preparing coronavirus virus-like particles, which includes the following steps: a) Construct a recombinant baculovirus carrying a coronavirus spike protein gene; b) Transduce the recombinant baculovirus from step (a) into an insect cell; c) The insect cells transduced with the recombinant baculovirus in step (b) are cultured in a serum-free medium to produce a coronavirus-like particle; The coronavirus-like virus particles include a coronavirus spike protein.

[0009] In the above method, the insect cell is a mosquito cell, and the mosquito cell line is selected from C6 / 36 cells or AP-61 cells.

[0010] Furthermore, in the above method, the coronavirus spike protein gene is derived from severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), or severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2).

[0011] Furthermore, in the above method, the coronavirus spike protein gene is a prefusion-stabilized full-length spike protein gene, and contains mutations at the furin cleavage site and 2P (two proline) mutations.

[0012] Furthermore, in the above method, the recombinant baculovirus further comprises: One hr1 pag1 mosquito promoter; A signal peptide of the Japanese encephalitis virus pre-membrane protein (prM signal peptide). The ribosome entry site within the 5'-untranslated region of a common aphid virus (RhPV 5'-UTR IRES; Rhir = RhPV 5'-UTR IRES); An enhanced green fluorescent protein (eGFP) gene; and Translate the stop codon.

[0013] In one embodiment, the serum-free culture medium in the above method comprises: The medium consisted of RPMI-1640 medium, tryptose phosphate broth (TPB), pluronic® F-68, peptone primatone, yeast extract, and a lipid mixture; wherein the concentrations of tryptose phosphate broth were 0.3%, Pluronic® F-68 were 0.2%, peptone primatone were 0.5%, yeast extract was 0.4%, and lipid mixture was 0.1%.

[0014] Simultaneously, a secondary objective of this invention is to provide a vaccine comprising coronavirus virus-like particles obtained by the preparation method described above. This vaccine is administered via nasal or intramuscular injection and induces a strong mucosal and systemic immune response without the need for adjuvants. This adjuvant-free vaccine not only simplifies the production process but also reduces the risk of potential side effects caused by adjuvants. In particular, when administered nasally, the vaccine directly stimulates the respiratory mucosal immune system, providing first-line defense and effectively preventing the virus from spreading externally and multiplying in the human body during the initial infection phase. Furthermore, due to the use of coronavirus virus-like particles produced using serum-free culture medium, this vaccine exhibits higher safety and consistency. This novel vaccine is expected to play a significant role in the prevention and control of SARS-CoV-2 and its variants, providing a new tool for global public health. Simple Explanation of the Diagram

[0015] Figure 1 shows the basic structure of the BacMos system and the production process of coronavirus virus-like particles (VLPs); Figure 2A shows the immunofluorescence staining results of SARS-CoV-2 spike protein in transduced cells; Figure 2B shows the secretion of monomeric full-length spinin; Figure 2C shows the secretion of MERS-CoV-2 spike protein (wild type and 2P mutant) in transduced cells; Figure 2D shows the expression of SARS-CoV (2003) spike protein (2P mutant) in transduced cells; Figure 3A shows the results of sucrose density gradient centrifugation of SARS-CoV-2 VLPs; Figure 3B shows electron microscope images of SARS-CoV-2 VLPs; Figure 4A shows the binding affinity of SARS-CoV-2 VLPs to different receptors; Figure 4B shows the inhibition assay of SARS-CoV-2 VLPs binding to human ACE2; Figure 5A shows the immunization schedule for mice; Figures 5B-D show the responses of SARS-CoV-2 S-specific total IgG, IgG1, and IgG2a after the second and third immunizations; Figure 6A shows the systemic SARS-CoV-2 S-specific IgA response; Figure 6B shows the SARS-CoV-2 S-specific secretory IgA reaction in bronchoalveolar lavage fluid and intestinal mucosa; Figure 6C shows the SARS-CoV-2 S-specific total IgG reaction in the bronchoalveolar lavage fluid mucosa; Figure 7A shows the effect of systemic neutralizing antibody response on wild-type virus strains; Figure 7B shows the effect of systemic neutralizing antibody response on the variant viral strain; Figure 7C shows the mucosal neutralizing antibody response against wild-type and Omicron virus strains in bronchoalveolar lavage fluid; Figures 8A-8B show the duration of systemic responses induced by nasal or intramuscular immunization with SARS-CoV-2 VLPs; Figures 9A-9C show the protective effect of SARS-CoV-2 VLPs nasal drop vaccination on K18-hACE2 transgenic mice; Figure 10 shows the results of immunofluorescence analysis of MERS-CoV VLP production and expression; Figure 11 shows the Western blot analysis of MERS-CoV spike protein secretion; Figure 12A shows the tangential flow filtration (TFF) purification and Western blot analysis results of MERS-CoV VLPs; Figure 12B shows the results of sucrose gradient purification and spectrophotometry of MERS-CoV VLPs; Figure 13 shows the expression of SARS-CoV spike protein in transduced AP-61 cells and the results of immunofluorescence staining analysis; Figure 14 shows the Western blot analysis of the secretory expression of SARS-CoV spike protein in transduced mosquito cells; Figure 15 shows a comparison of the optimal production of SARS-CoV-2 VLPs under different cell lines, culture conditions, and MOIs; Figure 16 shows the optimization analysis of MERS-CoV VLPs production in C6 / 36 cells using serum-free medium (SFM); Figure 17 shows the optimization analysis of SARS-CoV VLPs production in C6 / 36 cells using serum-free medium (SFM); Figure 18A shows the experimental design for evaluating the immunogenicity and protective effect of SARS-CoV-2 VLPs in a hamster model; and Figure 18B shows a comparison of viral RNA content in the lungs of hamsters after SARS-CoV-2 VLPs vaccination. Implementation

[0016] All technical and scientific terms used in this specification, unless otherwise defined, shall have the meaning commonly understood by one of ordinary skill in the art. The invention will be illustrated by the following embodiments, but are merely illustrative and not limiting; the invention is not limited to the following embodiments. Unless otherwise stated, all materials used in this invention are commercially available and readily available; the following are merely examples of where they can be obtained.

[0017] Example 1: Construction of Recombinant Baculovirus Referring to Figure 1, this embodiment uses a baculovirus / mosquito (BacMos) system to construct a recombinant baculovirus carrying the spike protein gene of MERS-CoV, SARS-CoV, or SARS-CoV-2. This spike protein gene is a pre-fused stable version containing furin cleavage site mutations and 2P mutations. The recombinant virus also contains the hr1 pag1 mosquito promoter, JEV prM signal peptide, RhPV 5'-UTR IRES, eGFP gene, and translation stop codon.

