Vaccine delivery system based on yeast surface double expression and application thereof

Abstract

The invention provides a nucleic acid construct for a yeast surface double expression vaccine delivery system. The nucleic acid construct comprises: (a) a promoter element active in a yeast cell; and (b) at least two heterologous polynucleotides fused to the yeast surface display polypeptides, respectively. The invention also provides corresponding polypeptide, recombinant yeast, subunit vaccine composition and application thereof. Vaccine potency assays in mice show that a double fusion expressed RBD Saccharomyces cerevisiae vaccine elicits more powerful humoral and mucosal immune responses than conventional Saccharomyces cerevisiae vaccines in which RBD is fusion expressed only on Aga2p. The invention shows a double-expression saccharomyces cerevisiae system with higher cost benefit, and the double-expression saccharomyces cerevisiae system not only has potential in the aspect of resisting COVID-19, but also can be applied to the development of vaccines for other infectious diseases.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering technology, and in particular to a vaccine delivery system based on double expression on yeast surface and application thereof. Background Art

[0002] In recent years, yeast-based vaccines, especially Saccharomyces cerevisiae vaccines, have shown great promise due to their excellent safety, cost-effectiveness and rapid growth rate. Saccharomyces cerevisiae has been used to develop potential oral vaccines against various infectious diseases. Traditional oral Saccharomyces cerevisiae vaccines usually rely on the Aga1p-Aga2p YSD (yeast surface display) system of α-agglutinin (composed of two subunits, Aga1p and Aga2p), in which the candidate antigen is fused with the Aga2p protein and covalently linked to the cell wall-associated Aga1p protein through two disulfide bonds. For example, the scheme disclosed in the following literature.

[0003] Chinese patent document CN113322271A discloses a COVID-19 subunit vaccine based on a yeast surface display system. The new vaccine is obtained by fusion-expressing the new coronavirus antigen gene RBD gene and the lectin receptor Aga2 subunit through the yeast surface display system, and displaying the new coronavirus surface antigen RBD on the yeast surface.

[0004] Chinese patent document CN113528564A discloses a nucleic acid construct for heterologously expressing SARSCoV-2 antigens on the surface of yeast cells, related polypeptides, recombinant yeast cells, vaccine compositions, oral dosage formulations, and methods for inducing antigen-specific immune responses to SARS CoV-2. SARS-CoV-2 antigens are derived from SARS-CoV-2 spike protein ("S protein"), S1 subunit of SARS-CoV-2 S protein ("S1 subunit" or "S1 protein"), and receptor binding domain ("RBD") of S1 subunit. The polypeptide displayed on the surface of yeast is an α-lectin polypeptide or a fragment thereof, such as an Aga2 peptide, which interacts with an Aga1 α-lectin subunit expressed on the surface of yeast cells (such as EBY100 cells) through a disulfide bridge.

[0005] It can be seen that the existing technologies are all displayed through Aga2p. It has been observed that fusing the target protein to Aga1p on the surface of yeast can increase the display efficiency by two times compared with fusing to Aga2p. However, there is limited research on the potential of using Saccharomyces cerevisiae based on Aga1p display antigen as an oral vaccine, its ability to co-express the target antigen with Aga1p, and whether it can effectively enhance immunogenicity. Summary of the invention

[0006] The present invention aims at improving the expression and immunogenicity of antigens displayed on the surface of yeast, and provides a new strategy of fusing double-expressed antigens on Aga1p and Aga2p, respectively, to solve the problems existing in the above-mentioned prior art.

[0007] In order to achieve the above technical objectives, the present invention adopts the following technical solutions: A nucleic acid construct for a vaccine delivery system with dual expression on the surface of yeast, the nucleic acid construct comprising: (a) a promoter element active in yeast cells; and (b) at least two heterologous polynucleotides fused to polypeptides displayed on the surface of yeast, respectively, wherein the heterologous polynucleotides encode SARS-CoV-2 antigens or active fragments or combinations thereof.

[0008] Preferably, the SARS-CoV-2 antigen or an active fragment or combination thereof is derived from the S protein of SARS-CoV-2.

[0009] Preferably, the SARS-CoV-2 antigen or its active fragment or combination is derived from the S1 subunit of the S protein of SARS-CoV-2.