[0018] 1. Gene Construction The spike protein gene of MERS-CoV, SARS-CoV, or SARS-CoV-2 was inserted into a baculovirus vector using the BacMos system. This gene was modified to include a furin cleavage site mutation (GSAS) and a 2P (proline) mutation (986 / 7P) to ensure stability in the prefusion conformation. The recombinant baculovirus construction contained the following components: - hr1 pag1: Mosquito promoter, used to initiate gene transcription; - JEV SP: Japanese encephalitis virus prM signal peptide, which promotes the expression of spike protein; - S1-S2: A prefusion-stabilized full-length spike gene containing mutations at the furin cleavage site and 2P (two proline residues) mutations; - Rhir: Ribosome entry site within the 5'-untranslated region of the common aphid virus (RhPV 5'-UTR IRES); - eGFP: Enhanced green fluorescent protein gene; - ▼: Translate the stop codon

[0019] Figure 1 illustrates the basic structure of the BacMos system and the production process of SARS-CoV-2 VLPs. The recombinant baculovirus (BacMos-monomeric S) contains the aforementioned DNA sequence and can effectively transduce mosquito cells, inducing the expression and secretion of SARS-CoV-2 spike protein.

[0020] 2. Virus preparation and transduction Recombinant baculovirus protein expression was performed in insect cells (C6 / 36 cell line, Aedes albopictus) using the BacMos system. A key innovation of this invention is the use of serum-free culture medium to culture the insect cells. Table 1 below provides a detailed comparison and characteristics of serum-free and traditional serum-containing culture media: Table 1. Comparison of serum-free and serum-containing culture media feature serum-free culture medium (Serum-free medium) Transitional cell culture medium (traditional serum-containing culture medium) (Transitional cell medium) Element Basic culture medium: RPMI-1640; 0.3% tryptose phosphate broth (TPB); 0.2% Poloxamer (Pluronic® F-68); 0.5% peptone primatone; 0.4% yeast extract (yeastolate); 0.1% lipid mixture. Basic culture medium: RPMI-1640; 10% serum (fetal bovine serum or bovine growth serum); Growth factors, etc. advantage 1. Safety: Animal serum may carry other unknown pathogens; serum-free culture avoids these potential risks. 2. Product consistency and controllability: Avoid batch-to-batch variations in serum and improve the reproducibility of VLP production; 3. Simplified purification process: Serum contains a large amount of protein and other components, requiring additional steps to remove them. 4. Reduce production costs: Serum is expensive, and most components of serum-free products can be aseptically processed using high temperature and high pressure. 5. To avoid batch-to-batch variations in serum and improve the reproducibility of VLP production; 6. Environmental and ethical advantages: Reduces the demand for animal-derived materials and responds to societal demands for animal protection and ethical considerations. 1. Applicable to the growth of most cell lines; 2. Optimized production conditions are readily available. shortcoming Different cell lines require different serum-free culture conditions, so it is necessary to try different approaches. 1. High price, increasing the cost of biological products; 2. There may be a risk of contamination at the source of the serum; 3. Serum has a complex composition and may become an allergen; 4. The products come from different batches and have significant quality differences.

[0021] It is worth noting that the serum-free culture medium composition used in this invention is particularly suitable for the production of SARS-CoV-2 VLPs and MERS-CoV VLPs using C6 / 36 (Aedes albopictus) cells, but may not be suitable for other mosquito-derived cell lines, such as AP-61 (Aedes pseudoscutellaris). This highlights the importance of optimizing culture conditions for specific cell lines. That is, the serum-free culture medium used in this invention ensures efficient cell growth and viral expression, which is beneficial for VLP vaccine production.

[0022] 3. Cultivation Strategies In practice, the present invention employs the following optimization strategy: 1. Use serum-containing medium for routine cell subculture to maintain stable cell growth. 2. Only switch to serum-free medium when VLPs production is required. 3. During the 12 days of VLP production, change the serum-free culture medium every 2-3 days. This strategy has the following advantages: 1. To maintain stable conditions for normal cell growth. 2. During the VLP production stage, both basic cell growth is maintained and VLP production is carried out in a serum-free state. 3. No subsequent serum removal step is required, thus reducing the cost of culture and purification.

[0023] By employing the aforementioned culture strategy, this invention successfully balances the needs of cell growth and VLP production, providing a reliable method for the efficient and low-cost production of high-quality VLPs. This method not only improves the yield and quality of VLPs but also significantly reduces production costs and potential contamination risks, laying the foundation for large-scale production of coronavirus virus-like particle vaccines.

[0024] Example 2: Identification of Coronavirus Virus Particles Figure 2A shows the immunofluorescence staining results of SARS-CoV-2 spike protein in transduced cells. The immunofluorescence staining results show the expression of SARS-CoV-2 spike protein in transduced cells. Transduced cells (top row) or mock cells (bottom row) were stained with Anti-S1 NT mAb1 antibody (left row), Anti-S1 NT mAb2 antibody (middle row), or Anti-S2 mAb (right row) and Hoechst 33342, respectively.

[0025] Figure 2B shows the secretion of monomeric full-length spike proteins. Western blot (WB) was used to analyze culture supernatants from BacMos-monomeric S-transduced mosquito cells and control cells to detect S1 (left and middle) or S2 (right). Polymeric and monomeric spike proteins (S) are indicated by arrows on the right, and the size (kDa) of the protein markers is shown on the left.

[0026] Figure 2C shows the results of the immunospot assay, which illustrates the secretion of MERS-CoV-2 spike protein (wild type and 2P mutant) in transduced cells.

[0027] Figure 2D shows the results of immunofluorescence staining and Western ink dot method, which shows the expression of SARS-CoV (2003) spike protein (2P mutant) in transduced cells.

[0028] Figures 2B-2C show the expression and secretion of spike proteins of different coronaviruses (including SARS-CoV-2, MERS-CoV-2 and SARS-CoV) in mosquito cells, and the production of these coronavirus virus-like particles (VLPs) was verified by immunofluorescence staining and immunoink dot method using the method described in Example 1.

[0029] Example 3: Production and purification of coronavirus virus-like particles (VLPs) This embodiment demonstrates a method for producing and purifying coronavirus virus-like particles (VLPs) using a baculovirus / mosquito (BacMos) system.

[0030] 1. Production of coronavirus virus-like particles (VLPs) 1.1 Expression System This embodiment uses a baculovirus-transduced mosquito cell line (C6 / 36) expressing a single S protein (spike protein) to produce coronavirus virus-like particles (VLPs). Specifically, the monomeric S protein, a pre-fusion version of the SARS-CoV-2 spike protein (S), with double mutations at the furin cleavage site and 2P, was expressed using the BacMos system.

[0031] 1.2 Assembly of coronavirus virus-like particles (VLPs) By using the multifunctional BacMos system to express a single spike protein, insect cells can efficiently assemble and release coronavirus-like particles, such as those of SARS-CoV-2, MERS-CoV, or SARS-CoV (2003), composed of approximately 40 nm spherical particles. Notably, unlike known techniques, in this embodiment, the BacMos system using a single vector to express the monomeric S protein (spike protein) is sufficient to efficiently assemble and produce functional SARS-CoV-2 VLPs that bind to the ACE2 (angiotensin-converting enzyme 2) receptor.

[0032] 1.3 Production Advantages The non-cytolytic nature of the BacMos system in this embodiment facilitates extended harvest time and simplifies downstream purification of coronavirus virus-like particles (VLPs). Furthermore, this method may allow for rapid adaptation to new variants of interest or other emerging beta coronaviruses.

[0033] 2. Purification of coronavirus virus-like particles (VLPs) 2.1 Sucrose gradient banding SARS-CoV-2 VLPs were isolated and purified using sucrose density gradient centrifugation. The specific steps are as follows: (1) Load the VLPs sample onto the sucrose density gradient. (2) Perform ultracentrifugation. (3) Collect the separated segments, a total of 12 segments from the top (No. 1) to the bottom (No. 12).

[0034] 2.2 Identification of purified coronavirus virus-like particles (VLPs) Western blot analysis was used to identify the purified VLPs. The specific steps are as follows: (1) Western blot analysis was performed on the 12 segments after centrifugation of the sucrose density gradient. (2) Detection was performed using anti-S1 monoclonal antibodies, such as anti-S1 mAb. (3) Observe and record the segments containing VLPs.

[0035] 2.3 Electron Microscopy Observation Transmission electron microscopy (TEM) was used to observe the partially purified VLPs to confirm their morphology and size.

[0036] Using the methods described in this embodiment, functional SARS-CoV-2, MERS-CoV, and SARS-CoV (2003) VLPs can be successfully produced and purified. These coronavirus virus-like particles (VLPs) maintain a stable pre-fusion conformation and possess functional antigenic epitopes capable of binding to the ACE2 receptor, laying the foundation for subsequent vaccine development.

[0037] Figures 3A and 3B show the characteristics of the coronavirus virus-like particles (VLPs) isolated in this embodiment. Figure 3 shows the separation of SARS-CoV-2 virus-like particles (VLPs) using a sucrose density gradient after ultracentrifugation. Fractions from 1 (top) to 12 (bottom) were separated by the sucrose density gradient and analyzed using Western ink dot assay with an anti-S1 monoclonal antibody (anti-S1 mAb). The protein size (kDa) of the marker (M) is shown on both sides. Figure 3B shows an electron microscope image of the SARS-CoV-2 virus-like particles (VLPs). Partially purified virus-like particles (VLPs) were examined using a transmission electron microscope (TEM). White arrows indicate virus-like particles. Scale bar is 50 nanometers (nm).

[0038] Example 4: Functional Analysis of Coronavirus Virus-like Particles (VLPs) This embodiment performed an ACE2 binding experiment, demonstrating that the coronavirus virus-like particles (VLPs) produced and purified in Example 3 have the ability to bind to the ACE2 receptor, indicating that they retain functional antigenic epitopes. Figure 4A shows the binding ability of SARS-CoV-2 VLPs to different receptors (hACE2, hDPP4, and MXRA8), while Figure 4B shows the inhibition assay of SARS-CoV-2 VLPs binding to human ACE2 (hACE2).

[0039] Figure 4A shows the test results for SARS-CoV-2 VLP-ACE2 binding. First, wells were coated with recombinant hACE2, hDPP4, or MXRA8. Mouse serum and SARS-CoV-2 VLPs were serially diluted and added to the pre-coated wells. The bound VLPs were then detected using an NT mAb (GTX635791) neutralizing monoclonal antibody. Figure 4A shows that as the concentration of SARS-CoV-2 VLPs increased, their binding to hACE2 increased significantly, while their binding to hDPP4 and MXRA8 was almost zero, demonstrating the specific binding of the coronavirus virus-like particles (VLPs) produced and purified in Example 3 to hACE2.

[0040] Figure 4B illustrates the binding inhibition assay of SARS-CoV-2 VLPs to ACE2. First, recombinant hACE2 protein was coated onto wells. SARS-CoV-2 VLPs, either untreated or pretreated with serially diluted positive control (sera from infected patient 1 and infected patient 2) or negative control (sera from healthy individuals 1 and healthy individuals 2), were added to the hACE2-coated wells. The bound VLPs were detected according to the method shown in Figure A. Figure 4B shows that untreated VLPs bound to hACE2, while the pretreated positive control serum showed significant binding inhibition, and the negative control serum showed no significant inhibition.

[0041] Example 5: Immunogenicity of Coronavirus Virus-like Particle Vaccine This example demonstrates the immunogenicity of coronavirus virus-like particles (VLPs) vaccines administered via nasal drop (IN) and intramuscular injection (IM).

[0042] 1. Immunization methods Immunization experiments were conducted using BALB / c mice. Mice were vaccinated with VLPs via nasal drop (IN) or intramuscular injection (IM), at doses of 2.5 μg or 10 μg, respectively. The immunization schedule consisted of a primary immunization and two booster immunizations, spaced 21 days apart.

[0043] 2. Systemic immune response 2.1 IgG reaction Following the second and third immunizations, serum levels of SARS-CoV-2 S-specific total IgG, IgG1, and IgG2a antibodies were measured. Results showed that the VLPs vaccine induced the production of high-affinity IgG antibodies and exhibited a balanced S-specific IgG2a (Th1) / IgG1 (Th2) response.

[0044] 2.2 Neutralizing antibody response The levels of neutralizing antibodies against the wild-type (WT) strain of SARS-CoV-2 in serum were assessed using a pseudovirus neutralization assay. Results showed that both the IN and IM pathways induced effective neutralizing antibody responses, with ID50 titers ranging from 100 to 1800. Notably, at high-dose (10 μg) immunization, the IN pathway induced 3.64 times more neutralizing antibodies than the IM pathway (mean ID50 titer of IN / IM = 1800 / 500).