[0010] More preferably, the SARS-CoV-2 antigen or its active fragment or combination is from the RBD region of SARS-CoV-2 or a combination thereof.

[0011] Further preferably, the yeast surface displayed polypeptide is an α-lectin polypeptide or a fragment thereof.

[0012] Preferably, the α-lectin polypeptide or fragment thereof is Aga1 peptide and / or Aga2 peptide.

[0013] More preferably, the heterologous polynucleotides are located in different yeast display vectors YIP and / or pCTCON2, respectively.

[0014] Also preferably, the heterologous polynucleotides are respectively under the control of different promoters, and the promoters are selected from TEF2, PGK1 and GAL promoters.

[0015] The present invention also provides a polypeptide or a mature form thereof, wherein the polypeptide is expressed by the above nucleic acid construct.

[0016] The present invention also provides a recombinant yeast comprising the above-mentioned nucleic acid construct or polypeptide, wherein the recombinant yeast displays the SARS-CoV-2 antigen or an active fragment or combination thereof on its cell surface.

[0017] The present invention also provides a subunit vaccine composition, which comprises an effective amount of the above-mentioned recombinant yeast or an extract thereof.

[0018] The present invention also provides the use of the above-mentioned nucleic acid construct, polypeptide, recombinant yeast or subunit vaccine composition in the preparation of a medicament for preventing or treating COVID-19.

[0019] The present invention has the following beneficial effects: The present invention innovatively proposed a new strategy of fusing dual-expressed antigens on Aga1p and Aga2p, respectively, to improve antigen expression and immunogenicity. As a result, a brewer's yeast vaccine strain that co-expressed the receptor binding domain (RBD) of the SARS-CoV-2 S protein was successfully obtained. Compared with the traditional brewer's yeast vaccine that only expressed RBD by fusion on Aga2p, the dual-fusion-expressed RBD brewer's yeast vaccine triggered a stronger humoral and mucosal immune response. This dual expression system can not only be used for the prevention and control of SARS-CoV-2 infection, but is also expected to be used in the development of vaccines for other diseases.

[0020] The present invention establishes a powerful dual-expression yeast system and an inducible dual-expression yeast system by modifying the saccharomyces cerevisiae expression plasmid and optimizing the expression method.

[0021] The present invention conducted a comparative analysis on the expression of various spike proteins of SARS-CoV-2 on the surface of Saccharomyces cerevisiae, and successfully generated a Saccharomyces cerevisiae vaccine strain with stable dual expression and inducible dual expression of RBD.

[0022] In the present invention, GAL and PGK1 promoters were selected to construct a more stable dual-expression RBD yeast strain to improve the expression level and immunogenicity of RBD. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 Different strategies for yeast-presented oral vaccines versus traditional vaccines are shown.

[0024] Figure 2 The figure shows the expression of different SARS-CoV-2 S proteins on the surface of Saccharomyces cerevisiae; 2A is a schematic diagram of the structural layout of S, 3RBD, 2RBD and RBD proteins; 2B is the flow cytometry analysis of the expression levels of different S proteins fused with Aga1p; 2C is the flow cytometry analysis of the expression levels of different S proteins fused with Aga2p.

[0025] Figure 3 The figure shows the display of RBD on the surface of Saccharomyces cerevisiae through different promoters; 3A is the flow cytometric analysis of the expression of RBD fused with Aga1p; 3B is a schematic diagram of the pCTCON2 plasmid and Saccharomyces cerevisiae structure used for RBD expression; 3C is the flow cytometric analysis of the expression of RBD fused with Aga2p before induction; 3D is the flow cytometric analysis of the expression of RBD fused with Aga2p 72 hours after induction.

[0026] Figure 4 The analysis of Saccharomyces cerevisiae expressing RBD is shown; 4A shows the vectors and schematic diagram of the Saccharomyces cerevisiae structure showing dual-expression and single-expression RBD; 4B evaluates the expression of RBD in Saccharomyces cerevisiae by flow cytometry analysis using anti-RBD antibodies. Among them, PCT-RBD and YIP-RBD / PCT-RBD groups were tested 72 hours after induction, while Empty, YIP-RBD, and YIP-RBD / PCT-PGK1-RBD groups were not tested; 4C quantifies the expression level of RBD in Saccharomyces cerevisiae by enzyme-linked immunosorbent assay (ELISA); 4D analyzes the expression of RBD in Saccharomyces cerevisiae by flow cytometry using ACE2; 4E uses anti-His antibody for Western blot analysis to confirm the expression of RBD fused to Aga2p; 4F evaluates the expression of RBD in Saccharomyces cerevisiae by immunofluorescence assay.