[0045] 2.3 Cross-neutralizing activity The cross-neutralizing activity of serum against SARS-CoV-2 variants (including Alpha, Delta, and Omicron) was further evaluated. Results showed that the VLPs vaccine induced broad-spectrum neutralizing antibodies against multiple SARS-CoV-2 variants.

[0046] 3. Mucosal immune response 3.1 Secretory IgA (sIgA) response Significantly elevated levels of S-specific sIgA antibodies were detected in bronchoalveolar lavage fluid (BALF) and the small intestine. Notably, only the IN pathway could induce mucosal sIgA production in BALF.

[0047] 3.2 Mucosal neutralizing antibodies Mucosal neutralizing antibodies against SARS-CoV-2 WT and Omicron pseudoviruses were detected in BALF.

[0048] 4. Persistent immunity The persistence of the immune response was assessed. The results showed that a significant systemic antibody response was still detectable 4 months after the third immunization.

[0049] In vivo protective effect The in vivo protective efficacy of VLPs vaccines was evaluated using a K18-hACE2 transgenic mouse model. Results showed that nasal drop administration of the adjuvant-free SARS-CoV-2 VLP vaccine protected mice from lethal doses of SARS-CoV-2 WT virus and provided cross-protection against the Omicron variant.

[0050] In summary, this embodiment demonstrates that the coronavirus virus-like particle vaccine produced and purified according to Embodiment 3 of this case possesses excellent immunogenicity, can induce strong systemic and mucosal immune responses, and provides effective protection against SARS-CoV-2 and its variants. In particular, intranasal administration showed superior immunogenicity compared to intramuscular injection, providing important evidence for the development of novel COVID-19 vaccines.

[0051] Figures 5A-5D show the production of S-specific antibodies (including total IgG, IgG1, and IgG2a) in mice after different vaccination methods, nasal drops (IN) and intramuscular injections (IM), and different numbers of vaccinations, used to evaluate the immune response efficacy of the coronavirus unadjuvanted virus-like particle nasal drop vaccine.

[0052] Figure 5A shows the immunization schedule (nasal drop (IN) and intramuscular injection (IM)) and blood collection time points in mice (BALB / c strain). Figures 5B-D show the responses of SARS-CoV-2 S-specific total IgG (5B), IgG1 (5C), and IgG2a (Figure 5D) after the second (grey) and third (black) immunizations, respectively. The results in Figures 5B-5D show that both nasal drop (IN) and intramuscular injection (IM) routes induced strong SARS-CoV-2 S-specific antibody responses.

[0053] Figure 5B shows the response of SARS-CoV-2 S protein-specific total IgG antibodies. The vertical axis represents antibody titer, and the horizontal axis shows different immunization routes (IN or IM) and doses (2.5 or 10 μg). Gray bars represent the results after the second immunization, and black bars represent the results after the third immunization. Figure 5B shows that both IN and IM immunizations induce significant IgG antibody responses. After the third immunization, antibody levels further increased. In particular, the high-dose (10 μg) IN route showed the best performance after the third immunization.

[0054] Figure 5C shows the response to SARS-CoV-2 S protein-specific IgG1 antibodies. The vertical axis, horizontal axis, and colors represent the same values ​​as in Figure 5B. The results indicate that IgG1 antibodies were produced in all immunized groups. Both the IN and IM pathways can induce IgG1 responses, but the IN group (especially the high-dose group) showed better performance after the third immunization.

[0055] Figure 5D shows the response to SARS-CoV-2 S protein-specific IgG2a antibodies. The vertical axis, horizontal axis, and colors represent the same values ​​as in the previous two figures. The results indicate that IgG2a antibodies were produced in all immunization groups. Notably, the IN pathway (especially the high-dose group) was more effective in inducing the IgG2a response, particularly after the third immunization.

[0056] Figures 6A-6C illustrate how nasal drop (IN) immunization with SARS-CoV-2 virus-like particles (VLPs) induces mucosal and systemic immune responses. Figure 6A shows systemic SARS-CoV-2 S-specific IgA responses after the second (grey) and third (black) immunizations. Figure 6B shows SARS-CoV-2 S-specific secretory IgA responses in the bronchoalveolar lavage fluid (BALF) (left) and small intestine (right) mucosa after the second boost. Figure 6C shows the SARS-CoV-2 S-specific total IgG response in the bronchoalveolar lavage fluid mucosa after the second boost. The results in Figures 6A-6C show that nasal drop inoculation of SARS-CoV-2 VLPs induces strong mucosal and systemic immune responses. In particular, the IN route induces significant S-specific IgA responses in serum, bronchoalveolar lavage fluid (BALF), and the small intestine. Notably, only the IN route induces mucosal sIgA production in BALF, which is crucial for preventing viral infection at the respiratory tract inlet.

[0057] Figures 7A-7C illustrate systemic and mucosal neutralizing antibody responses. Figure 7A shows the systemic neutralizing antibody response against wild-type (WT) virus strains after the second (grey) and third (black) immunizations. Figure 7B shows the systemic neutralizing antibody response against variants of concern (VOCs) after the second boost. Figure 7C shows the mucosal neutralizing antibody response against wild-type (black) and Omicron (grey) virus strains in bronchoalveolar lavage fluid (BALF) after the third boost. The results in Figures 7A-7C demonstrate that SARS-CoV-2 VLPs vaccines can induce strong systemic and mucosal neutralizing antibody responses. In particular, the IN pathway performed exceptionally well in inducing broad-spectrum neutralizing antibodies against wild-type and multiple variants, including Alpha, Delta, and Omicron. In addition, IN immunization can induce mucosal neutralizing antibodies against wild-type and Omicron strains in BALF, which is very important for the prevention and spread of upper respiratory tract infections.

[0058] Figures 8A-8B illustrate the duration of systemic responses induced by SARS-CoV-2 virus-like particle nasal drop (IN) or intramuscular injection (IM) immunization. Systemic SARS-CoV-2 S-specific total IgG (see Figure 8A) and neutralization response (see Figure 8B) are shown at 0.5 months and 4 months after the third injection. The results in Figures 8A-8B demonstrate that SARS-CoV-2 VLPs vaccines can induce a durable systemic immune response. In particular, significant levels of S-specific IgG and neutralizing antibodies were still detectable 4 months after the last immunization. The IN route outperformed the IM route in maintaining a long-term immune response, which is crucial for providing durable protection.