[0027] Figure 5 The humoral and mucosal immune responses induced by RBD-expressing Saccharomyces cerevisiae are shown; 5A is the time of mouse immunization and serum sampling; 5B is the RBD-specific IgG and IgA serum antibodies at different time points after immunization; 5C is the RBD-specific IgA fecal antibodies after three immunizations; 5D is the pseudovirus neutralization test after three immunizations, and each data point represents one animal. For statistical analysis, t-tests were performed to compare the empty vector group and the PBS group. The significance levels are indicated as follows: ns: not significant; *: p < 0.05; **: p < 0.01; ***: p < 0.001; ****: p < 0.0001.

[0028] Figure 6 The cellular immune response analysis of spleen cells from mice immunized with Saccharomyces cerevisiae expressing RBD is shown; 6A shows the measurement of cytokine secretion in mice (on day 52) that received three oral immunizations after RBD stimulation, and the RBD-stimulated cytokine concentrations were subtracted from the non-stimulated control for accurate measurement; 6B shows the cytokine secretion levels after RBD stimulation by heat map; 6C shows the heat map of total cellular cytokine response by intracellular cytokine staining analysis; 6D shows the flow cytometry analysis by intracellular cytokine staining to assess the total cellular cytokine response, and the levels of IFN-γ, TNF-α, and IL-4 secreted by CD4+T and CD8+T cells were quantitatively assessed. The RBD-stimulated cytokine concentrations were subtracted from the non-stimulated control for accurate measurement. DETAILED DESCRIPTION

[0029] The following is further described in detail through specific implementation methods. However, it should be pointed out that the following embodiments of the present invention are only for better illustrating the content of the present invention, but do not mean that the content of the present invention is limited to the examples. Therefore, those skilled in the art can make non-essential improvements and adjustments to the implementation scheme according to the above invention content, which still belongs to the protection scope of the present invention and is subject to the protection scope of the attached claims.

[0030] The term "new coronavirus infection" as used in the present invention refers to an infection caused by the new coronavirus, which the World Health Organization has named "COVID-19". Therefore, in the present invention, "new coronavirus infection" and "COVID-19" are synonymous terms that can be used interchangeably.

[0031] The term "new coronavirus" used in the present invention is called acute respiratory syndrome coronavirus 2, or SARS-CoV-2, which is a virus that causes new coronavirus infection. "New coronavirus" and "SARS-COV-2" are synonymous terms that can be used interchangeably. The new coronavirus is a beta coronavirus of the Coronavirus family. It is a single-stranded positive-strand RNA virus with an envelope structure, containing four main structural proteins: three membrane proteins, namely, spike protein (S), small envelope protein (E) and membrane glycoprotein (M), and one nucleocapsid protein (N) that binds to viral RNA. Among them, the S protein is a homotrimer that can recognize and bind to host cell surface receptors. It plays a key role in mediating the fusion of the viral envelope and the cell membrane. It is not only the main immunogen of the virus, but also the main target for the development of vaccines and antiviral drugs. The S protein consists of two subunits, S1 and S2. The S1 subunit is divided into the N-terminal domain (NTD) and the C-terminal region containing the receptor binding domain (RBD). The S2 subunit contains the basic elements required for the membrane fusion process, such as the fusion peptide, two 7-peptide repeat sequences HR (hepeat repeat region, HR) and the transmembrane region. Similar to SARS-CoV, SARS-CoV-2 enters the cell through two processes: RBD binds to angiotensin converting enzyme 2 (ACE2) on the surface of the host cell, and S2 protein mediates membrane fusion.

[0032] The present invention used plasmids YIP and pCTCON2 (Addgene, USA) for vector construction, and Escherichia coli JM109 competent cells (Takara, Japan) for constructing expression vectors. Saccharomyces cerevisiae BJ5464 (BNCC, Suzhou, China) was used as the host cell for S protein surface display and cultured in YPD medium (2% peptone, 1% yeast extract, and 2% glucose) at 30°C. For yeast screening, SC-Ura medium (0.67% yeast nitrogen base without amino acids, 0.01% adenine, arginine, cysteine, leucine, lysine, threonine, and 0.005% aspartic acid, histidine, isoleucine, methionine, phenylalanine, proline, serine, tyrosine, valine, containing 2% agar) and SC-Trp-Ura medium (0.67% yeast nitrogen base without amino acids, 0.01% adenine, arginine, cysteine, leucine, lysine, threonine, and 0.005% aspartic acid, histidine, isoleucine, methionine, phenylalanine, proline, serine, tyrosine, valine, containing 2% agar) are used. Plasmid components, vector components, yeast cell culture and other techniques are well known to those skilled in the art.

[0033] The present invention uses GraphPad Prism 7.0 software for statistical analysis. All data are analyzed using a two-sided independent Student's t test. These software and analysis methods are well known to those skilled in the art.

[0034] If the technology, instruments, equipment and materials used in the present invention are not particularly specified, any other technology, instruments, equipment and materials known to those skilled in the art that can achieve the same purpose may be used; even if the technology, instruments, equipment and materials used in the present invention are particularly specified, it does not mean that the present invention can only use these technologies, instruments, equipment and materials, which only represent the preferred embodiments of the present invention, and those skilled in the art can still use any other technology, instruments, equipment and materials known to those skilled in the art that can achieve the same purpose.

[0035] Example 1 Construction of recombinant plasmid and dual expression Saccharomyces cerevisiae 1.1 Construction of recombinant plasmid and surface display in Saccharomyces cerevisiae The SARS-CoV-2 Delta variant S gene (NC_045512.2) and its receptor binding domain (RBD: R317-F539), RBD dimer (composed of two tandem repeats of RBD, 2RBD), and RBD trimer (composed of three tandem repeats of RBD, 3RBD) were synthesized and integrated into the yeast surface display plasmids YIP and pCTCON2. The constructed plasmids were then transformed into competent E. coli JM109 cells. After plasmids were extracted from the transformed E. coli, electroporation was performed to introduce the plasmids into competent Saccharomyces cerevisiae BJ5464 cells. These yeast cells were cultured on selective plates at 30°C for about three days to promote colony formation. Once single clones of PCT-RBD, YIP-RBD / PCT-RBD, and YIP-RBD / PCT-PGK1-RBD yeast strains were obtained, they were cultured in SC-Trp-Ura liquid medium (without 2% agar), respectively. In addition, YIP-RBD clones were cultured in SC-Ura liquid medium (without 2% agar) at 30°C overnight. PCT-RBD and YIP-RBD / PCT-RBD yeast strains were cultured in SC-Trp-Ura liquid medium containing 2% lactose instead of glucose and induced at 20°C for 72 h. Saccharomyces cerevisiae BJ5464 containing empty YIP and pCTCON2 plasmids served as negative controls.

[0036] 1.2 Displaying SARS-CoV-2 S proteins of different lengths on the surface of Saccharomyces cerevisiae Due to the limited secretion efficiency of Saccharomyces cerevisiae, the expression of foreign proteins in yeast is restricted to a certain extent, especially for proteins larger than 30 kDa, whose secretion is challenging. Therefore, we first compared and analyzed the expression levels of S proteins of different lengths.

[0037] In the present invention, Saccharomyces cerevisiae BJ5464 was used for protein expression. As shown in FIG2A , different strategies were used to express S, 3RBD, 2RBD, and RBD fused with Aga1p or Aga2p. As expected, successful expression of 3RBD, 2RBD, and RBD fused with Aga1p was observed ( Figure 2 B), in which RBD showed the highest expression level, while the expression level of S protein was significantly lower. When S, 3RBD, 2RBD and RBD were fused with Aga2p ( Figure 2C) All four proteins were successfully expressed, with RBD having a very high cellular display rate, with an average positive cell rate of 58.63%, exceeding the previously reported rate for displaying SARS-CoV-2 RBD on Aga2p in Saccharomyces cerevisiae.