[0059] Figures 9A-9C illustrate how intranasal vaccination with SARS-CoV-2 virus-like particles (VLPs) protects K18-hACE2 transgenic mice from SARS-CoV-2 infection. Figure 9A details the timeline of mouse immunization (nasal drop (IN) or intramuscular injection (IM)) and exposure to the pathogen (i.e., challenge). Mice were immunized with SARS-CoV-2 virus-like particles (10 μg per mouse) via nasal drop (IN) or intramuscular injection (IM) and received booster doses at 21 and 42 days post-immunization (dpi). Two weeks after the last immunization, K18-hACE2 mice were intranasally infected with 20 μl of SARS-CoV-2 prototype virus in PBS containing 10⁴ PFU. Figure 9B shows the viral RNA load detected by qRT-PCR in lung and brain tissue samples obtained from each group (n=5) of mice (nasal drop vaccine (IN VLPs), intramuscular vaccine (IM VLPs), and no vaccine (NV)) three days after SARS-CoV-2 infection. Figure 9C shows the survival results of K18-hACE2 transgenic mice (n=4). The results in Figures 9A-9C show that nasal drop vaccination with SARS-CoV-2 VLPs effectively protects K18-hACE2 transgenic mice from fatal SARS-CoV-2 infection. In particular, the viral load in the lungs and brain of mice in the IN VLP group was significantly reduced. These results strongly suggest that nasal drop vaccination with SARS-CoV-2 VLPs provides strong protection and is expected to be an effective strategy for preventing COVID-19.

[0060] This invention demonstrates, through a series of experiments, the effectiveness and superiority of SARS-CoV-2 virus-like particles (VLPs) produced based on the BacMos system as a nasal drop vaccine. The experimental results and their significance will be described in detail below.

[0061] Traditional vaccines are primarily administered via intramuscular injection. However, many pathogens, including SARS-CoV-2, enter the body mainly through mucosal surfaces. Therefore, intranasal vaccination presents an attractive alternative for mucosal vaccine delivery. Experiments have also demonstrated that intramuscular COVID-19 vaccines are insufficient to control SARS-CoV-2 replication and viral shedding in the upper respiratory tract and may lead to asymptomatic transmission or milder symptomatic infection. Conversely, intranasal vaccines have shown the potential to induce sterilizing immunity against mucosal pathogens.

[0062] In this study, the inventors demonstrated that a mosquito cell line (C6 / 36) transduced with a baculovirus expressing a single S protein (spike protein) can efficiently assemble and release SARS-CoV-2 (see Figures 2A, 2B, 3A, 3B), MERS-CoV (see Figure 2C), and SARS-CoV (2003) VLPs (see Figure 2D) consisting of approximately 40 nm spherical particles. Furthermore, the minimum molecular requirements for coronavirus VLP assembly can vary; for example, in the prior art, co-expression of multiple structural proteins is necessary for coronavirus VLP assembly in mammalian and other insect cells. In contrast, this invention innovatively utilizes the BacMos system, which expresses a single S protein via a single vector, to efficiently assemble and produce functional SARS-CoV-2 VLPs that bind to the ACE2 receptor (see Figures 4A, 4B). This breakthrough not only simplifies the production process but also allows for rapid adaptation to new variants of interest or other emerging β-coronaviruses. Furthermore, the BacMos system of this invention is non-cytolytic, which facilitates extended harvest time and simplifies downstream purification of VLPs.

[0063] Furthermore, unlike intramuscular injection, the nasal drop vaccine provided by this invention is needle-free and does not require a sterile environment. This not only improves patient compliance and reduces professional training costs, but also minimizes the risk of bloodborne diseases. More importantly, it can induce mucosal and systemic immunity. Although challenges such as the mucus barrier, epithelial barrier, and lack of compatible immune adjuvants have hindered the efficacy of nasal vaccination, the nanoparticle device provided by this invention offers an innovative solution to these challenges. Specifically, the nanoparticle vaccine of this invention enhances the permeability of antigens across the nasal mucosal barrier, ensuring entry into the nasal cavity immune stimulation site, while resisting vaccine degradation, prolonging retention time in the nasal environment, and stimulating a long-term immune response. In addition, the nanoparticles of this invention have an intrinsic adjuvant effect, which can enhance the immunogenicity of subunit vaccines.

[0064] In this invention, the inventors developed novel insect-derived SARS-CoV-2 VLPs. These VLPs (virus-like particles) were generated from a monomer-based construct (a pre-fused version of the SARS-CoV-2 spike protein (S) with double mutants at the furin cleavage site and 2P) and were evaluated as potent nasal vaccine (IN) candidates. Experimental results showed that this VLP vaccine could simultaneously induce mucosal and systemic immunity in a mouse model. This dual immune response has potentially significant implications for the prevention of SARS-CoV-2 infection and transmission.

[0065] The self-assisted VLPs of this invention, administered via nasal drops (IN) or intramuscular injection (IM) in two doses (2.5 μg or 10 μg per dose), effectively elicited high-affinity IgG titers (see Figure 5B), a balanced S-specific IgG2a / IgG1 response, and potent neutralizing antibodies (ID50 titers versus values ​​of 100 and 1800) (see Figure 7A), all against wild-type (WT) viral strains. Notably, after three doses, the titers of S-specific total IgG, IgG2a, IgG1, and neutralizing antibodies were significantly enhanced in the nasal drop group (see Figures 5C, 5D, and 7A). In contrast, no significant enhancement of these titers was observed in the intramuscular injection group after the third dose. This result highlights the superiority of nasal drop administration of the VLPs vaccine of this invention. Furthermore, the Th1 (IgG2a) response induced by this invention (see Figure 5D) may be related to CD8+ T cell immune surveillance, thereby enhancing viral clearance. More importantly, compared with intramuscular injection (IM) vaccination, nasal drop (IN) vaccination induced a 3.6-fold increase in neutralizing antibody (NT) response at high doses (mean ID50 titer of IN / IM = 1800 / 500) (see Figure 7A), further demonstrating the superiority of the nasal drop vaccine of the present invention.

[0066] Secretory IgA antibodies (sIgA) are the main effector of mucosal immunity, and their ability to combat SARS-CoV-2 infection is superior to that of IgG. Studies have shown that resistance to SARS-CoV-2 mucosal infection is closely related to the presence of sIgA in animal models. In this invention, the inventors discovered that nasal drop (IN) immunization with VLPs not only induced systemic neutralizing antibodies against wild-type (WT)-S and variant viral strains (VOCs) (Alpha, Delta, and Omicron)-S pseudoviruses in serum (see Figure 7B), but also observed mucosal neutralizing antibody responses against WT-S and Omicron-S pseudoviruses in bronchoalveolar lavage fluid (BALF) (see Figure 7C). This finding highlights the potential of the VLPs vaccine of this invention in inducing comprehensive immune protection.