[0038] 1.3 Expression of SARS-CoV-2 RBD protein on the surface of Saccharomyces cerevisiae using different promoters In order to enhance the expression of RBD, the present invention studied the effects of different promoters on the surface display of RBD in Saccharomyces cerevisiae. As shown in FIG3A, TEF2, PGK1 and GAP promoters were used to construct YIP plasmids for RBD expression. The results showed that all three promoters could effectively express RBD fused to Aga1p, among which the GAP promoter showed the highest expression level. In FIG3B, the PCTCON2 plasmid was constructed using TEF2, PGK1 and GAL promoters for RBD expression. TEF2 and PGK1 are constitutive promoters, while GAL is an inducible promoter. The expression level of RBD and the proportion of positive cells were measured at 0 hours (before induction) and 72 hours after induction. As shown in FIG3C, PGK1 and TEF2 promoters can express RBD fused to Aga2p without induction, among which the PGK1 promoter showed higher expression levels and positive cell rates compared with the TEF2 promoter group. The average positive cell rate of the PGK1 promoter was 82.07%, and the average positive cell rate of the TEF2 promoter was 69.73%. As shown in Figure 3D, no significant increase in the positive cell rate of the PGK1 and TEF2 promoter groups was observed after 72 hours of galactose induction. However, without induction, the expression of the GAL promoter was minimal; however, after a 72-hour induction period, the positive cell rate reached an impressive 60.63%, exceeding the PGK1 and TEF2 promoters. Therefore, the PGK1 promoter with the best constitutive expression and the GAL promoter with the best inducible expression were used in the subsequent construction of the dual expression system.

[0039] Example 2 Identification of dual-expression Saccharomyces cerevisiae 2.1 Flow cytometric analysis and immunofluorescence assay Based on the construction of Example 1, the samples were collected at 1.0 OD 600 Yeast cell pellet at nm (approximately 10 7cells) and washed three times with 500 µL sterile phosphate-buffered saline (PBS). Subsequently, the yeast pellet was incubated with 1:100 dilution of human anti-SARS-Cov-2 RBD protein antibody (kept in this laboratory) and ACE2-hFc (Atagenix, France) for 1 hour at 4°C. For flow cytometric analysis, yeast cells were then incubated with 1:500 dilution of goat anti-human IgG conjugated to 633 (Invitrogen, USA) for 30 minutes at 4°C. Yeast cells were resuspended in 500 µL sterile PBS and analyzed by flow cytometry (BDVerse, BD). For immunofluorescence assay, yeast cells were incubated with 1:100 dilution of FITC-conjugated goat anti-human IgG (Sigma, GRE) for 30 minutes at 4°C. Afterwards, yeast cells were resuspended in 500 µL sterile PBS, and 5µL of yeast cell pellet was used for subsequent analysis.

[0040] 2.2 Quantification of RBD by enzyme-linked immunosorbent assay (ELISA) To evaluate the expression of S protein on the yeast surface, an indirect ELISA was performed to assess the binding of monoclonal antibodies (mAbs) to yeast cells. 600 The yeast culture was harvested at 400 nm and washed twice with PBS. The cells were then resuspended in 100 µL of HRP-conjugated anti-RBD IgG solution serially diluted in PBS containing 1% bovine serum albumin (BSA) and incubated at room temperature for 1 hour to allow antibody binding. After incubation, the cells were washed three times with PBS to remove unbound antibodies. Next, 100 µL of HRP substrate 3,3′,5,5′-tetramethylbenzidine (TMB) (Solarbio, Beijing, China) was added to the cell suspension and incubated at room temperature for 5 minutes to allow the color reaction to develop. The reaction was stopped by adding 100 µL of 2 mol / L H2SO4. The absorbance of the supernatant was measured at 450 nm using a Biotek Synergy H1 microplate reader (USA) to quantify the extent of antibody binding as an indicator of S protein expression on the yeast surface.

[0041] 2.3 Western blot analysis For western blot analysis, place the cells at 1.0 OD 600The yeast cell pellet collected at nm was washed three times with 500 µL sterile PBS. The pellet was resuspended in 5 µL of 5× SDS loading buffer (Epizyme Biomedical, Shanghai, China) and 20 µL sterile distilled water. The suspension was boiled for 15 min to prepare the sample. The sample was then separated using SDS-PAGE and transferred to a PVDF membrane (Invitrogen, USA). The membrane was blocked in TBST buffer (0.1% Tween-20, 150 mM NaCl, and 10 mM Tris, pH 8.3) containing 5% skim milk for 1 hour at room temperature to prevent nonspecific binding. After blocking, the membrane was incubated with mouse HRP-conjugated anti-His antibody (Proteintech Group, Inc., USA) at room temperature for 1 hour. The membrane was washed three times with TBST for 10 minutes each. Finally, protein bands were detected using ECL substrate (Bio-Rad, USA) and visualized using the ChemiDoc imaging system (Bio-Rad, USA).