[0067] The nasal drop, adjuvant-free prototype SARS-CoV-2 VLPs vaccine of this invention protected K18-hACE2 mice from lethal SARS-CoV-2 challenge (see Figures 9A, 9B, 9C) and provided cross-protection against SARS-CoV-2 Omicron variant challenge (not shown). These results demonstrate that nasal drop (IN) immunization with VLPs can establish systemic and mucosal immunity, thereby providing critical protection against SARS-CoV-2 variant viral strains (VOCs). The nasal drop (IN) vaccine can induce pan-neutralizing antibodies against variant viral strains, a property that is particularly attractive for preventing the spread of new COVID-19 variants. Specifically, nasal drop (IN) immunization with VLPs provides high levels of neutralizing IgA in the upper respiratory tract, serving as first-line defense against Omicron subvariant infection and transmission.

[0068] Notably, while both nasal (IN) and intramuscular (IM) administration of SARS-CoV-2 VLPs can trigger an immune response, the stimulatory effect of IN is significantly stronger. Specifically, mucosal sIgA in BALF is stimulated only via the IN route (see Figures 6A, 6B, and 6C). This study suggests that sIgA can function by interfering with viral infection at both entry and exit points. In contrast, intramuscular injection of wild-type SARS-CoV-2 VLPs did not produce a neutralizing titer against the SARS-CoV-2 WT strain (data not shown).

[0069] However, when SARS-CoV-2 VLPs with a stable pre-fusion conformation were administered via nasal drop (IN) or intramuscular injection (IM), highly effective neutralizing antibodies against SARS-CoV-2 WT and VOCs (Alpha and Omicron) were observed. These results further demonstrate that the SARS-CoV-2 VLPs S (2P) candidate vaccine of the present invention can prevent infection with different variant virus strains.

[0070] Furthermore, this invention utilizes a strategy of enhancing mucosal immunity by altering the physicochemical properties of nanoparticles, including controlling the size range of 20-200 nm in diameter and using positively charged nanoparticles, as well as partially targeting specific cells through carbohydrates and other components, and combining mucosal adjuvant activity. Specifically, antigens encapsulated in carefully designed particles (such as the VLPs of this invention) can be more effectively recognized by the innate immune system and can act as a stronger inducer of mucosal immune responses compared to soluble counterparts. The mosquito cell-derived VLPs used in this invention are associated with specific ligands, can target microfolded cell (M cell)-specific molecules, and are absorbed by M cells in the nasal cavity and bronchial-associated lymphoid tissue, and then transported across the epithelial barrier. The highly repetitive surface antigenic epitopes of these VLPs are associated with effective pathogen-associated molecular patterns, bind to pattern recognition receptors of innate and adaptive immune cells (e.g., Toll-like receptors, nod-like receptors, and RIG1-like receptors), and produce strong in vivo immunogenicity.

[0071] More importantly, since VLPs are inherently immunogenic, they can serve not only as multivalent scaffolds for three-dimensional antigen display but also as adjuvants to enhance immune responses. Therefore, the functional SARS-CoV-2 VLPs with specific ligands of this invention adhere well to the nasal epithelium, cross the epithelial barrier, and induce strong mucosal and durable systemic immunity (see Figures 8A and 8B).

[0072] In summary, this invention provides a novel method for producing functional SARS-CoV-2 VLPs with inherent self-help properties and excellent safety, achieved through the innovative BacMos monomeric S (WT strain) transduced mosquito cell system of this invention. Studies of this invention show that nasal drop inoculation of these SARS-CoV-2 VLPs leads to the production of S-specific IgG and IgA antibodies, along with broadly neutralizing antibodies effective against various SARS-CoV-2 variants. More importantly, this vaccination regimen induces mucosal cross-neutralizing activity against the Omicron variant and establishes durable systemic immunity. Particularly noteworthy is that the nasal drop vaccination of this invention is superior to the traditional intramuscular injection route in evoking an immune response.

[0073] Given the crucial role of mucosal transmission in SARS-CoV-2 infection, this invention successfully transforms nasal drop administration of self-assisted SARS-CoV-2 VLPs into a potential clinical mucosal vaccination strategy, supported by ample scientific data. This method not only promises to enhance the control of SARS-CoV-2 but may also provide new insights for the prevention of emerging coronavirus infections.

[0074] Example 6: Production and Identification of MERS-CoV VLPs This embodiment demonstrates a method for producing MERS-CoV VLPs using a baculovirus / mosquito (BacMos) system and the identification results.

[0075] 1. Production of MERS-CoV VLPs A recombinant baculovirus carrying the MERS-CoV spike protein gene was constructed using the BacMos system. This spike protein gene was a pre-fused stable version containing a furin cleavage site mutation and a 2P mutation. The recombinant virus also contained the hr1 pag1 mosquito promoter, the JEV prM signal peptide, the RhPV 5'-UTR IRES, the eGFP gene, and a translation stop codon.

[0076] 2. Expression and secretion of MERS-CoV VLPs As shown in Figure 10, immunofluorescence staining results demonstrated successful expression of MERS-CoV spike protein in transduced C6 / 36 cells. Transduced cells (top row) and control cells (bottom row) were stained with Anti-MERS-CoV S RBD monoclonal antibody (antibody targeting the receptor-binding domain (RBD) of MERS-CoV spike protein; NT Ab: neutralizing antibody) (left column), Anti-MERS-CoV S ECD monoclonal antibody (antibody targeting the extracellular domain (ECD) of MERS-CoV spike protein; NT Ab: neutralizing antibody) (middle column), and Rabbit poly anti-MERS-CoV S multiclonal antibody (rabbit-derived multiclonal antibody targeting MERS-CoV spike protein) (right column), respectively, and nuclear staining was performed using Hoechst 33342. Scale bar: 30 μm.

[0077] Figure 11 illustrates the secretion of MERS-CoV spike protein in transduced cells. Culture supernatants from BacMos-monomeric MERS-CoV S transduced mosquito cells (T) and control cells (m) were analyzed using Western blot. Spike protein was detected under both non-reducing and denaturing conditions, and three antibodies were used for detection: Anti-Pan CoVs (pan-coronavirus antibody) (ECD), Anti-MERS-CoV S RBD (receptor-binding region targeting MERS-CoV spike protein), and Anti-MERS-CoV S ECD (extracellular region targeting MERS-CoV spike protein). The figure shows the protein bands detected under different conditions.