[0042] As shown in the figure, the present invention constructed a dual-expression yeast strain and a single-expression yeast strain in Saccharomyces cerevisiae by using YIP and PCTCON2 plasmids as controls for the traditional system, and an empty plasmid as a negative control (Figure 4A). In order to verify the expression of the constructed yeast strain, the present invention performed flow cytometry analysis, enzyme-linked immunosorbent assay (ELISA), protein blotting and immunofluorescence analysis. As shown in Figures 4B and 4C, flow cytometry analysis and ELISA showed that the expression level of RBD in the dual-expression Saccharomyces cerevisiae strains (YIP-RBD / PCT-RBD and YIP-RBD / PCT-PGK1-RBD) was significantly higher than that in the single-expression strains. The average positive rates were 98.6% for YIP-RBD, 65.4% for PCT-RBD, 99.4% for YIP-RBD / PCT-PGK1-RBD, and 99.1% for YIP-RBD / PCT-RBD. The corresponding EC 50The values were 0.1268 µg / mL for YIP-RBD, 0.1418 µg / mL for PCT-RBD, 0.05901 µg / mL for YIP-RBD / PCT-PGK1-RBD, and 0.05072 µg / mL for YIP-RBD / PCT-RBD. In addition, flow cytometry using ACE2 confirmed that both single-expressed and double-expressed RBDs retained their affinity for ACE2 (Figure 4D). Western blot analysis showed that the molecular weight of RBD fused with Aga2p was approximately 50 kDa, consistent with previous reports (Figure 4E). Immunofluorescence microscopy showed distinct green fluorescence in the fusion proteins expressed in yeast cells with double-expressed and single-expressed RBDs, while no detectable green fluorescence was observed in negative control cells cultured under the same conditions (Figure 4F). In summary, we successfully constructed a dual-expression yeast display system for efficient expression of the RBD of SARS-CoV-2.

[0043] Example 3 Mouse Animal Experiment 3.1 Oral immunization of mice Animal studies were reviewed and approved by the Experimental Animal Ethics Committee of the Chongqing Academy of Animal Sciences (Permit Number: SYXK (Yu) 2022-0010). The cultured and induced yeast cells were washed twice with sterile PBS and adjusted to a final concentration of 0.3 OD 600 nm / µL. Six-week-old SPF female BALB / c mice used in the experiment were provided by Beijing Vital River Laboratory Animal Technology Co., Ltd. On days 0 and 1, mice (n = 4 per group) were orally immunized with 150 OD 600 nm of yeast cells. Subsequently, immunizations were performed on days 21-22 and 42-43, respectively. Yeast cells containing the empty expression plasmids YIP and pCTCON2 were used as negative controls.

[0044] 3.2 ELISA quantification of IgG and IgA responses in immunized mice Blood samples were collected from the mice on days 10, 31, and 52 after the primary immunization, and then the sera were separated to detect antibody levels and subsequently stored at -20°C.

[0045] 96-well microplates were coated with recombinant S protein (AtaGenix, Wuhan, China) at a concentration of 2 µg / mL and incubated overnight at 4°C. After washing with PBS containing 0.05% Tween 20 (PBST), the plates were blocked with PBST containing 1% BSA for 2 hours at room temperature. Serum and fecal samples were diluted 20-fold and added to the plates, followed by incubation at 37°C for 1 hour. HRP-conjugated goat anti-mouse IgG (Sigma, GRE) and HRP-conjugated goat anti-mouse IgA (Abcam, UK) were used as secondary antibodies and incubated at 37°C for 1 hour. The plates were then incubated with TMB substrate (Solarbio, Beijing, China) and the absorbance was quantified at OD450nm.