[0078] Furthermore, Figure 10 shows the successful expression of MERS-CoV spike protein in transduced C6 / 36 cells, with obvious fluorescent signals observed by different antibodies (RBD, ECD, and full-length S protein). Figure 11 shows that the signal of MERS-CoV spike protein was detected in the culture supernatant of transduced cells (T), but not in control cells (m). That is, the transduced C6 / 36 cells successfully secreted the monomeric full-length spike protein of MERS-CoV, and this protein could be detected by antibodies targeting different regions (such as RBD and ECD). In addition, pan-coronavirus antibodies (Anti-Pan CoVs) showed that the secretion of these spike proteins has universality across coronaviruses, indicating that the system of the present invention successfully generated biologically functional MERS-CoV spike protein.

[0079] 3. Purification and identification of MERS-CoV VLPs As shown in Figure 12A, MERS-CoV VLPs were concentrated using a MidiKros Module Tangential Flow Filtration (TFF) system (300,000 molecular weight cutoff, Spectrum Repligen, Rancho Dominguez, CA, USA). Each fraction of the TFF (ori: input, ft: flow through, conc.: after TFF) was analyzed by Western blot and detected using an anti-MERS-CoV ECD monoclonal antibody (NT Ab) under non-reducing conditions.

[0080] As shown in Figure 12B, the concentrated VLPs were then loaded onto a sucrose gradient (20 / 60%). After another round (3 hours) of centrifugation at 250,000 x g, eight fractions (from top to bottom) were collected and dot blot analysis was performed using the Anti-MERS ECD monoclonal antibody (NT Ab).

[0081] Based on the results in Figures 12A and 12B, it can be seen that this invention has successfully developed an efficient method for the concentration and purification of MERS-CoV virus-like particles (VLPs). Tangential flow filtration (TFF) significantly concentrated the VLPs, as can be seen from the Western blot analysis in Figure 12A, where the concentrated sample (conc.) exhibited the strongest signal. Subsequent sucrose density gradient centrifugation further separated the VLPs, and the speckle analysis in Figure 12B shows that the VLPs were mainly concentrated in fragments 4, 5, 6, and 7. This two-step method not only effectively concentrated the VLPs but also achieved high-purity separation, providing high-quality MERS-CoV VLP samples for subsequent vaccine development and related research. In other words, this method demonstrates great potential in the production and purification of coronavirus VLPs.

[0082] Example 7: Production and Identification of SARS-CoV VLPs This example demonstrates a method for producing SARS-CoV VLPs using a baculovirus / mosquito (BacMos) system and the identification results.

[0083] 1. Production of SARS-CoV VLPs A recombinant baculovirus carrying the SARS-CoV spike protein gene was constructed using the BacMos system. This spike protein gene was a pre-fused stable version containing a furin cleavage site mutation and a 2P mutation. The recombinant virus also contained the hr1 pag1 mosquito promoter, JEV prM signal peptide, RhPV 5'-UTR IRES, eGFP gene, and a translation stop codon.

[0084] 2. Production and expression of SARS-CoV VLPs As shown in Figure 13, immunofluorescence staining results indicate that SARS-CoV spike protein was successfully expressed in transduced AP-61 cells. Transduced cells (top row) and control cells (bottom row) were stained with Anti-SARS-CoV / SARS-CoV-2 S1 monoclonal antibody (NT Ab) (left column), Anti-SARS-CoV-2 RBD monoclonal antibody (middle column), and Anti-SARS-CoV / SARS-CoV-2 S2 monoclonal antibody (right column), respectively, and nuclear staining was performed using Hoechst 33342. Scale bar: 30 μm.

[0085] 3. Secretion of SARS-CoV VLPs Figure 14 also illustrates the secretion of SARS-CoV spike protein in transduced cells. Western blot analysis was performed on culture supernatants of BacMos-monomeric SARS-CoV S-transduced mosquito cells (T) and control cells (M), using Anti-SARS-CoV / SARS-CoV-2 S1 monoclonal antibody (NT Ab) (left panel) and Anti-SARS-CoV / SARS-CoV-2 S2 monoclonal antibody (right panel) under non-reducing conditions. The protein molecular weight markers (kDa) are indicated on the left.

[0086] These results demonstrate that the method of this invention successfully expressed SARS-CoV spike proteins in AP-61 cells, and that these proteins could be secreted into the culture medium. Immunofluorescence staining and Western blot analysis both confirmed the successful production of SARS-CoV VLPs.

[0087] Example 8: Optimization of SARS-CoV-2 VLP Production This example demonstrates the optimized production results of SARS-CoV-2 VLPs under different cell and culture conditions.

[0088] As shown in Figure 15, this invention compares the production of SARS-CoV-2 (S2P) VLPs in AP-61 and C6 / 36 cells. 1. For AP-61 cells, three different culture media were used: L15+FBS, L15+SFM, and VP+SFM, and three different MOIs (viral transduction doses) were tested: 20, 40, and 80. 2. For C6 / 36 cells, two culture media were used: RPMI+FBS and RPMI+SFM, and three MOIs were also tested: 20, 40, and 80.

[0089] VLP yield in culture supernatant was analyzed using Western blot and detected using SARS-CoV-2 RBD monoclonal antibody (NT Ab) under non-reducing conditions. Development was performed using Amersham ECL Select (RPN2235) (high sensitivity).

[0090] The results showed that the yield of SARS-CoV-2 (S2P) VLPs varied under different cell lines, culture media, and MOI conditions. This provides an important reference for further optimizing the production of SARS-CoV-2 VLPs. Specifically, the yield of SARS-CoV-2 (S2P) VLPs was optimal in C6 / 36 cells using RPMI+SFM (serum-free medium); and under RPMI+SFM conditions, the signal intensity gradually increased with increasing MOI (20 to 40 to 80), indicating that VLP yield increased with increasing MOI. In summary, the optimal production conditions for SARS-CoV-2 (S2P) VLPs are: cell line: C6 / 36, culture medium: RPMI+SFM (serum-free medium), MOI: 80. This combination of conditions produced the strongest signal, indicating the highest VLP yield, providing an important reference for large-scale production of SARS-CoV-2 (S2P) VLPs.

[0091] Example 9: Optimization of MERS-CoV VLP Production This embodiment demonstrates the optimized production results of MERS-CoV VLPs. As shown in Figure 16, this invention optimized the production of MERS-CoV (S2P) VLPs in C6 / 36 cells using SFM medium. Different MOIs (1.69, 3.38, 6.75, 13.5, 27, 54) and different harvest times (3, 5, 7, 10, 12, 14 days post-transduction) were tested. Detection was performed using an Anti-Pan CoVs ECD antibody, with an ECL select development time of 3 seconds.