[0046] 3.3 Pseudovirus neutralization test Virus neutralization assay was performed using a luciferase reporter gene integrated into the SARS-CoV-2 Delta variant pseudovirus. Mouse sera were diluted 16, 32, 64, 128, 256, and 512 times and incubated with an equal volume of pseudovirus at room temperature for 1 hour. 293T-hACE2 cells were digested, counted, and then cultured at 2×10 4 Cells were seeded at a concentration of 10 cells / mL in a 96-well cell culture plate. The cells were incubated at 37°C, 5% CO2 for 12-16 hours, and luciferase activity was measured on a BioTek Synergy H1 multi-mode microplate reader using D-luciferin as a substrate. The pseudovirus neutralization percentage was calculated using the following formula: pseudovirus neutralization percentage (%) = (RLU value of pseudovirus control wells - RLU value of test wells) / (RLU value of pseudovirus control wells - RLU value of cell control wells). The half-maximal inhibitory concentration was determined by analyzing the data using GraphPad Prism software (GraphPad Software Inc., USA).

[0047] To investigate whether Saccharomyces cerevisiae expressing dual RBDs as oral vaccines provide superior immune responses than conventional Saccharomyces cerevisiae expressing single RBDs, mice were orally immunized with the obtained Saccharomyces cerevisiae dual-expression and single-expression strains, while the negative control group was treated with empty vector and PBS (Figure 5A). Blood samples were collected on days 10, 31, and 52 after the initial immunization to quantify the levels of RBD-specific IgG and IgA. The results showed that there was no significant difference in the levels of RBD-specific IgG and IgA in the serum of mice orally immunized with dual-expression and single-expression RBD Saccharomyces cerevisiae after the first immunization (day 10) compared with the empty vector and PBS groups (p>0.05). However, after the second immunization on day 31, the levels of RBD-specific IgG and IgA in the serum increased significantly, and further increased after the third immunization on day 52 (Figure 5B). Due to individual differences, the IgG and IgA levels of some mice increased more slowly, while those of others increased more rapidly. Therefore, the difference between the YIP-RBD / PCT-PGK1-RBD group and the PCT-RBD group became less obvious after the third immunization compared with the second immunization. Fecal samples were collected on day 52 after the third immunization to measure the levels of RBD-specific IgA, and the results were consistent with those of serum analysis (Fig. 5C).

[0048] Plaque reduction neutralization test (PRNT) was used to measure the neutralizing antibody titer in the sera of mice orally administered with S. cerevisiae. Serum samples were collected on day 52 after the third immunization for subsequent testing. The results showed that the level of neutralizing antibodies in the serum after immunization with S. cerevisiae expressing RBD was significantly increased compared with the PBS or empty vector group. Notably, the YIP-RBD / PCT-RBD group showed the highest antibody titer, reaching 94.585±33.215 (Figure 5D). Our results indicate that oral YIP-RBD / PCT-RBD dual-expressing S. cerevisiae induces stronger humoral and mucosal immune responses than traditional single-expressing S. cerevisiae, thereby providing stronger protection against SARS-CoV-2.

[0049] 3.4 Cytokine expression Splenocytes (1.0×10 6Cells were cultured at 4% paraformaldehyde (400 μg / well) in a medium containing 10% fetal bovine serum, 100 U / mL penicillin, 100 µg / mL streptomycin, 1 mM sodium pyruvate, and 50 µM β-mercaptoethanol (Gibco, USA). Cells were stimulated with 2 µg / mL RBD (AtaGenix, Wuhan, China) for 72 h at 37°C and 5% CO2. After stimulation, cell culture supernatants were collected for ELISA analysis to evaluate the expression levels of cytokines. Various cytokines were detected using commercial ELISA kits: interferon gamma (IFN-γ) and interleukin 4 (IL-4) were quantified using ELISA kits purchased from Invitrogen (USA); interleukin 6 (IL-6), interleukin 10 (IL-10), and tumor necrosis factor alpha (TNFα) were quantified using ELISA kits purchased from Boster (Wuhan, China). Lymphocytes were then washed with sterile PBS and stained with anti-CD8 and anti-CD4 antibodies (Biolegend, USA) for flow cytometric analysis after fixation and permeabilization. Subsequently, intracellular staining was performed by fixing and permeabilizing cells to enable detection of anti-IFN-γ, anti-TNFα, and anti-IL-4 antibodies (Biolegend, USA).

[0050] To evaluate T cell responses induced by oral immunization with RBD-expressing Saccharomyces cerevisiae, mice were euthanized on day 63 after primary immunization, and splenocytes were isolated and stimulated with RBD. As shown in Figures 6A-6B, cytokine secretion levels were measured after RBD stimulation. The results showed that the YIP-RBD / PCT-RBD group exhibited significantly higher levels of cytokine secretion, especially increased levels of cytokines IFN-γ and TNF-α secreted by Th1 cells, compared with the cytokines IL-6, IL-10, and IL-4 secreted by Th2 cells observed in the YIP-RBD, PCT-RBD, and YIP-RBD / PCT-PGK1-RBD groups. In addition, as Figure 6 As shown in C-6D, comparison of intracellular cytokine staining of lymphocytes in different groups showed that the YIP-RBD / PCT-RBD group showed a significantly higher abundance of specific T cells, indicating a stronger T cell immune response compared with the YIP-RBD, PCT-RBD, and YIP-RBD / PCT-PGK1-RBD groups. Overall, CD4+ T cells showed a more obvious response compared with CD8+ T cells, especially CD4+ IFN-γ+ and CD4+ TNF-α+ T cells showed a more obvious response than CD4+ IL-4+ T cells.

[0051] In summary, oral immunization with a Saccharomyces cerevisiae strain dually expressing YIP-RBD / PCT-RBD elicited a stronger cellular immune response than conventional single-expressing yeast strains and showed Th1 cell differentiation similar to that observed in individuals infected with SARS-CoV-2.

[0052] The above is only an embodiment of the present invention. The commonly known technical common sense in the scheme is not described in detail here. The ordinary technicians in the relevant field are aware of all the common technical knowledge in the technical field to which the invention belongs before the application date, can obtain all the existing technologies in the field, and have the ability to apply the conventional experimental means before that date. The ordinary technicians in the relevant field can improve and implement this scheme in combination with their own abilities under the enlightenment given by this application. Some typical commonly known technologies should not become obstacles for ordinary technicians in the relevant field to implement this application. It should be pointed out that for those skilled in the art, without departing from the structure of the present invention, they can also make some adjustments and improvements, which should also be regarded as the scope of protection of the present invention, which will not affect the effect of the implementation of the present invention and the practicality of the patent. The scope of protection claimed by this application shall be based on the content of its claims, and the specific implementation methods and other records in the specification can be used to interpret the content of the claims.

Claims

1. A nucleic acid construct for a vaccine delivery system for dual expression on the yeast surface, the nucleic acid construct comprising: (a) a promoter element active in yeast cells; and (b) at least two heterologous polynucleotides fused to polypeptides displayed on the yeast surface, respectively.

2. The nucleic acid construct of claim 1, wherein the heterologous polynucleotide encodes a viral antigen, preferably a SARS-CoV-2 antigen or an active fragment or combination thereof, more preferably an S protein from SARS-CoV-2, more preferably a SARS-CoV-2 S protein S1 subunit, and most preferably the RBD region of SARS-CoV-2 or a combination thereof.

3. The nucleic acid construct of claim 1 or 2, wherein the yeast surface displayed polypeptide is an α-lectin polypeptide or a fragment thereof. 4 . The nucleic acid construct according to claim 3 , wherein the α-lectin polypeptide or fragment thereof is an Aga1 peptide and / or an Aga2 peptide.

5. The nucleic acid construct according to any one of claims 1 to 4, wherein the heterologous polynucleotides are located in different yeast display vectors YIP and / or pCTCON2, respectively.

6. The nucleic acid construct of any one of claims 1-5, wherein the heterologous polynucleotides are respectively under the control of different promoters, and the promoters are selected from TEF2, PGK1 and GAL promoters.

7. A polypeptide or a mature form thereof, wherein the polypeptide is expressed by the nucleic acid construct according to any one of claims 1 to 6.

8. A recombinant yeast comprising the nucleic acid construct according to any one of claims 1 to 6 or the polypeptide according to claim 7, wherein the recombinant yeast displays the viral antigen product expressed by the heterologous polynucleotide or an active fragment or combination thereof on its cell surface.

9. A subunit vaccine composition comprising an effective amount of the recombinant yeast or an extract thereof according to claim 8.

10. Use of the nucleic acid construct of claims 1-6, the polypeptide of claim 7, the recombinant yeast of claim 8 or the subunit vaccine composition of claim 9 in the preparation of a medicament for preventing or treating COVID-19.

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