[0092] The results showed that when using SFM medium in C6 / 36 cells, the yield of MERS-CoV VLPs varied with increasing MOI and prolonged harvest time. This provides an important reference for the large-scale production of MERS-CoV VLPs. Specifically, under an MOI of 54, VLP production was clearly observed from day 3, and the yield remained stable from day 3 to day 14, exhibiting long-term stable production characteristics. This characteristic not only helps to increase the total yield but may also reduce production costs, which is of great significance for industrial production.

[0093] Example 10: Optimization of SARS-CoV VLP Production This embodiment demonstrates the optimized production results of SARS-CoV VLPs. As shown in Figure 17, the production of SARS-CoV (S2P) VLPs was optimized in C6 / 36 cells using SFM medium. Different MOIs (5, 10, 20, 40, 80, 160) and different harvest times (3, 5, 7, 10, 12, 14 days post-transduction) were tested. Detection was performed using Anti-SARS-CoV / SARS-CoV-2 S antibody, with ECL select imaging time of 1 minute.

[0094] The results showed that different MOIs and harvest times significantly affected the yield of SARS-CoV VLPs when using SFM medium in C6 / 36 cells. These data provide important reference for the large-scale production of SARS-CoV VLPs. Specifically, the cumulative yield of VLPs increased significantly with prolonged culture time, especially under MOIs of 80 and 160, continuing until day 14, exhibiting long-term stable production characteristics. This continuous production model provides the possibility for optimizing harvesting strategies and increasing total yield, helping to improve production efficiency and reduce costs.

[0095] Example 11: Protective Immunization Experiment of SARS-CoV-2 VLPs in a Hamster Model This embodiment evaluated the immunogenicity and protective effect of SARS-CoV-2 VLPs in a hamster model.

[0096] As shown in Figure 18A, the experimental design was as follows: Hamsters (N=2) were inoculated with 10 μg of VLPs via nasal drop (IN) or intramuscular injection (IM), or PBS was used as a negative control for intranasal administration. One week after completing the two doses of vaccination, the vaccinated hamsters were co-housed with hamsters challenged with wild-type (WT) SARS-CoV-2 virus for one day to conduct the challenge experiment.

[0097] Figure 18B shows the viral RNA content in the lungs of hamsters in each group on day 3 post-infection (dpi). Results include the following groups: Unvaccinated hamsters were housed together with hamsters that had been challenged with the virus. Nasal drop vaccine co-housing group: Hamsters vaccinated with SARS-CoV-2 VLPs via nasal drop (IN) were co-housed with challenged hamsters; Intramuscularly administered vaccine co-hospital group: Hamsters vaccinated with SARS-CoV-2 VLPs via intramuscular injection (IM) were co-housed with challenged hamsters; Control group: Hamsters that served as the source of infection, measured on day 4 post-infection.

[0098] The results showed that, compared with the control group, the viral RNA levels in the lungs of hamsters vaccinated with VLPs (regardless of whether the inhalation or intramuscular route) were significantly reduced. This indicates that SARS-CoV-2 VLPs can induce protective immunity against SARS-CoV-2 in hamster models by both nasal drop and intramuscular injection. Notably, the viral RNA levels in the lungs of hamsters vaccinated with VLPs via nasal drop were lower than those in the intramuscular injection group.

[0099] Specifically, Figure 18B shows the viral RNA levels in the lungs of hamsters in the control group, the unvaccinated group, the nasal drop vaccine group, and the intramuscular vaccine group. By comparing the viral RNA levels in different groups, the significant effect of VLP vaccination (especially nasal drop administration) on reducing viral replication can be clearly seen.

[0100] These results further confirm the effectiveness of the SARS-CoV-2 VLPs vaccine of this invention, particularly the superiority of the nasal drop administration route in reducing viral load in the upper respiratory tract. This not only helps reduce individual infection symptoms but may also reduce the risk of virus transmission, providing a new strategy for controlling the spread of SARS-CoV-2.

[0101] In summary, this embodiment demonstrates the protective efficacy of the SARS-CoV-2 VLPs vaccine of this invention in a hamster model, particularly highlighting the advantages of the nasal drop administration route. The experimental results clearly show that, compared to the unvaccinated control group, hamsters vaccinated with VLPs (especially the nasal drop group) exhibited significant protection against SARS-CoV-2 infection. This provides important experimental evidence for the potential of this invention in clinical applications and also offers a new direction for developing more effective COVID-19 prevention strategies.

[0102] The foregoing description has fully and clearly illustrated a method for preparing coronavirus-like virus particles and a vaccine comprising them according to the present invention. It must be emphasized that the detailed description above is a specific illustration of feasible embodiments of the present invention; however, these embodiments are not intended to limit the patent scope of the present invention. All equivalent implementations or modifications that do not depart from the spirit of the present invention should be included within the patent scope of this case.

Claims

1. A method for preparing coronavirus virus-like particles, comprising the following steps: a) constructing a recombinant baculovirus carrying a coronavirus spike protein gene, wherein the recombinant baculovirus includes the hr1 pag1 mosquito promoter, the Japanese encephalitis virus pre-membrane protein signal peptide, the RhPV 5'-UTR IRES (RhPV 5'-UTR IRES; Rhir = RhPV 5'-UTR IRES) ribosome entry site in the 5'-untranslated region of aphid virus, an enhanced green fluorescent protein (eGFP) gene, and a translation stop codon; b) transducing C6 / 36 cells using the recombinant baculovirus of step (a); c) culturing the C6 / 36 cells transduced with the recombinant baculovirus in a serum-free medium to produce coronavirus virus-like particles; wherein, The coronavirus spike protein gene is derived from the full-length spike protein gene of SARS-CoV, MERS-CoV, or SARS-CoV-2, and the full-length spike protein gene contains a furin cleavage site mutation and a 2P mutation; wherein, the serum-free medium is a medium based on RPMI-1640 medium, supplemented with 0.3% tryptose phosphate broth (TPB), 0.2% poloxamer F-68, 0.5% peptone primatone, 0.4% yeast extract, and 0.1% lipid mixture.

2. A vaccine comprising coronavirus virus-like particles produced using the preparation method described in claim 1.

3. The vaccine as described in claim 2, wherein, The vaccine is administered via nasal drops or intramuscular injection.

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